SIMPÓSIO INTERNACIONAL SÔBRE VENENOS ANIMAIS

INTERNATIONAL SYMPOSIUM ON ANIMAL VENOMS

INSTITUTO BUTANTAN

17 a 23 de julho de 1966

FASCÍCULO 1

ÍNDICE — INDEX Pág.

I. Dados biográficos de VITAL BRAZIL. XI

Biographical data of VITAL BRAZIL.

II. Sessão inaugural — Inaugural meeting.

"Discurso introdutório” — Mário Machado de Lemos, Secretário da

Saúde Pública e da Assistência Social, representante do Governo do

Estado de São Paulo . Xll

"Transcendence of VITAL BRAZIL s work — Bernardo A. Ifous-

say (Argentina) . XIII

“Conferência inaugural” — A. Vallejo-Freire (Brasil) . XVII

III. Animais venenosos — Venomous animals

1. Venomous Marine Animais of Brazil — /i. IV. Halstead (E.U.A.) 1

2. Taxonomia de A NT II O ZOA (COELENTER ATA) bra¬

sileiros: distribuição e frequência em águas brasileiras — I). D.

Corrêa (Brasil) . 27

3. Toxic Marine Invertebrates — Venomous and Noxious Fishes

of Fresh Water — P. Sawaya (Brasil) . 31

4. La fonction venimeuse chez les araignés — /. Vellard ( Bolívia) 35

5. El araneísmo en el mundo tropical y subtropical — R. Gajardo-

Tobar (Chile) . 45

SciELO

10 11 12 13 14 15

VIII

ÍNDICE

Bág. 1

6 .

Biological significance of cutaneous secretions íd loads and frogs

- B. Lutz 1 Brasil) .

55 j|

Taxonomy and distribution of arrow-poison frogs in Colondiia

D. M. Cochran (E.l .A.) .

61 1

C) .

Modes of evolution in New World Opisthoglyph Snakes - ./. R.

Bailey (E.l .A.) .

67 1

Poisonous snakes of Surinam — F. D. Brongersma (Holanda) . .

73 1

10 .

Ecology of roek-viper (Vipera xanthina raddei Boettger) in lhe

natural surroundings of Armênia — /. S. Darevsky (URSS) . . .

81 1

11 .

Unterlagen zur Õkologie, Ethologie und Evolution der Baum-

schlangen — R. Mertens (Alemanha) .

85 ]

12 .

Biology and ecology of venomous animais in Israel — A. S/iulov

(Israel) .

9.3

13.

Observations on lhe biology of sea-snakes (HIDROPHIIDAE) with

remarks on their systematics — K. Klemmer (Alemanha) ....

K )1

14.

Le cycle sexuel des serpents venimeux — //. Sainl Girons (França)

105

15.

The ovarian cycle of Natrix rhombifera — an apparently general-

ized cycle of snakes of temperate latitudes — 7'. W. Bctz (E.l .A.)

115

16.

Vertebrate hormones as defence substances in Dytiscides -

//. Schildknecht (Alemanha) .

121

17.

Pesquisas de citologia quantitativa. XIX — DNA e volume nu¬

clear nos tecidos somáticos dos vertebrados — G. Schreiber, N. B.

Melucci, S. E. Gerken, Y. X. Sand Ana, L. A. Fallieri, F. 0.

Amorim (Brasil) .

135

18.

Evolution and sex chromosomes in SERPENTES — II . Bcçak,

M . L. Bcçak e II . Nazareth (Brasil) .

151

19.

Karyotypes of South-American ARANEIDA — /17. 0. Diaz e F.

A. Saez (Uruguai) .

153

20.

Evolution of Vertebrates Genomes — S. Ohno (E.l.A.) ....

155

21 .

PENTASTOMIDA of snakes. Their parasitological role in man

and animais — A. Faia (Bélgica) .

167

1 IV.

PATOLOGIA 1)0 ENVENENAMENTO E PREVEÇÃO de acidentes -

Pathology OE ENVENOMATION and PREVENTION oe accidents

22.

The diagnosis, symptoms, treatment and sequella of envenomation

by Crotalus adamanteus and genus A gkistrodon — N. C.

McCollough (E.U.A.) .

175

10 11 12 13 14 15

ÍNDICE

IX 1

I‘dg. 1

23.

The influence of snake venom on fihrinogen conversion and fi-

brinolysis — F. Kornulík (Checoslováquia) .

179 !

2 !.

Clinicai manifestations of snake hite hy Vipera xanlhina pules-

tinae (Werner) and their pathophysiological hasis — /'. Efrati

(Israel) .

189

1 25.

Biochemical and pathological aspects of hemorrhagic principies

in snake venoms with special reference to Habu (T rimeresurus

flavoviridis) venom — A. Ohsaka, T. Omori-Satoh. II. Kondo,

S. Kondo & R. Murata (Japão) .

193

26.

Latrodectismo y loxoscelismo en Chile. Incidência, características

clínicas, pronóstico, Iratamiento y prevención — //. Schenone

(Chile) .

207

27.

The clinico-pathology and treatment of snakebite in Southern

and Central África — I). S. Chapman (África do Sul) .

213

i 28.

Poisonous snake biles in Germany — //. Lieske (Alemanha) ..

227

1 29.

Diagnosis of Snake Rite — E. R. Tretheuie & R. Raivlinson

(Austrália) .

235

30.

Comments of the moderator G. Rosenfeld (Brasil 1 .

241

V. Imunologia — Immunology

31.

The preparation and purifieation of antivenoms — P. A. Chris-

tensen (África do Sul) .

245

j 32.

Antivenin testing at different venom leveis — P. Krag e M. II eis

Bentzon (Dinamarca) .

251

1 o 9

DD .

The role of enzymes in the processes responsible for lhe toxicity

of snake venoms (an immunological study) — 0. Zwisler (Ale-

manha) .

281

34.

Venom and antivenin specificity: modem concept — A. do

Amaral (Brasil) .

293

1 35.

Venom and anti venom potency estimation — P. A. Christensen

(África do Sul) .

305

36.

Cross immunological reactions in snake venoms — C. Purana-

nanda, P. Lauhatirananda, S. Ganthavorn (Tailândia) .

327 |

37.

Problems in delermination of antihemorrhagic potency of Habu

(Trimeresurus flavoviridis) antivenine in the presence of multiple

hemorrhagic principies and their antihodies — A. Ohsaka. II.

Kondo, S. Kondo, M. Kurokaua and R. Ma rata (Japão) ....

331

| 38.

Immunologic Studies of Coral Snake Venom — P. Cohen and

E. li. Seligmann, Jr. (E.U.A.) ..

339 I

2 3 4

SciELO

10 11 12 13 14 15

BIOGRAPHICAL DATA OF VITAL BRAZIL

Vital Brazil Mineiro ria Campanha was born on April 28. 1865 in Campanha.

State of Minas Gerais. He stndied al lhe Faculty of Medicine in Rio de Janeiro

from 1886-1891. and received his doctor’s degree based on a thesis on “The

function of the spleen”.

As sanitary inspeetor of the Public Health Service in the State of São Paulo,

he organized campaigns against typhoid fever, plague, smallpox, diphteria, cholera-

morbus and mainly tetanus, from 1898-1895.

From 1895-1897 he practiced medicine in Botueatu and performed his first

experiments vvith the venoms of poisonous snakes, principally rattlesnakes and

jararacas.

From 1897-1900 he became a member of the Instituto Bacteriológico in São

Paulo, directed by Adolpho Luiz, where besides his bacteriological studies, he

succeeded in immunizing dogs and goats against raltlesnake- and jararaca venom.

and experimentally prepared an anti-venom sera. On November 8. 1899 he was

made head of the State laboratories at the Butantan Farm, in order to prepare

sera against the plague. Finally the Instituto Serumtherapico was officiallv inau-

gurated under the direction of Vital Brazil. in the farm, on February 23, 1901.

Following his retirement in 1919, he established the Instituto Vital Brazil

in Niterói, returning in 1921 to the Instituto where he commissioned as Director

unlil 1927.

On May 8. 1950 he died in Bio de Janeiro, 85 years old.

Vital Brazil was an excellent organizer, an exceptional and well-known re-

searcher. He surrounded himself with a well-chosen staff and planned «midin®

jrrinciples for lhe Instituto. Together with the Federal Government he worked

out lhe free transportation syslem of poisonous snakes. In 1914 he obtained,

from the State Government the permission to erect new buildings, which are used

up to present.

Some hundred original papers, monographs and lectures on his research work

were puhlished.

As the first one he proved the specificity of the anti-venom sera; observed

the parcial immunization against jararaca venom by immunization through the

yellow fever vinis; he made intense studies on immunology, improved the methods

for venom and serum dosage; discovered that serum, even expired, may and should

be used, however in higher than prescribed doses.

SciELO

10 11 12 13 14 15

II.

SESSÃO INAUGURAL — INAUGURAL MEETING

DISCURSO INTRODUTÓRIO

MÁRIO MACHADO DE LEMOS

Secretário de Estado da Saúde Pública e da Assistência Social

Representante do Govêrno do Estado de São Paulo

Nesta solenidade, em que se inaugura o "Simpósio Internacional Sôbre Ve¬

nenos Animais", é oportuno expressar, com a maior ênfase, o integral apoio e a

grata satisfação do (iovêrno do Estado de São Paulo, por dois aspectos funda¬

mentais: 1) pelas suas perspectivas científicas de alto interesse para todos os

povos; 2| pela honrosa deferência que hoje, aqui. se concretiza na escolha dêste

local para a sua realização, isto é. no Estado de São Paulo e exatamente neste

Órgão da Secretaria da Saúde, com a presença de cêrca de 200 cientistas de

quase todos os continentes, alguns detentores de Prêmio Nobel.

E foi nesta mesma Casa que Vital Brazil desenvolveu, neste campo específico,

as suas atividades de pesquisa, autêntico galardão de benemerência para nosso

país. E hoje, quando se comemora o seu centenário, por uma feliz coincidência,

o Instituto Butantan. convertido em Fundação, inicia uma nova etapa de profun¬

das transformações estruturais e administrativas, a fim de que possa desenvolver-se

adequadamente, na plenitude de seus objetivos. E lerá, para isto, todo o apoio

do Chefe do Executivo, ora empenhado na reformulação, em lermos mais racionais

e decisivos, de todos os problemas fundamentais de saúde pública do Estado.

Êste Simpósio, pelo alcance e magnitude de seus propósitos, inclui-se entre

as recomendações do Plano Decenal de Saúde Pública da Aliança para o Pro¬

gresso da Carta de Punta dei Leste, referendada pelo Grupo de Estudos dos Mi¬

nistros da Saúde que se reuniu em Washington, sob os auspícios da Organização

Sanitária Pan-Americana.

Em meu nome, na qualidade de Secretário de Estado da Saúde Pública e

da Assistência Social e. sobretudo, em nome do Governador Laudo Natel, a quem

tenho a honra de representar, formulo, com o maior aprêço. o nosso agradeci¬

mento à Comissão Organizadora do conclave e as nossas boas-vindas a todos os

congressistas nacionais e estrangeiros, neste espetáculo soberbo de humanitarismo

e de confraternização científica de todos os países em proveito de todos os povos.

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10 11 12 13 14 15

Mem. Inst. Butantan

Slmp. Internae.

33(1) :XIII-XVI, 1966

B. A. HOUSSAY

XIII

TRANSCENDENCE OF VITAL BRAZIL’S WORK

B. A. HOUSSAY

Instituto de Biologia y Medicina Experimental , Buenos Aires, Argentina

This First International Symposium on Venomous Animais justly takes placr

in São Paulo, as it is organized in honour oí the outstanding vvork and illustrious

personality of Vital Brazil, founder of the Instituto Butantan, which has been and

still is an example in its íield in the world. It was the first in South America

to introduce lhe specific treatment and prophylaxis of accidents in man and do-

inestic animais by snake, scorpion and spider venom. His life should 1 >e an

example for the future generations and his work rendered it a glory to Brazil

and South America.

The great human achievements are obtained hy scientific studies and lheir

application. There is no pure and applied Science, but only Science and ap-

plication of Sciences. The hasic investigation is the fountain of all progress of

the applied Science and of the technology which unrelentlessly transforms lhe

world. In the past century a movement of nationalist affirmation and of faith

in its destiny was observed in Brazil. Numerous Brazilians changed lheir family

names to names of regions and rivers, such as Americano, Brazil. Amazonas and

Tocantins, etc., or else to tliose of ancient tribal chiefs, such as Tibiriçá, Juracy,

Tamandaré, etc. Therefore, Mr. Santos Pereira gave his firstborn the name of

Vital Brazil Mineiro da Campanha. Vital, for his strengh of life, Brazil. for his

country, Mineiro because he carne of the State of Minas Gerais, Campanha be-

cause this was the city where he was born. His life honoured these names, due

to his own enduring and intelligent endeavour.

Born on April 28. 1865 and deceased on May 8, 1950. with 85 years of

age. This way, in such a large life span. he could look back on the success of

the work he hegan.

He studied humanities in São Pa ulo (1880 to 1885), then enrolled in me¬

dicine in Rio de Janeiro, where he studied from 1886 to 1891. working to pa\

for his studies; hy contest he won lhe job of technician of physiology and presenteei

a final theses on ‘‘Physiology of the spleen”.

Back in São Paulo, he hold the job of physician of the police force, sanitarv

inspector (1892 to 1895), worked in epidemies of cholera, yellow fever, cow-pox.

typhoid fever and diphtheria. He also worked successfully as a general practitioner

during the years (1895-1896) in Botucatu and was well avvare of the medicai and

sanitarv problcms of his country.

Those were limes of great medicai progress and useful application of bacte-

riology and serumtherapy. This promising field awoke the enthusiasm of lhe

young serious and hard working physicians, wherefore Vital Brazil. in 1897. en¬

rolled as a technician in the Instituto Bacteriológico de São Paulo under the

2 3 4

SciELO

10 11 12 13 14 15

XIV

TRANSCENDENCE OF VITAL BRAZIL'S WORK

direclion of Adolfo Lutz, one of lhe most eminent scientists Brazil ever had.

There Vital Brazil made his firsl attempts at the immunization against snake

venoms. In 1889 an epidemic of bubonic plague erupted in São Paido. Vital

Brazil was sent to stiuly il and prepare an anli-plague serum in an improvised

laboratory in Butantan. He fell gravely ill with plague, but fortunately reeovered

to take charge of his job.

With him in this epidemic worked also Oswaldo Cruz, and it is interesting

to point out that from there on Vital Brazil founded Butantan and Oswaldo Cruz

founded Manguinhos.

In 1901, on advice of Adolfo Lutz, the Institute of Serumtherapy of the

State of São Paulo was created in Butantan, under the direction of Vital Brazil,

who directed it from 1901 to 1909, and later from 1924 to 1927.

Called by the government of the State of Rio de Janeiro, he founded in

1919, in Niterói, the Institute of Hygiene, Serumtherapy and Veterinary, which

hears his name and where his sons worked with him.

Under his direction were undertaken the prophylaxis and treatment of plague,

typhoid fever, cow-pox, tetanus, diphtheria and zoonoses or diseases of domestic

animais.

However, his most important work was the fight against ophidism and his

studies on the snake, scorpion and spider venoms, the preparation of specific sera

against lhe snake (1901), scorpion (1918) and spider (1925) venoms. He also

studied the cutaneous batrachian venoms (1925).

The most original and successful work of Vital Brazil was lhe fight against

ophidism and animal venoms, where he set a model treatment for all America

and one of lhe best of lhe world.

Vital Brazil proved the specificity of the antivenomous sera, as he showed that

the antitoxic sera prepared against the Asiatic poisons were inefficient against

the poisons of the South American snakes. He obtained specific sera against the

latter and showed that there are three types of clinicai poisoning by the venoms

of Bothrops, C rotalu s and Ela p s, the symptoms of which can he treated

with specific sera. There are some common ones to the poisons of the Bothrops

and the antipoisonous serum against one species has paraspecific effect up to a

certain point on the poisons of other Bothrops. The serum against the venom

of our South American C r o t alu s has a certain paraspecific effect against the

venom of the North American Crotaliis.

These knowledges made Butantan prepare several sera: monovalent antibo-

thropic, polyvalent anlibothropic, anliophidic for Central America, anticrotalic,

antielapidic. The more commonly used sera are two: anticrotalic and anliophidic

prepared against the venoms of Bothrops and Crotulus durissus terrificus.

To obtain the venom necessary to prepare these sera a great number of

serpents were needed. The Institute established an exchange system, delivering

one ampule of serum for each snake specimen sent by the farmers. These were

given also a snare to capture the snakes and a wooden box lo ship them. The

railrooads carry them for free. This method was also tried in Argentine, however,

at presenl an additional amount is paid in money for each snake received.

The serumtherapy is responsible for a decrease in mortality. Of 20,000 men

bitten each year, lhe mortality decreased from 25-30% to a mere 2%, and these

due to late or insuffieienl treatment.

1, i SciELO

XVI

TRANSCENDENCE OF VII AL BRAZIL'S WORK

In 1943, I had the privilege to join in a tribute justly paid to Vital Brazil

when a new building was inaugurated in the Instituto Vital Brazil, in Niterói,

and I today rejoice to repeat textually:

“Vital Brazil is a glory lo South America and his name should be remem-

bered as that of Oswaldo Cruz, among lhose who initiated the true immunological

Science in Latin America”.

“My studies have allowed me to measure the great value of the extensive

worlc of Vital Brazil ou venoms. His demonstration of antitoxic specificity of

the antipoisonous sera obliges to consider him, with justice, as a founder of the

South American antiophidic serumtherapy, while high authorities wrongly assured

lhe efficiency of antitoxic sera prepared against venoms in índia”.

“It gives me a great satisfaction and a true honour to express in public all

appreciation and respecl his work and example inspire me, and cordially join

the intended tribute.

Great and eminent persons are found today in the Science of Brazil, however,

their way was Iargely paved by the initial work of Oswaldo Cruz and Vital Brazil”.

j, | SciELO

Mem. Inst. Butantan

Simp. Internac.

33(1) : XVII-XX, 1966

A. VALLEJO-FREIRE

XVII

CONFERÊNCIA INAUGURAL

A. VALLEJO-FREIRE

Diretor do Instituto Butantan

Presidente do Simpósio Internacional Sôbre Venenos Animais

Autoridades,

Presidente de Honra,

Meus senhores, minhas senhoras:

0 Instituto Butantan sente-se orgulhoso e agradecido de poder encerrar as

comemorações do centenário do nascimento de seu fundador, Vital Brazil, com a

especial homenagem que representa a presença de cientistas, vindos dos mais lon¬

gínquos recantos para participar do Simpósio Internacional Sôbre Venenos Ani¬

mais, que se inicia com esta cerimônia.

Conhe ao grupo de pesquisadores desta instituição a sugestão de reunir nesta

oportunidade especialistas em animais peçonhentos e peçonhas animais.

Ao constituir-se a comissão organizadora, que temos o privilégio de presidir

e ao estabelecer os primeiros contactos com cientistas do exterior, foi do Prof.

Bernado Houssay, nosso presidente de honra, que recebemos o mais efusivo apoio,

insistindo mesmo em participar das homenagens a Vital Brazil, não só pelo respei¬

to à obra por êle realizada, mas também pela amizade pessoal que a êle dedicava.

Senhores simposistas, o programa estabelecido e o resultado da consulta feita

a cada um de vós. Nêle certamente encontrareis incluídas muitas das vossas su¬

gestões.

A aceitação e o interesse manifestados ultrapassaram as nossas previsões de

tal modo, que o número de participantes interessados permitiria a organização

de uma reunião mais ampla, com caráter de congresso, o que coloca em destaque

a importância e a atualidade do assunto a ser tratado. É provável que, por êste

motivo, o tempo colocado à vossa disposição para exposição e debate seja exíguo,

levando-se em conta a importância dos trabalhos inscritos no ternário, mas estamos

certos de que o convívio amistoso entre os especialistas durante tôda a semana do

Simpósio será compensador e proveitoso.

Não é nossa intenção, nestas palavras de saudação, em cpie vos damos as

boas-vindas em nome do Instituto Butantan, discorrer demoradamente sôbre a

vida de Vital Brazil, que, em seus aspectos marcantes, foi focalizada pelo Prof.

Houssay. Parece-nos, no entanto, apropriado traçar um nítido perfil do passado

vivido por Vital Brazil que, a nosso ver. poderá nesta oportunidade servir para

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SciELO

10 11 12 13 14 15

XVIII

CONFEIíK.NCIA inaugurai-

u’a melhor análise da importância presente dos estudos sôlire os animais peço-

nhentos e de suas peçonhas e antever do forma mais adequada as perspectivas

do futuro.

Vários fatores, dos (piais dois parecem ler sido do maior significado, contri¬

buíram para alterar profundarncnle o equilíbrio demográfico no Estado de São

Paulo no fim do século passado e nos primeiros anos do século XX: o término

da escravatura, em 1888, desorganizando o trabalho do campo e o grande e rá¬

pido desenvolvimento da lavoura do café nas férteis terras do planalto paulista.

Em pràticamente dez anos, êsle Estado, que não contava com uma população

muito superior a um milhão de habitantes, recebeu um milhão adicional de pes¬

soas. sendo que não menos de 800.000 europeus. Em tão reduzido espaço de

tempo deve ler sido esta uma das maiores migrações para as proximidades do

trópico. Esta avalanche humana invadiu principalmente as zonas rurais e provo¬

cou o início de grande desenvolvimento das zonas urbanas. São Paulo, a cidade

que hoje vos hospeda, contava então com 60.000 habitantes e, doze anos depois,

atingia 240.000, isto é, 400% de aumento de população.

Aão foi sem conseqüências para a saúde pública esta explosão demográfica.

Pagou-se alto tributo em vidas humanas; às moléstias transmissíveis propagadas

pela contínua chegada de navios abarrotados de emigrantes imediatamenle enca¬

minhados para as zonas rurais, juntou-se o recrudescimento de infecções e infes¬

tações de várias naturezas, endêmicas nesta região, que tomavam caráter epidê¬

mico com a chegada de grande número de indivíduos suscetíveis, falhos de imu¬

nidade adquirida. A peste bubônica, a cólera, a varíola, a difteria, a escarlatina,

a febre amarela, as febres Iíficas, com pouca diferença de tempo incidiram inten¬

sa e gravemente sôbre a grande massa de população flutuante.

As medidas de ordem sanitária e profilática, devidas à aplicação de recursos

práticos introduzidos na era pasteuriana, aliadas à imunidade progressivamente

adquirida pela população, foram suficientes para restabelecer o equilíbrio sanitário.

É bem conhecida a ativa e destacada participação de Vital Rrazil nestas cam¬

panhas de saúde pública e que o levaram à criação do Instituto Butantan.

Permanecia, entretanto, para os pioneiros, constante e incontrolável por quais¬

quer meios conhecidos ou recursos médicos, o risco de morte por acidentes devidos

ao envenenamento por picada de animais venenosos, principalmente serpentes. A

mortalidade por acidentes ofídieos atingia 3 por 1.000 da mortalidade geral no

Estado de São Paulo.

As campanhas anliofídicas com o auxílio da soroterapia específica trouxeram,

sem dúvida, a solução parcial do problema do ofidismo, mais tarde igualmente

aplicada ao escorpionismo e ao araneísmo.

A utilização das terras para cultura e o progressivo extermínio dos ofídios,

provocados pelo homem, a destruição das matas, a moderna mecanização da la¬

voura, enfim, a alteração da geografia provocada pelo homem, deveria teoricamente

trazer como consequência a eliminação do problema nas áreas rurais mais inten¬

samente cultivadas; porém, a experiência veio mostrar que é o contrário que se

verifica: a ruptura do equilíbrio biológico em certas regiões — como é o caso

de São Paulo — proporciona uma seletiva multiplicação, devida às suas caracte¬

rísticas biológicas e conduz a uma ofio-fauna predominantemente constituída de

espécies peçonhentas, principalmente daquelas que se nutrem de preferência de

roedores e que se reproduzem mais intensamente nas vizinhanças de campos cul¬

tivados.

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Mem. Inst. Butantan

Simp. Internac.

33(1) :XVII-XX, 1966

A. VALLEJO-FREIRE

XIX

Não lom diminuído o envio de animais peçonhentos ao Instituto, principal¬

mente ofídios. Apenas, como curiosidade, vos informamos que, até a presente

data, um milhão de exemplares foram recebidos polo Instituto Butantan.

Acreditamos, senhores simposislas, aproximar-se para a humanidade um mo¬

mento de histórica importância, em que a experiência pioneira, a solução encon¬

trada por Vital Brazil no planalto paulista, no limite do trópico, servirá como

modelo de organização de proteção às populações, que agora invadem com re¬

cursos e técnicas modernos, mas de forma maciça, o mundo tropical, para utili¬

zá-lo em benefício da humanidade e afastar as preocupações da superpopulação

de outras áreas.

Momentos semelhantes aos verificados no começo do século, nesta região, es¬

tão se repetindo em vários países e regiões da terra, com os mesmos riscos e

idênticos problemas. Sentimos no Instituto Butantan, os apelos que se renovam,

vindos de novas regiões conquistadas pelo progresso, no caminho para o oeste,

rumo ao centro e ao norte do país, assim como de outros países, do nosso e de

outros continentes. Nos dois hemisférios, a moderna civilização, na contínua aven¬

tura do homem, segue a natural tendência para caminhar em direção ao Equador.

Não há dúvida que, inexoràvelmente, a grande fusão da humanidade irá proces¬

sar-se, em futuro próximo, nas zonas semitropicais e tropicais, até agora não den¬

samente povoadas, mas em intenso desenvolvimento, inicialmente no continente

americano e posteriormente no africano. Ora, nestas regiões, à medida que, do

norte ou do sul das zonas subtropicais, se caminha para o trópico, rumo ao Equa¬

dor, aumentam progressivamente o número e a variedade de espécies de animais

peçonhentos, principalmente da ofio-fauna.

O estudo, pois, dos animais peçonhentos e dos venenos animais, passa a ser

não mais de interêsse local ou regional, mas sim internacional. As soluções não

serão ràpidamente encontradas, a não ser que se realize um esforço conjugado e

que os métodos da moderna ciência, com a sua estreita aliança com a tecnologia,

nos proporcionem novos conhecimentos básicos sôbre muitos aspectos, que são o

motivo principal dêste Simpósio. Bessalta a urgência de, em extensas áreas, in¬

dependente dos artificiais contornos políticos das nações, hem estudar tôdas as

espécies de interêsse médico, para difundir a sua adequada identificação e co¬

nhecer, de forma global, a distribuição geográfica das espécies de maior impor¬

tância. Minto há a ser esclarecido sôbre a biologia e ecologia de animais vene¬

nosos e até, paradoxalmente, sôbre a maneira de obter a sua multiplicação arti¬

ficial controlada, visando à obtenção dos grandes volumes de veneno indispensáveis

ao estudo das suas propriedades ou para utilização como antígenos na obtenção

de antídotos.

É na fisiopatologia, na bioquímica e na farmacologia, principalmente, que a

pesquisa pode ser especialmente proveitosa e altamente esclarecedora. É de todo

interêsse intensificar os estudos sôbre a maneira de ação dos venenos o conheci¬

mento adequado da sua estrutura química, o isolamento dos princípios ativos, de

ação direta ou indireta, que compõem a complexa natureza dos venenos animais

e bem determinar as suas ações lesivas para o organismo.

Muito esforço ainda deve ser feito para aprofundar o conhecimento sôbre

os venenos e chegar à obtenção de conceitos mais adequados, que permitam a

padronização internacional daqueles de, maior importância e dos sôros mono e

polivalentes dotados de maior potencial de neutralização dos princípios realmente

responsáveis pela ação letal dos venenos.

SciELO

10 11 12 13 14 15

CONFERÊNCIA INAUGURAL

Nós nos consideraríamos imensamente satisfeitos se o csíôrço agora dispendido

para a realização dêste Simpósio puder, de alguma maneira, contribuir para al¬

cançar êste objetivo. Esperamos que possais tirar benefícios dêste convívio que

hoje se inicia e que êste primeiro Simpósio Internacional seja apenas o começo

de uma série de outros encontros, semelhantes e periódicos, para revisão dos re¬

sultados alcançados e estímulo para o vosso dignificante trabalho.

2 3

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ANIMAIS VENENOSOS

VENOMOUS ANIMALS

Mem. Inst. Butantan

Simp. Internac.

33(1):l-26, 1966

BRUCE W. I1ALSTEAD

1. VENOMOUS MARINE ANIMALS OF BRAZIL

BRUCE W. HALSTEAD

World Life Research Institute, Colton, Califórnia, U.S.A.

Our knowleclge of lhe venomous marine animais of Brazil dates back to the

Renaissance and the writings of Willem Piso and George Marcgrave, who puhlished

an excellent hiological description of the venomous spotted eagle-ray Aetobatus

narinari in their Historia Naturalis Brasiliae in 1648. This particular de¬

scription is noteworthy because it inchtded a brief general description of lhe

venom apparatus on the tail of the ray. Despite this early beginning of in-

vestigations on lhe venomous marine animais of Brazil, there was a gap of several

centuries hefore these organisms received very much scientific attention. Some

of the more comprehensive references by Brazilian workers that deal with the

subject are by Diniz (1905) and Gonsalves (1907) on venomous fishes; Silvado

(1911) on the noxious fishes of the Rio de Janeiro Bay; Fonseca (19171 on

the venomous fishes of Brazil; Fróes (1932, 1933a. h) on venomous fishes and

particularly the toadfish Thalassophryne ; and variouS“other workers. most

of whose puhlications also deal with venomous fishes, viz: Carvalho, 1947; Costa,

1958; Fonseca, 1952; Santos, 1952. In view of this most auspicious occasion

of the International Symposium on Animal Venoms in Commemoration of

the Centennial of Vital Brazil liere at the Instituto Butantan, it should be re-

cognized that the Instituto Butantan lias played a significant role in these studies

on venomous marine animais.

In discussing the venomous marine fauna of Brazil, one must first take into

consideration the vast Coastal expanses of the country whieh extend from latitude

4°20’45” N. to latitude 33°45’09” S., or 38 degrees of latitude, a coastline of

4,603 miles. The river systems of Brazil are lhe most extensive in the world,

and lhey exert a profound effect on the marine flora and fauna. The Brazilian

marine fauna is comprised of tropical Atlantic and West Indian (or Caribbean)

forms, as well as the temperate western Atlantic species. Caribbean species occur

as far south as Bahia (Lagler, Bardach, and Miller, 19621.

It is lhe purpose of this paper to briefly review the phylogenetic distribulion

of the venomous marine animais of Brazil. the nature of their venom organs and

venoms, the clinicai effects whieh lhey produce, and lhe treatment of their slings.

The term “venomous marine animal is defined as a marine animal thal is equipped

with a venom apparatus, i.e., a traumagenic device associated with a poison or

venom gland, capable of producing an envenomation. Organisms that are poisonous

lo eat have not been inchtded in this discussion. The organisms have been ar-

ranged according (o their phylogenetic position.

2 3

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VENOMOUS MARINE ANIMALS OF BRAZIL

INVFRTFBKATFS

Phylum COELENTERATA: Hydroids, jellyfishes, sea anemones, co¬

ra LS.

Class HYDROZOA: Hydroids.

Family MILLEPORIDAE : Millepora alcicornis Linnaeus. Stinging coral or

íire coral (USA). — Caribbean Sea, northern coast of Brazil.

Family OLINDIADIDAE: Olindias sarnbaquiensis Müller. Stinging medusa

(USA), relojinho (Brazil). — Coast of tropical Brazil.

Family PENNARIIDAE: Pennaria cf. liarella (Ayres). Stinging hydroid

(USA). —Coast of Maine to Brazil.

Family PHYSALIIDAE: Physalia physalis Linnaeus. Portuguese man-o-war,

bluebottle (USA), caravela (Brazil). — Tropical Atlantic.

Family PLUMULARIIDAE : Lytocarpus philippinus (Kirchenpauer). Feather

hydroid (USA). — Circumtropical.

Family RHIZOPHYSIDAE : Rhizophysa eysenhardti Gegenbaur. Stinging

hydroid (USA). — Warm waters of all oceans. Rhizophysa fili-

formia (Forskal). Stinging hydroid (USA). —- Warm waters of all

oceans.

Class SC Y PH O ZOA: Jellyfishes.

Family CARYBDEIDAE: Carybdea olaia Reynaud. Sea wasp (USA). —

Circumtropical. Carybdea marsupialis (Linnaeus). Sea wasp (USA).

-—- Tropical Atlantic. Tamoya haplonema Müller. Sea wasp (USA).

—- Tropical Atlantic.

Family CHIRODROPIDAE : Chiropsalmus quadrumanus (Müller). Sea wasp

(USA). — Tropical Atlantic.

Family LINUCHIDAE: Linuche unguiculata (Schwartz). Thimble jellyfish

(USA). — Tropical Atlantic and Pacific Oceans.

Family LYCHNORHIZIDAE: Lychnorhiza lucerna Haeckel. Água viva

(Brazil). — Coast of Brazil, French Guiana.

Family NAUS1THOIDAE: Nausithoê punctata Kõlliker. Stinging alga

(USA, juvenile form). — All warm seas.

Family PELAGIIDAE: Chrysaora quinquecirrha (Desor). Sea nettle (USA).

Tropical Atlantic and Pacific Oceans.

Family ULMARIDAE: Aurelia aurita (Linnaeus). Moon Jelly (USA). —

All warm seas.

Class A NTIIO ZOA: Corais and sea anemones. There are no reports of

stinging anthozoans froin Brazilian waters.

2 3

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Mem. Tnst. Butanlan

Simp. Internac.

33(1): 1-26, 1966

BRUCE W. HALSTEAD

Biology — HYDItOZOA: Induded within the HYDKOZOA are the

hydroids (e.g., A glaopheni a) , stinging or hydroid corais (e.g., Al illepora),

and lhe íree-floating siphonophores (e.g., P hy s alia). The hydroids are gener-

ally found attached to a suhstratum in shallow waters, from low tide levei down

lo depths of 1,000 melers or more. The ecological conditions in which hydroids

are found are extremely variable and lhey fluctuate according to the species.

Some species have a wide distrihution, whereas others are restricted to definite

latitudes. Generally, hydroids are more abundant in temperate and cold zones.

The forrn of the colony may he radically altered by such environmental factors

as wave shoek, currents, and temperatures. Colonies usually are small or moderate

in size. hui some species may atlain a length of 2 m. Because of the sessile habits

of hydroids, eommensalism with other animais is of frequent occurrence. Since

hydroids attach themselves to pilings, rafts, shells, rocks, or algae, and display

their fine moss-like growth, they are sometimes mistaken for seaweed. Colonial

hydroids are. comprised of two kinds of polyps or zooids: the feeding hydranth

that takes in food for the colony, and the reproductive polyp or gonangium. Tlie

nutritive polyps have a crown of tentacles and a central mouth that leads into

the stomach cavity to which all the other polyps are connected by the coenosarc

which encloses the common enteron. The nematocysts nettle or stinging cells are

restricted to the tentacles. Food is procured hy the use of the nematocysts.

Hydroids reproduce hy budding; the free-swimming, solitary medusae that

separate from the reproductive polyp, produçe ova that result in attached hydroids.

Medusae are more difficult to obtain than are the plant-like hydroids, but they

may be taken in fine-meshed plankton nets. The medusae may be likened to a

tiny umbrella with a short handle, the manubrium, which contains the mouth.

Tentacles provided with stinging cells hang from the velum or margin of the

umbrella. Medusae swim in a jerky fashion by spasmodic _contractions of the

umbrella. fn the medusae, the sexes are separate.

the order HYDlíOCOKALLINA or hydroid corais of which M illepora is

iiíe best-known genus, is widely distributed throughout tropical seas in shallow

water to the depth of 30 m. Hydroid corais are important in the development

of coral reefs, for they form upright, clavate, blade-like, or branching calcareous

growths, or encrustations over other corais and objects. They vary in color from

white to yellow-green; because of their variable appearance, they are sometimes

difficult to recognize. The order is characterized by a massive exoskeleton of lime

carbonate, lhe surface of which is covered with numerous minute pores. There

are two sizes of pores: the larger gastropores, which are 1 to 2 mm apart, and

the smaller dactylopores, irregularly interspersed about the gastropores. The sur¬

face of lhe coral between the pores has a pilted appearance. The entire stony

mass is traversed by a complex system of branched canais which communicate

with the pores. From the gastropores protrude the feeding gastrozooids, equipped

with a hypostome and capitate tentacles. Dactylozooids, extending from the dactylo¬

pores, are mouthless; however, they are provided with tentacles, which are believed

to have a protective and tactile funetion. According to Hyman (1940), mille-

pores have two or three types of powerful nematocysts located on the polyps, polyp

bases, and in the general coenosarc. Apparently most of the M illepora have

the ability to sting, but the venomousness of the sting varies from one species to

the next (Boschma, 1948).

The order Siphonophores, of which P hy s alia is a pertinent example, is

highly polymorphic free-swimming or floating colonies composed of several types

of polypoid or medusoid individuais attached to a floating stem. Siphonophores

SciELO

10 11 12 13 14 15

VENOMOUS MARINE ANIMALS OF BRAZII,

are pelagic animais, inhabiting lhe surfaee of lhe sea. They dcpend largcly upon

eurrents, wind, and lides for lheir movemenl. They are widely distributed as a

group, but most abundant in warm waters. The f loat or pneumatophore of /' h y -

sal ia is greally enlarged, and il is represenled by an i nverted, modified, medusan

bell, whereas lhe remainder of lhe coenosarc is correspondingly reduced. Phy-

salia may atlain a large size with a float 10 lo 30 cm in length. From lhe

underside of lhe floal hang gastrozooids, dactylozooids, and lhe reproductive gono-

dendra with lheir gonophores or hudding medusoids. The female gonophores are

medusoid and may swim free, but lhe rnale reproductive zooid remains attached

to lhe float. The gastrozooids or feeding polyps are without tentacles. Some of

lhe tentacled dactylozooids are small; however, several of lhe large dactylozooids

are equipped with very elongate “fishing” tentacles.

The number of fishing tentacles varies with lhe species of Phy salia. In

lhe Pacific forrn, P. utriculus, there is a single fishing tentacle; but in lhe Atlantic

species, P. physalis, there are multiple fishing tentacles. Extending along the

entire length of the large daclylozooid, a band of specialized tissue covers di-

verticulae of lhe gaslrovascular cavity of lhe tentacle. These fishing tentacles or

large dactylozooids may be found in the water to a depth of more than 30 m

and, because of lheir almost transparent appearance, constitute a definhe hazard

lo the nnsuspecting swimmer. Upon contraction, the remainder of the tentacle

shortens more completely than does lhe superficial band, and this causes the band

to be thrown into loops and folds thal are known as “stinging batteries”. The

nemalocysts are contained in cnidoblasts located in the superficial epithelium of

lhe battery. The toxin, a structureless fluid within the nemalocyst capsule, bathes

the surfaee of the nematocyst tubule (Lane, 1960).

According to Parker (1932), scores of these fishing filaments may extend

dovvn into the water from a single Phy salia. Parker lias observed that the

nematocyst heads occur at regular intervals along the side of the filament op-

posile the point from which lhe main muscle plate takes its origin. Each full-

sized head contains about 500 large nematocysts and about 2,000 small ones. In

one extended filament measuring 9 m in length, the nematocyst heads were dis¬

tributed at intervals of approximately 3 cm apart. According to these figures,

each fishing filament contained about 750,000 nematocysts. When one considers

lhe large number of fishing filaments on each Phy salia, he finds a formidable

venorn apparatus.

When the animal is moving through lhe water, the fishing tentacles undergo

a conlinuous rhythmic movemenl, alternately conlracting and relaxing. Thus there

is a constant sampling of lhe water beneath the pneumatophore. Jf the tentacle

hrushes against a prey organism, the nematocysts are stimulated, and they trigger

the immediate release of the coiled nematocyst thread. The fully uncoiled thread

may he several hundred times as long as lhe diameter of lhe parent capsule.

The extreme length of the tubule. together with its chitinous barbs and spines,

constitute a highly effeetive entanglement. II the tip of the cnidal thread penetrates

the victim, lhe toxin is eonveyed directly into lhe body of the prey through the

hollow thread. Lane (1960) found that lhe thread can penelrate even a surgical

glove.

Lane further observed that the magnitude of the response to contact with

lhe victim is proportional lo the area of contact helween tentacle and prey. A

small copepod may elicit lhe discharge of 20 to 50 adjacent nematocysts, whereas

contact with a larger animal might evoke a discharge of several hundred lhousand

nematocysts. Genlle stimulation of lhe nematocyst results in a rapid release of

Mem. Inst. Butaman

Simp. Internac.

H3(1):1-26, 1966

BRUCE \V. HALSTEAD

the nematocyst llircacl. Iiut does not dislodge lhe parent capsule from its position

in the epithelium. Vigorous resistance by the ])rey restilts not only in greatly

increasing the number of cnidae but also in disloding many of lhem from the

epithelium. Dislodged nematocysts are replaced hy cnidohlasts that differentiate

outside the stinging battery but suhsequently come to occupy a definitive position

iir the battery epithelium.

It is interesting that the loggerhead turtle, Caretta caretta, has been reported

to feed on P h y s alia. The potency of the toxin and the ability of the P h y -

s a I i a nematocyst to penetrate even a surgical glove make this a gastronomic

feat of no small accomplishment (Lane, 1960).

SCYPHOZOA : All Scyphozoans or jellyfishes are marine and the majority

are pelagie. A few species are known to inhahit depths of 2,000 fathoms or more.

In lhe adull stage, most jellyfishes are free swimming. Because their swimming

ability is relatively weak, jellyfishes are greatly influenced in their movements

hy currenls, lides, and wind. Scyphozoans are widely distributed throughout all

seas. Many medusae reveal that they are affected hy light intensity in that lhey

surface during the morning and late afternoons and descend during lhe midday

and in the darkness, whereas other react in just the opposite manner. A des-

cension is usually made during periods of stormy wealher. Swimming is ac-

complished hy rhythmic pulsations of the bell, and this aclion determines the

vertical rather than the horizontal progress of the animal. Jellyfishes display a

remarkable ability to withstand considerahle temperature and salinity changes.

They are carnivorous, some of the Iarger species being capahle of capturing and

devouring large crustaceans and fishes. Jellyfishes display a wide variety of

sizes, shapes, and colors; many of them are semitransparent or glassy in appearance

and often liave hrilliantly colored gonads, tentacles, or radial canais. In some

species, they may vary in size from a few millimeters to more than 2 m across

lhe bell, with tentacles more than 36 m in length, as in Cyanea capillata.

Kegardless of their size, jellyfishes are very fragile; many of them contain

less than 5 percent of solid organic matter. Scyphomedusae liave an eight-notch

marginal bell, but lack a velum; the gonads are connected with the endoderm.

Heproduction is hy an alternation of generations, as in the hydroids. although

the polyp stage is reduced. Jellyfishes have a complex system of hranched radial

canais, and numerous oral and marginal tentacles.

The cubomedusae are among the most venomous marine creatures known.

The genera, Chiropialmus and Carybdea, contain some of the more

dangerous species of the group. lhey range in size from a small grape to that

of a large pear. Cubomedusae are widely distributed throughl all the warmer

seas. They generally seem to prefer the quiet shallow waters of protected bays

and esluaries and sandy bottoms, although some species have been found in the

open ocean. During lhe summer montlis, the immature forms which slay on the

bottom, reach maturity. The adulls may then be found swimming at lhe surface.

Light-sensitive cubomedusae, however, descend to deeper water during the bright

sim of the middle of the day and come to the surface during early morning, late

aflernoon, and evening.

Morphology of the venorn apparatus — The venom apparatus of coelenterates

consists of lhe nematocysts or stinging cells which are largely located on their

tentacles. These nematocysts are situated within the outer layer of tissue of the

tentacle. Each of the capsule-like nematocysts is contained within an outer

capsule.-like device called lhe cnidoblast. Projecting at one point on lhe outer

2 3

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11 12 13 14 15

VENOMOUS MARINE ANIMALS OF BRAZIL

surface of the cnidolilast is the trigger-like cnidocil. Contained vvilhin the fluid-

filléd capsular nematocyst is lhe hollow, coiled, thread lube. The opening through

whicli the thread luhe is everted is closed prior to discharge hy a liddike device

ealled lhe operculum. The fluid within the capsule is the venom. Stimulation of

the cnidocil appears to produce a change in the capsular Wall of the nematocyst

causing the operculum to spring open like a trap door, and the thread tulie con-

veying the venom is everted. The sharp tip of lhe thread tube penetrates the

skin of lhe victim and the venom is thereby injected. When a diver comes in

contact with the tentacles of a coelenterate, he brushes up against the cnidocils

of literally thousands of these minute stinging organs.

Clinicai, characteristics — The symptoms produced by coelenterate slings vary

according to the species, the site of the sting, and the person. In general, those

caused by hydroids and hydroid corais (Millepora), are primarily local skin

irritations. P hy s alia stings may be very painful. Sea anemones and true

corais produce a similar reaclion, but may be accompanied by general symptoms.

Symptoms resulting from scyphozoans vary greatly. The sting of most scyphozoans

is too mild to be noticeable, whereas Carybdea and C h i r o p s ai m u s are

capable of inflicting very painful local and generalízed symptoms. C hir o -

p s a lm u s is probably lhe most venomous marine organism known and may

produce death within 3 to 8 minutes in humans.

Symptoms most commonly encountered vary from an immediate mild prickly,

or stinging sensation like that of a nettle sting, to a burning, throbbing or shoot-

ing pain which may render the victim unconscious. In some cases, the pain is

restricted to an arca within the immediate vicinity of lhe contact, or il may

radiate to the groin, abdómen, or armpit. The area coming in contact with the

tentacles usually becomes reddened, followed by a severe inflammatory rash, blis-

tering, swelüng, and minute skin hemorrhages. In severe cases, in addition lo

shock, there may be muscular cramps, abdominal rigidity, diminished touch and

temperalure sensation, nausea, vomiting, severe backache, loss of speech, frothing

at the mouth, sensation of conslriclion of the throat, respiratory difficully, paralysis,

delirium, convulsions, and death.

Treatment — Treatment must be directed toward accomplishing three ob-

jectives: relieving pain, alleviating effects of the poison, and controlling primary

shock. Morphine is effective in relieving pain. Intravenous injections of calcium

gluconate have been recomraended for the control of muscular spasms. Oral

histaminics and topical cream are useful in treating the rash. Dilule ammonium

hydroxide, sodium bicarbonate, olive oil, sugar, ethyl alcohol, and olher types of

soothing lolions have been used with varying degrees of success. Artificial

respiration, cardiac and respiratory stimulants, and other forms of supportive

measures may be required. There are no known specific antidotes.

Pharmacology — See Chemistry section.

Chemistry — Coelenterate venom contains a number of quaternary ammonium

compounds, of which tetramine is lhe most active. The venom also contains

5-hydroxytryptamine, histamine and histamine releasers, and several proteins of

relatively low molecular weight. The paralyzing and lethal effects of lhe toxin

appear to be caused largely by the proteins which may act directly on eholinergic

neurons. The localized dermatological signs and symptoms may be attributable

to 5-hydroxitryptamine, histamine and histamine-releasing substances (Russell, 1965;

Halslead, 1965). The chemistry of most coelenterate venoms lias not been studied.

2 3

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11 12 13 14 15

Mem. Jnst. Butantan

BRUCE W. HALSTEAD

Simp. Internac. '

33(1): 1-26, 1966

Phylum E C H IN 0 D E R M ATA: Starfishes, sea urchins, etc.

Family ARBACIIDAE; A rbacia lixula (Linnaeus). Sea urcliin (USA), ouriço

do mar (Brazil). — Tropical Atlantic and Mediterranean Sea.

Family DIADEMATIDAE : Diadema antillarum Philippi. Black sea ureliin,

needle-spined urchin (USA). — Tropical Atlantic, West Indies.

Family TOXOPNEUSTIDAE: Lytechinus variegatus Lamarck. Sea urchin

(USA), ouriço do mar (Brazil). — West Indies, North Caroline, south

lo Brazil.

Biology — Sea urchins are free-living echinoderms, having a globular, egg-

shaped, or flattened body. The viseera are enclosed within a hard shell or test.

formed by regularly arranged plates, carrying spines articulating with tubercles

on the test. Between tbe spines are situated three-javved pedicellariae, which are

of interest to the venomologist. In some species of sea urchins, the spines are

also venomous. Tube feet are arranged in 10 meridian series rather than in

furrows. A double pore in lhe test corresponds lo each tube foot. The intestine

is long and coiled, and an anus is always present. The gonads are attached by

mesenteries lo the inner aboral surface of the test. The mouth, situated on the

lower surface, turns downward, and is surrounded by five strong teeth incorporated

in a complex structure termed ‘‘Aristotle’s lantern”. Their power of regeneration

is great, but autotomy, as observed in the asteroids, does not occur. By means

of spines on the oral side of lhe test. sea urchins move slowly in the water. The

tube feet are utilized to climb vertical surfaces. Some forms have the ability

to burrow into crevices in roeks, while others cover themselves with shells, sanei,

and bits of debris.

Some urchins are nocturnal, hiding under rocks during lhe day and coming

out to feed at night. Echinoids tend lo be omnivorous in their feeding habits,

ingesting algae, mollusks, foraminifera, and various other types of benthic or-

ganisms.

Sea urchins are dioecious, hermaphroditism occurring only as a rare auomally.

Sexual dimorphism is generally absent. Spawning usually takes place during the

spring and summer in the Northern Hemisphere, but somewbat earlier in the

more Southern latitudes. The reproduetive periods of echinoids have been discussed

at great length by Hyman (1955). Several species of European and tropical

echinoids serve as important sources of food lo man. Only the gonads are eaten,

either raw or cooked. The hathymetric range of echinoids is great. extending

from the intertidal zone to great depths.

Morphology of the venom apparatus — The venom apparatus of sea urchins

is believed to consist of their hollow venom-filled spines, and the globiferous pe¬

dicellariae. However, usually only one or the other is present within a sin°le

species of sea urchin.

The spines of sea urchins vary greatly from group lo group. In most

instances lhe spines are solid, have blunt, rounded tips, and do not constitule a

venom organ. However, some species have long, slender, hollow, sharp spines,

which are extremely dangerous to handle. The acute tips and lhe spinules permit

ready entrance of the spines deep into the flesh, but because of their extreme

brittieness, they break off readily in the wound and are very difficult to with-

draw. The spines in Diadema may attain a length of a foot or more. lt

2 3

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11 12 13 14 15

VENOMOUS MARINE ANIMA LS OF BRAZIL

is believecl lhat the spines of some of these species secrele a venom, but lliis

lias not heen experimentally demonstratep. The aboral spines of Asthenosoma

are developed itilo special venom organs carrying a single large gland. The

poinl is sharp and serves as a means of introducing the venom.

Pedicellariae are small, delicate, seizing organs which are found scattered

among the spines of the shell. There are several different lypes of pedicellariae.

One of these, because of its globe-shaped head. is called the globiferous pedicel¬

lariae. and serves as a venom organ. Thcy are comprised of l\vo paris, a terminal.

swollen, conical head, which is armed wilh a set of calcareous pincer-like valves

or jaws, and a supporting stalk. The head is altached to the stalk either directly

by the muscles, or by a Iong flexible neck. On the inner side of each valve is

found a small elevation provided wilh fine sensory hairs. Contact with these

sensory hairs causes the valves lo elose instantly. The outer surface of each

valve is covered by a large gland which in Toxopneustes has two ducts that

empty in the vicinity of a small looth-like projection on the terminal fang of

the valve. A sensory hristle is located on the inside of each valve. Contact

wilh these bristles causes the small muscles at the base of the valve to eontract,

thus closing the valves and injecting the venom into the skin of lhe victim.

One of the primary functions of pedicellariae is that of defense. When lhe

sea urchin is at resl in calm water, the valves are generally extended, moving

slowlv about, awaiting prey. When a foreign body comes in contact with them,

it is immedialely seized. The pedicellariae do not release their hold as long as

the object moves, and if it is too strong to be held, the pedicellariae are torn

from the test, or shell, hut continue to bite the object. Detached pedicellariae

may remain alive for several hours after being removed from the sea urchin.

Clinicai characteristics — Penetration of lhe needle-sharp sea urchin spines

may produce an immediate and intense hurning sensation. The pain is soon

followed by redness, swelling, and an aching sensation. Numbness and muscular

paralysis have been reported. Secondary infections are not uncommon.

The sting from sea urchin pedicellariae may produce an immediate, intense,

radiating pain, faintness, numbness, generalized muscular paralysis. loss of speech,

respiratory distress, and in severe cases, death. The pain may diminish after

about 15 minutes and completely disappear within an hour, but paralysis may

continue for six hours or longer.

Treatment — Insofar as the. venom is concerned, sea urchin slings should

bc handled in a manner similar lo any other venomous sting. However. altention

is directed to lhe need for prompt removal of the pedicellariae frotn the wound.

When pedicellariae are detached from the parent animal, lhey frequently continue

to be active for several hours. Duríng this time they will introduce venom into

the wound.

The extreme brittleness and retrorse barbs of some sea urchin spines present

an added mechanical problem. Nielly (1881) recommended that grease be ap-

plied, stating that this woidd allow lhe spines to be scraped off quite easily.

Cleland (1912), Earle (1940), and olhers, are of the opinion that some sea

urchin spines need not be removed, as they are readily absorbed. Absorption

of the spines is said lo he complete within 21 lo 48 hours. However, Earle

(1911 I later pointed out lhat lhe spines of Diadema setosum are not readily ab¬

sorbed, and months later roenlgenological examination may reveal them in the

wound. It is recommended that the spines of Diadema be removed snrgically.

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BRUCE W. HALSTEAD

Vharmacology — lhe only attempt lo evalute lhe general j)harmacological

properties of gloliiferous pedicellarial venom of sea urehins lias been made by

Mendes, Abbud, and l iniji (1963). Saline extracts were prepared from liomo-

genates of globiferous pedieellariae of Lytechinus variegatus and tested on ac-

cepled cholinergic effector systerns, viz. gninea pig ileum, ral uterus, amphibian

liearl. longiludinal muscle of a hololhurian, lhe protractor muscle of a sea urcliin

lanlern, and lhe blood pressure of dogs. The response obtained ivas consistent

wilh that of a dialyzable acetycholinelike subslance which lhe researchers conclnded

lo be in pedicellarial venom.

Chemistry — Unknown.

Phylum MOLL I SCA : S.vails, bivalves, octoruses, etc. — There are no

reports of human encounlers wilh venomous mollusks in Brazilian waters.

Phvi.um ANNELIDA : Segmente d worms.

Family AMPHINOMIDAE: Chloeia viridis Schmarda. Sea mouse, bristle worm

(I SA). — Tropical America, both sides. Eurythoê brasiliensis Hansen.

Brislle worm (USA). — Coast of Brazil. Eurythoê complanata (Palias).

Bristle worm (USA). — Circumtropical.

Hiology — The polychaetes are divided into Iwo major groups: lhe Erran-

tiu, which includes most of lhe free-moving kinds, and lhe Sedentária or tube-

dwelling and burrow-inhabiting species. The polychaetes that have been in-

criminaled as toxic are largely errant fornis.

Polychaetes have cylindrical bodies and are metameric, having numerous

somiles — each bearing a fleshy paddle-like appendage, or parapodia, that bears

niany selae. The head region has tentacles. There is no clitellum. Sexes are

usually separate. There are no permanent gonads and fertilization is commonlv

externai. Polychaetes have a trochophore larval stage, and there is a sexual

budding in some species.

Most polychaetes are free living; a few are ectoparasitic. They have a bathy-

metric range from lhe tide line to depths of more than 5,000 melers. A few

species are pelagic. Several of the polychaetes inhabit freshwater. Polychaetes

are largely carnivorous. Some of the burrowing worms feed on bottom detritus

whereas the tube dwellers subsist on plankton. Generally, polychaetes spend their

existence crawling under rocks, burrowing in the sand or mud, in and around

lhe base of algal growths; or they construct tubes, which they leave at periodic

intervals in search of food. The majority of polychaetes range in size from 5

lo 10 cm. However. some of the syllids are only 2 mm in length; whereas the

gianl Australian species Onuphis teres and Eunice aphroditois mav attain a meter

or more in Iensth.

Morphology oj the venom apparatus — Setae: The members of the poly-

chaete genera Chio ei a. E urythoê, Hermodice, and others, possess

elongate pungent chitinous brislles or selae which project from the parapodia.

The parapodia are a pair of lateral appendages extending from each of the body

segments. The structure appears as a more or less laterally compressed fleshy

projection of the body Wall. Each parapodium is biramous; it consists of a

dorsal portion, the notopodium, and a ventral part. the neuropodium. Each

division of lhe parapodium is supported internally by one or more chitinous rods.

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VENOMOUS MARINE ANIMALS OF BRAZII,

termed acicula, to which are altached some oí lhe parapodial muscles. Each of

lhe distai ends of lhe two parapodial divisions are invaginated to form a setal

sac or pockel in which lhe projecting setae are situated. Each sela is secreted

hy a single cell at the hase of lhe setal sae. Generally the setae of polyehaetes

projeet some distance beyond the end of the parapodiiim. However, Eurythoê

and H ermo d ice have lhe ability to retraet or extend their setae lo a re-

markable degree. When lhe living worm is at rest, the setae appear to be quite

shorl and harely in evidence; hut when irritated, the setae are rapidly exlended

and the worm appears to be a mass of bristles.

The severity of symptoms reported in some of the clinicai accounts lends

credence to the belief thal both Eurythoê and II e r m odicc possess venomous

setae. The setae of both E. complanata and II. carunculata appear to be hollow.

and at limes seem to be filled with fluid. A seta of E. complanata lias a series

of retrorse spinules along the shaft, whereas the seta of II. carunculata is without

spinules and has a needlelike appearance. The setae of C h I o i e a are said to be

nonvenornous (Pope, 1963), hut are listed as a “stinging” worm hy others (Phillips

and llrady, 1953; Steinbeck and Ricketts, 19411. Examination of histological

sections of lhe parapodia of both species failed to reveal any glandular elenients.

However, the material examined was poorly preserved and it is quite possible

that glandular struclurès might have heen present and were not rccognized. Final

decision on this matter awaits further histological study.

Clinicai characteristics — Bristle worm ( C h l o e i u , E urythoê, II e r -

mo d ice) stings may result in an intense inflammalory reaction of lhe skin.

consisting of redness, swelling, hurning sensation, numhness, and itehing. Accord-

ing to Mullin (1923), llermodice carunculata is ahle lo inflict a “paralyzing

effect” with its setae. Contacts with the setae of bristle worrns have heen likened

to handling the spines of prickly pear caclus or nellle stings. Severe eomplications

may result in secondary infections, gangrene, and loss of the affected part. The

clinicai cffects of bristle worm stings have also heen discussed hy Baird (1864),

Paradice (1924), Levrat (1927), Roughley (1940. 1947), Pope (1917. 1963),

LeMare (1952), Phillips and llrady (1953), Halstead (1956. 1959). and Gillett

and McNeil (1962).

Treutmcnt — The Ireatment of worm biles and bristle worm stings is largely

symptomalic, There are no specific antidotes. Secondary infections may occur

which require the use of antibiotic therapy. The setae of bristle worrns can best

lie removed frorn lhe skin with adhesive tape, and the ammonia or alcohol should

hc applied to the area to alleviate lhe diseomforl.

Eharmacology — l nknown.

Chemistry — l nknown.

VERTERRATES

The Brazilian marine fauna does not appear to he especially rich in venomous

fishes. The families TRACHINIDAE, SIGANIDAE, SCATOPHAGIDAE, MONO-

DACTYLIDAE, HISTIOPTERIDAE, and several of the more important genera of

venomous SCORPAENIDAE are not represenled in Brazilian waters.

Despite the faet thal some of the earliest literature in biotoxicology deals with

venomous fishes, the bulk of our knownledge on the mor|>hology of the venom

organs of fishes has heen published during lhe past two decades. ( nfortunalely,

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BRUCE W. HALSTEAD

Simp. Internac.

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niost of ihis literature deals with European, North American, or tropical Indo-

Pacific species. The oídy endemic Brazilian species that have lieen studied lo

any extent are members of lhe genus Thalassophryne of lhe family

BATRACHOIDIDAE. The field of piscine venomology offers a vast field of im-

tappped opportunity to the researcher. There is urgent need for a thorongh

systematic investigation of the venomous fishes of Brazil in particular.

Class CHONDRICHTHYES: SharKS, RAYS, etc.

Horned SHARKS -—- Venomous sharks are limited to those species which possess

dorsal fin spines, namely, members of the families HETERODONTIDAE, SQUALI-

DAE, and DALATIIDAE. Although a number of species within these three

families are suspected of having venom organs, only two species, Hetodontus

facisci and Squalus acanthias, have been studied lo even a limited extent, and

only the latler is found in Brazilian waters. Clinicai reports from horned sharks

are based on 5. acanthias, and most of these reports are from Europe.

Family SQUALIDAE: Squalus acanthias Linnaeus. Spiny dogfish (USA),

galhudo (Brazil). —- Atlantic and Pacific Oceans. Squalus fernandinus

Molina. * Spiny dogfish (USA), galhudo (Brazil). — Circumpolar and

widespread throughout boreal and cool temperate latitudes of the South¬

ern Hemisphere.

Biology — Squalids are widely distributed throughout subarctic, temperate,

tropical, and subantarctic seas.

Most dogfish are somewhat sluggish in their movements, traveling singly or

in schools, and somewhat erratic in their migrations. Their bathymetric range

extends from the surface to depths of 100 fathoms or more. They are not

pelagic, preferring relatively shallow protected bays. The migration of squalids

seems to be governed by thermal changes, showing a preference for water lem :

perature from 7 o to 15°C. Squalids are viviparous, giving birth to their young

from late summer through the winter in some regions but earlier in olhers. Dog¬

fish are voracious and include a variety of fishes in their diel: capelin. herring,

menhaden, mackerel, hake, eod, haddock. They also feed on coelenterates, mol-

lusks, crustaceans, and worms. Squalids have been used lo a considerable extent

for fertilizer and as a source of vitamins A and D. Dogfish are of considerable

economic importance because of the damage that they do to fishing gear.

Morphology oj the venom apparalus — The venom apparatus of horned

sharks is comprised of the dorsal fin spines and the associated glandular tissue.

The dorsal stings are situated adjacenl lo the anterior margins of each of the two

spines in most of lhe horned sharks. Some of the dalatiid sharks have only a

single somewhat rudimentary fin spine or the spines are entirely absent. There-

fore, there is some question as to whethcr a true venom organ is present in this

Iatter group.

The anterior fin spine in Squalus acanthias is only slightly curved anlero-

posteriorly, whereas the posterior spine is more curved and in lateral view is

somewhat sabreshaped. The two sides are slightly convex and the longitudinally

grooved posterior aspect of the spine forms the base of the triangle. The spine

4 Squalus fernandinus is reported as venomous, but there is no Information regarding

the nature of íts venom apparatus or venom.

2 3

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VENOMOUS MARINE ANIMALS OF BRAZIL

is grooved only in its exposed portion, and lhe groove becomes more shallow

toward the lip. The glandular tissue appears as a glistening white substance

situated in lhe shallow posterior groove, or interdentate depression, of the spine.

Microscopic examination of the sting in cross section reveals it to be trigonal

in shapé and comprised of thrce principal layers: an outer layer of integument

which covers a thick wall of hard vasodentine and an inner core of carlilage.

Careful examination of the structure reveals that the glandular cells are situated

in lhe epithelial portion of the integumentary layer in the area of the antero-

lateral glandular grooves and in the interdentate depression. The glandular cells

are sparsely scaltered in the anteroglandular-groove area, hut are heavily con-

centrated in lhe interdentate depression. The glandular cells are of two basic

types. Some of lhese are polygonal-shaped, clear, finely granular cells having

slightly pycnotic nuclei, which appear to be of lhe mucin type. However, the

venom cells in hematoxylin and triosin preparations are oval-shaped, containing

homogenous brown-staining material with accumulations of finely granular material.

Venom production is apparently by a holocrine type of secretion. Morphological

studies on Squalus have been conducted largely by Evans (1921, 1923, 1943).

Clinicai characteristics — The symptoms consist of immediate intense, stab-

bing pain which may continue for a period of hours. The pain may be ac-

companied and followed by a generalized erythema and severe swelling of the

affected part. Tenderness of the affecled part may continue for several days.

According to Coulière (1899), dogfish stings may be fatal. The only clinicai

reports are by Evans (1920, 1923, 1943).

Treatment — Wounds produced by spined sharks are usually of lhe puncture

wound variety. Since shark spines do not have an enveloping integumentary

sheat, and the bulk of the glandular tissue is located near the base of the spine,

it would be a rare instance for the glandular tissue to become embedded in the

wound of lhe victim. In the most instances, effects resulting directly frorn the

action of the venom are of minor concern. Nevertheless it is advisable to irrigate

lhe wound with saltwater and to do whatever debridement may be necessary if

the tissues have been lacerated. If there is little or no bleeding, tlien moderate

bleeding should be encouraged. The pain is usually mild in comparison with

most stingray stings, but opiates may be needed. The extremity should be sub-

merged in hol water for a period of 30 minutes or more at as high a temperature

as the victim can tolerate without doing further injury. The addition of sodium

chloride or magnesium sulfate to the water is optional. Suturing may be required.

Antitetanus agents should be administered. Secondary infections from shark spines

may sometimes occur, and antibiotic therapy may be needed. Elevation of lhe

injured limb is recommended.

Pliarmacology — Unknown.

Chemistry — Unknown.

Stingrays

Stingrays constitute an important group of venomous fishes in that they are

probably lhe most common cause of fisli stings. The Suborder MYLIOBATOIDEA

includes lhe seven ray families: DASYATIDAE, stingrays or whiprays; POTAMO-

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38 ( 1 ): 1 - 26 , 1966

BRUCE W. HALSTEAD

TRYGONIDAE, river rays; GYMNURIDAE, butterfly rays; UROLOPHIDAE, round

stingrays; MYLIOBATIDAE, eagle or ba! rays; RHINOPTERIDAE, cow-nosed rays;

and MOBULIDAE, devil rays or mantas. (The caudal spines of the MOBULIDAE

when present, are generally quite rudimentary and will not be considered further

in tliis presentation) . Witb the exception of lhe POTAMOTRYGONIDAE, wbich

arc confined to the rivers of South America, most stingrays are marine, inhabit-

ing sballow Coastal waters, bays, brakish water lagoons, but may enter river

mouths and freshwater rivers. Most reports on stingray attacks and venom organs

are based on either European or North American species. Very little is known

regarding the venom organs of most of lhe stingray species of Brazil.

Family DASYATIDAE: Dasyatis americana Hildebrand and Schroeder. South¬

ern stingray (USA), raia (Brazil.) — Western Atlantic, New Jersey

to Rio de Janeiro, Gulf of México. Dasyatis centroura (Mitchell). Rough-

tail stingray — Atlantic Ocean, Mediterranean Sea. Dasyatis guttatus

Btoch and Schneider. Stingray (USA). — West Indies, Gidf of México

to Southern Brazil. Dasyatis sabina (Lesueur). Atlantic stingray (USA).

— Western Atlantic from Chesapeake Bay to Brazil, Gulf of México.

Dasyatis sayi (Lesueur). Bluntnose stingray. — Western Atlantic from

New Jersey lo Southern Brazil.

Family GYMNURIDAE: Gymnura altavela (Linnaeus). Spiny butterfly ray

(USA). — Tropical and temperate Atlantic Ocean. Gymnura micrura

(Btoch and Schneider). Smoolh butterfly ray (USA). — Western Atlan¬

tic from Chesapeake Bay to Brazil, Gulf of México.

Family MYLIOBATIDAE: Aetobatus narinari (Euphrasen). Spotted eagle ray

(USA). — Tropical and warm temperate waters of the Atlantic, Red

Sea, Indo-Pacific. Myliobatis jreminvillei Lesueur. Bullnose ray (USA).

— Western Atlantic, from New York to Brazil.

Family RHINOPTERIDAE: Rhinoptcra bonasus (Mitchell). Cownose ray (USA).

— Western Atlantic, from Southern New England to Brazil.

Family UROLOPHIDAE: Urolophus jamaicensis (Cuvier).

(USA). — Western tropical Atlantic.

Yellow stin

gray

liiology — Rays are common inhabitants of tropical, subtropical, and warm

temperate seas. With the exception of the family POTAMOTRYGONIDAE, which

is confined to freshwater, rays are essentially marine forms, some of which may

enter brackish, of freshwaters, freely. Rays are swimmers of moderate depths, and

are most common in shallow water. A deep sea species has recently been reported

from the Central Pacific Ocean. Sheltered bays, shoal lagoons, river mouths, and

sandy areas between patch reefs are favorite habitats of rays. They may be

observed lying on top of the sand, or partially submerged, with only their eyes,

spiracles, and a portion of the tail exposed. Rays burrow into the sand and

mud, and excavate the bottom with the use of their pectoral fins, by which

means they obtain the worms, molluscs, and crustaceans upon which they feed.

Morphology of the venom apparatus — The venom apparatus of stingrays

is an integral part of lhe caudal appendage. The venom organs of stingrays

have been dividéd into four anatomical types based upon their adaptability as

a defense organ. This subject was discussed by Halstead and Bunker (1953).

SciELO

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VENOMOUS MARINE ANIMALS OF BRAZIL

Gymnurid type: This is the mosl weakly-developed type of stingray venom

apparatus. The caudal appendage in gymnurid rays are cylindrical, tapering,

and greatly reduced in size. The sling is small, seldorn exceeding 2.5 em in

length, and usually situated in lhe middle or proximal ihird of the tail. The

striking ability of the organ is relatively feehle.

Myliobatid type: The venom organs of myliohatid rays are better adapted

as a striking organ than those found in gymnurid rays. The caudal appendage

is cylindrical and tapers oul to a long whip-like tail. The sting is generally

situated on the proximal portion of the basal third of lhe tail and is moderate

to large in size, ranging from about 5 lo 12 cm or more in lenglb. Allbough

myliobatid rays can inflict serious wounds, the striking force of the sting is less

than that of the dasyatid rays largely because of the proximal location of the

sting on the caudal appendage.

Dasyatid type: The venom organs of dasyatid rays are better adapted as

a striking organ than are those of myliobatid rays. The caudal appendage is

cylindrical and tapers oul to a long whip-like tail. The sting in some species

may be very large, attaining 37 cm or more in lenglh, and is located in lhe

distai portion of the basal or middle third of lhe tail. The more distai location

of the sting improves the striking force of the sting.

Urolophid type: The venom organs of urolophid rays are probably the most

highly developed of any of the sting rays. The caudal appendage is relatively

short, very muscular, and is not as a whip-like structure, but rather the tail be-

comes compressed distai lo the sting and forms a more or less distinct caudal

fin. The sting is usually located in the middle or distai third of the tail and is

moderate in size, seldorn exceeding 5 cm in length. The powerful muscular

structure of the tail and lhe distai location of the sting make this a highly ef-

ficient defensive weapon.

Although there is considerable variation in the morphology of the venom

organs of various stingray species there is a basic pattern which all of the species

examined thus far appear to follow. For the purpose of this review only a

general description of the stingray will be given.

The venom apparatus of stingrays consists of a bilaterally retroserrate spine

and its enveloping integumentary sheath. The spine is an elongate tapering

structure that ends in an acute sagitate tip. The spine is composed of an inner

core of vasodentine which is covered by a thin layer of enamel. lt is firmly

anchored in a dense collagenous network of the dermis on lhe dorsum of the

caudal appendage. The dorsal surface of the spine is marked by a number of

shallow longitudinal furrows. These furrows are usually more pronounced on the

hasal portion of lhe spine and disappear distally. The serrate edges of lhe spine

are termed the dentate margins. Medial to each dentate margin, on the ventral

side, is a longitudinal groove, the ventro-lateral-glandular groove. The grooves

are separated from each other by the median ventral ridge of lhe spine. Con-

tained within the grooves of an “unsheathed” or traumatized sting is a strip of

gray tissue. The tissue lying within the ventrolateral glandular grooves consists

of glandular epithelium and blood vessels. This is the primary venom producing

area of the sting. In most stingray species there is a thickened wedge-shaped

portion of the integument on lhe dorsum of the caudal appendage ventral to the

sting which is known as the cuneiform area. Toxicological studies of the cunei-

form integument indicate that the glandular cells of this area also secrcte venom.

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Mem. Inst. Butantan

BRUCE W. HALSTEAD

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Mieroscopic examination of histological cross sections ol an intact sting reveals

that it is roughly diamond shape and consists of a broad T-shaped dentinal

strueture coinpletely enveloped by a layer of integument. The integumcnt is

comprised of Iwo layers. The inner layer, lhe dermis, consists of areolar con-

nective tissue and vascular channels. The outer layer. lhe epidermis, is composed

of modified squamous epilheliiim conlaining tnany glandular cells. A cross section

of lhe ventrolateral-glandular groove lias been termed lhe glandular Iriangle.

Glandular activity is generally mosl concentrated in lhe epidermis in the im-

mediate vicinity of the ventrolateral-glandular grooves whieb is believed to be the

principal site of venom produetion. There is no histological evidenee of a venom

duel. Venom produetion is by a holocrine lype of lysis.

Clinicai characteristics -— Pain is the predominant symptom and usually

develops immediately or within a period of ten minutes following the attack. The

pain lias been variouslv described as sharp, shooting, spasmodic or throbbing in

character. The freshwater slingrays are reputed to cause extremelv painful wounds.

More generalized symptoms of fali in blood pressure, vomiting, diarrhea, sweat-

ing. rapid heart beat. muscular paralysis, and death have also been reported.

Stingray wounds are either of the laceration or puncture type. Penetration

of lhe skin and underlying tissue is usually accomplished without serious damage

lo lhe surrounding struetures, but withdrawal of the sting may result in extensivo

tissue damage due to the reeurved spines. Swelling in lhe vicinity of lhe wound

is a eonstant finding. The area about the wound at first lias an ashy appearance,

later becomes cyanotic and lhen reddened. Although stingray injuries occur mosl

frequently aboul the ankle joint and fool as a result of stepping on lhe ray,

inslanees have been reported in whieh the wounds were in the ehest.

T reatme.nl — The following recommendations are based on the clinicai

investigations of 1,725 cases of stingray attacks during lhe past 15 years by

Husseli (1951. 1965) and his associates, who have had more first-hand experience

in this field than any other group.

Efforts of treatment should be promptly and vigorously instituted. The treat-

inent is directed loward alleviating lhe pain, comhaling the effects of the venom.

and preventing secondary infection. Successful residis are in large measure de-

pendent upon lhe rapidity with whieh treatment is instituted. The victim should

immediately irrigate the wound with the eold saltwater at hand. This procedure

facilitates removal of the venom, and the cold water tends to act as a vaso-

eonstrictor thus reducing lhe amount of absorption of the poison, while serving

as a mild anesthetic agent. A tourniquet may be applied immediately above the

stab site but must be released every few minutes in order to preserve circulation.

The wound should be explored carefully for evidenee of pieces of the sting’s in-

legumentary sheath. All pieces of integumentary sheath must be eompletely

removed or envenomation will continue and the results of lhe treatment will be

greatly impaired. As soon as the wound has been thoroughly cleansed, the injured

member should be soaked in boi water. The water should be maintained at as

high a lemperature as ibe jiatient can tolerate without ]irodueing further injury

to the lissues. Soaking should continue for a period of 30 to 90 minutes. The

addition of magnesium sulfate lo lhe water is sometimes desirable because of its

mild anesthetic properties. The addition of other anesthetic and antiseptic agents

is optional. Following the soaking procedure, the wound should be debrided,

cleansed, and closed \viIh dermal sutures. The use of antitetanus agents is recom-

2 3

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VENOMOUS MARINE ANIMALS OF BRAZIL

mendecl. Antibiotic agents niay be required. The use of intramuscular or intra-

venous demerol has been found effective in controlling the ]>ain. Phe primary

shock so often seen immediately following the injury usually responds satisfactorily

to routine supportive measures. However, the secondary shock resulting directly

from lhe aclion of lhe venom on lhe cardiovascular system may require immediate

and vigorous therapy. Treatmenl should he directed toward maintaining cardio¬

vascular tone and the prevention of any furlher complicating factors. Elevation

of the injured member is advisable.

The use of potassium permanganate, ammonia, and cryotherapy I Mullins,

Wilson, and Best, 1957) is not only useless but may even have adverse effects.

They are not recommended for the treatmenl of stingray stings. For furlher

reading on lhe treatment of stingray attacks see Bayley (1940), Evans (1943),

Halstead and Bunker (1953), Russell and Levvis (1956), Halstead (1959), Rus-

sell (1959), and Halstead and Mitchell (1963).

1‘liarmacology — The most complete studies on the pharmaeological properties

of stingray venom have been conducted by Russell and bis associales working

primarily with the venom of Urolophus halleri.

Stingray venom has a deleterious effecl on the vertebrate cardiovascular ap-

paratus. The action on the blood vessels appears lo be diphasic. Low concentrations

of the venom give rise to simple peripheral vasodilatation or vasoconstriction.

With massive doses the venom causes vasoconstriction without a preliminary period

of dilatation. The most obvious effecl. and perhaps the more important one, is

lhat of vasoconstriction. This effect has been observed in all the blood vessels

examined. Some of the most serious effects vvere lhose directly upon lhe heart.

The most consistent change seen in the electrocardiographic pattern of cais lhaI

were injected with small amounts of the venom was bradycardia with an increase

in the PR interval giving a first, second, or third degree atrioventricular block.

The second degree block was usually followed by sinus arrest. Reversal of lhe

srnall dose effect occurred within 30 seconds following the end of the injection.

When cats were given larger amounts of the venom, they showed in addition to

the PR interval change, almost immediate ST, T wave change indicative of

ischemia, and in some animais, true muscle injury. High concentrations of the

venom eaused marked vasoconstriction of the large arteries and veins as well as

the arterioles. lhe direct effects on the heart muscle are quite drastic. 1 he

venom produces changes in the heart rate and amplitude of systole, and may

cause complete, irreversible, eardiac standstill. lt appears that stingray venom

affects lhe normal pacemaker. The new rhythm evoked following cardiac stand¬

still is frequently irregular and is believed to be elaborated outside the sino-atrial

node (Russell and van Harreveld, 1954; Russell, Barritt, and Fairehild, 1957).

Stingray venom depresses respiration. Although part of lhe respiratory de-

pression is secondary to the cardiovascular changes, the venom may have a direct

effect on the respiratory centers of the medulla.

Stingray venom produces many changes in the behavior of animais. Some

of these changes can be attributed to the direct effects of lhe venom on the

central nervons system. In mammals the venom occasionally produces couvulsive

seizures, but the mechanism of these seizures is not apparent. They may be due

in part lo cardiovascular failure. Seizure patterns were not reported in electro-

encephalograms from anesthetized animais (Russell et al., 1958). The venom

does not seem to have a deleterious effect on neuromuscular transmission (Russell

and Long, 1960; Russell and Bohr. 1962). When the venom is injected into the

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Mcm. Inst. Butantan

BRUCE W. HAL.STEAD

Simp. Internae.

33(l):l-26, 1966

lateral ventricles of mammals it produces transient apathy, aslasia, and licking

motions (Russell and Bohr, 1962). Mice injected with lethal doses of venom

developed hyperkinesis, prostration, marked dyspnea, blanching of the ears and

retina, and exophthalmos. These signs were followed by complete, atonia, gasping

respiratory movements, coma, and death. A similar syndrome was observed in

cats and moiikeys inclnding alaxia. dilated pupils, increased salivalion, mictnration

defecation, marked atonia, cyanosis, and hypoactive or absent deep and superficial

reflcxes. One monkey exhibited a conic-clonic generalized motor seizure ac-

companied by hypersalivation, twitching of the head. and marked dilatation of

lhe pupils (Russell et al ., 1958; Russell, 1965).

Chemislry — There is very liltle information available regarding the chemistry

of stingray venom?. The most specific data are lhose provided by Russell et al.

(1958), Russell, Fairchild, and Michaelson (1958), and Russell (1965). The

freshly prepared water exlract of crude venom prepared from Urobatis halleri is

described as clear, colorless, or faintly gray in color. The pH vvas 6.76. The

crude extract loses its toxieity within 4 to 18 hours upon standing at room

temperature hut is more stable at lower temperatures or in 20 lo 40 percent

glycerol. The venom will not lolerate lyophilization. The total protein content

was found to be approximately 30 percent, total nitrogen 3 percent, and total

carbohydrate 3 percent. Ten amino acids have heen found to he present. With

the use of disc electrophoresis, they have identified 15 fractions in extracts from

the venomous integumentary sheath of U. halleri. Extracts prepared from sponges

lhat had heen stahhed with fresh stings were found to contain 10 fractions.

Further studies on these extracts using gel filtration (Sephadex G 100 and G200)

süjggested that the loxic protein, or proteins, may have a molecular weighL in

excess of 100,000. The fraction of lhe toxin having the greatest lethality was

found to have two or three distinct bands when suhjected to disc electrophoresis.

Crude venom extracts have heen shown to contain serotonin, 5-nucleotidase. and

phosphodiesterase. Protease and phospholipase activity were absent.

Cl.ASS OSTEICHTHYES : CATFISHES, ScORPtOXFISHES, ToADFISHES, etc.

Catfishes — The suborder SILUROIDEA includes a group of fishes hav¬

ing a wide variety of sizes and shapes. Their hody shape may vary from short

to greatly elongate, or even eel-like. The head is extremely variahle, sometimes

very large, wide or depressed, again very small. The mouth is not prolractile hut

the lips are sometimes greatly developed, usually with long barbeis, generally with

at leasl one pair from rudimentary maxillaries, often one or more pairs aboul the

ehin. and sometimes one from each pair of nostrils. The skin of these fishes is

thick and slimy, or with bony jjlates. There is an absence of true scales. Aboul

one thousand species are included within this group, most of whieh are found in

the fresh-water streams of the tropics, hut a few species are marine. Considering

the large number of catfish species, amazingly little is known regarding the

morphology of their venom organs or the nature of their venom. Most of the

published literature on catfish venom organs deals with North American fresh-

water species. Papers on these species have heen published by Reed 11900, 1906,

1907, and 1924), and Halstead, Kuninobu, and Hebard (1953). There are no

reports on the venom apparatus of any Brazilian catfish.

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VENOMOUS MAHINE ANIMALS OF BRAZIL

Family ARIIDAE: Genidens genidens (Cuvier and Valenciennes). Catíish (ISA),

inandi (Brazil). — Brazil. Netuma barbus (Lacépède). Sea calfish (USA),

bagre marinho (Brazil). — East coast of Soulh America, from Guianas to

Argentina. Scideichthys albicans (Cuvier and Valenciennes). Calfish (USA),

liagre marinho (Brazil). — Brazil.

Biology The ariid catfishes are a large group of subtropical and tropical

marine fishes which are worldwide in distrihution. They are active fishes and

unlike their freshwater counterparts in lhat they are constantly on the move, fre-

quently in large schools. They resemhle most other catfishes in appearance hui

differ from other s[)ecies in thal the anterior and posterior nostrils are close lo-

gether, the Vitter covered hy a valve. Sea catfish have an interesting hahit in

which the male catfish incuhates their eggs hy placing lhem in its mouth. The

inale places up to 50 eggs or more in his mouth for a period of two months.

After the eggs are halehed out, the yoiiug fish remain in the mouth for an ad-

ditional period of two wecks.

Morphology <>) lho venom upparalus — The venom apparalus of catfishes

consists of the dorsal and pecloral stings and the axillary venom glands. The

dorsal and pecloral spines are comprised of modificd or coalescent sofl rays which

have hcconie ossified. and so constriieted lhat they can he locked in the cxtcndcd

position at the will of the fish. The maltire dorsal spine is a stoutly-elongate,

compressed. tapered. slightly arehed, osseous structure hearing a series of retrorse

dentations along the anterior and posterior snrfaces. and having an aeute sagittale

lip. The spine is generally enveloped hy a thin layer of sparsely pigmenled skin.

lhe inlegumcntary sheath, which is continuous with lhat of lhe softrayed portion

of lhe fin. There is no externai evidenee of a venom gland. The shaft of lhe

pecloral spine is similar lo lhe dorsal spine in its general morphology.

Microscopic examination reveals thal lhe levei of lhe middle third, lhe sling

mav he divided inlo thrce distinct zones: a peripheral integumentary sheath, an

intermediate osseous portion, and a central canal. The integumentary sheath is

comprised of a relativo ihick outer layer of epidermis and a thin layer of dermis.

The glandular cells which comprise the venom gland are most concentrated at

lhe anterolateral and posterolateral margins of the sting where they are sometimes

elumped two or thrce cells deep within lhe cpidermal layer. lhe venom glands

of most of the catfish species lhat have been studicd appear as a cellular shect

wedged between the pigment layer and the stratified squamous cpithelium of lhe

epidermis. The microscopic anatomy of the dorsal and pecloral stings are similar

in appearance. The axillary pore, which is lhe outlet of lhe axillary gland, is

located jusl helow the vertical conter of lhe posthumeral process of lhe cleithrum.

The gland is enclosed within a capsule of fihrous connective lissue, and is divided

inlo three or four lohcs which are furlher subdivided inlo a variahle nurnher of

lohidcs. The lobules are eomposed of large secretory cells. It is helieved lhat

Ihis gland may eontribule to the venom supplíed to lhe pecloral stings in those

species of catfishes in which the axillary gland is presenl.

Clinicai charactcristics - The pain is generally descrihed as an instantaneous

slinging, throbbing, or scalding sensation which may he localized or may radiate

up the affected limh. Some of the tropical species, such as Pio to sus, are

capable of producing violent pain, which may last for 48 hours or more. The

area about lhe wound becofnes pale immcdiately after heing stung. The pallor

is soon followed hy a cyanotic appearance, and then hy redness and swelling.

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Mem. Inst. Butantan

Slmp. Internac.

33(l):l-26, 1966

BRUCE W. HALSTEAD

In some cases Ihe swelling may be very severe, accompanied !>y numlmess and

gangrene of llie area about lhe wound. Shock may he present. Improperly

treated cases frequently result in secondary Imcterial infections of the wound.

Some species of catfishes may produce wounds which may take weeks to lieal.

hui in most instances lhe wounds are of minor consequenee. Deaths have heen

reported from the stings of some of lhe tropieal catfishes.

Treatment — Symptomatic. There are no specific antidotes. lollow lhe

same procedure as uSed in the treatment of stingray wounds.

1’harmacology — l nknown.

Chemistry — l nknown.

ScoHPioNKtsHES — The vasl family of SCORPAEN1DAE closely resemhle

Y are distinguished hy a suhorhital stay whieh is

The suhorhital stay joins the other hones of the

lich covers the whole head. The excessive number

lhe sea liasses from whieh they are distinguished hy a s

present in the scorpionfishes. The suhorhital stay joins

head lo forni a coat of mail which covers the whole head.

of spines about the head are characteristic of the members of this family. Seorpion-

fishes vary greatly in their form and eoloration. A few species may attain large

size and many are eonsidered to be valuahle foo<] fishes. Hepresentatives of llie

family are widely distributed throughout all trojrical and lemperate seas. and several

species oecur in Aretie waters. The family SCORPAENIDAE, as the name implies,

ineludes some of the most dangerous species of venomotts fishes known.

Family SCORPAENIDAE*: Scorpaenu bra.iiliensis Cuvier. Barlifisll (USA), peixe

escorpião (Brazil). - Western Atlantic, from Virginia to Bahia (Brazil).

Scorpaenu grandicornis Cuvier and Valenciennes. Lionfish, long-horned scorpion-

fish (USA), peixe-escorpião (Brazil). — Western Atlantic, Florida to Brazil.

Scorpaenu plumieri Bloch. Spotted scorpionfish, sculpin (USA), peixe-escor¬

pião (Brazil). -— Western Atlantic, from Massachussetts to Brazil.

Iliology — Members of the genus Scorpaenu are for lhe most part

shallow-water bottom dwellers, found in bays, along sandy beaches, rocky coast

line, or coral reefs. Their hahit of concealing lhemselves in creviees, among debris,

under rocks, in seaweed, together with their protective eoloration which blends

them almost perfectly into their surrounding cnvironment, makes them difficult

lo see. Scorpaenids are generally captured hy hook and line, and in many regions

they are a popular and important food fish. When they are removed from the

water they have lhe defensive hahit of erecting their spinous dorsal fin and flar-

ing out their armed gill covers, pectoral, pelvic, and anal fins. lhe pectoral fins,

although dangerous in appearance, are unarmed. Hinton (1962) and Breder

(1963) have reported observations ou lhe defensive behavior of Scorpaenu gultatu

and S. plumieri respectively. Whenever an object comes in close proximity to

5. guttata, the dorsal spines are immediately erected and the fish moves swiftly

toward the object so as to deliver a sharp blow lo the object with the side of its

head or with its dorsal sting. In the case of S. plumieri it was observed that

lhe fish was generally quiescent, hui when touched with a stick, it would settle

* Although only three species of scorpaenids are llsted here, it is believed that any

members of the genus Scorpaena inhabiting Brazilian waters are venomous. The

species given in this list are the only three species listed in venomological literature.

2 3

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VENOMOUS MAKINE ANIMALS OF BRAZIL

(lown tighlly on the sarul and somelimes arch its back. The heacl of the fisli

was directed slightly downward and the opercula expanded. If the intrusion

continued the fish would suddenly change its stance and expose large yellow

patches on the pecloral fins. Prior to this, all of the exposed surface of the

fish was somewhat drab, hut with the change in stance the pectorals would be

suddenly flipped over displaying the hrightly colored undersurface. The pale

dots near the base of the pectorals became a liright iridescent blue, and tbe inter-

radial light-colored patches along the margin to mid-part of the fins were bright

yellow. The dark area around the blue spots became an intense black and the

fin margin gray. If the provocation continued, lhe fish would repeatedly ram

or butt the intruding object.

Morphology oj lhe venom apparatus — The venomous members of the family

SCORPAENIDAE can be classified into three basic types on the basis of the

morphology of 1 bei r venom organs: (1) l } t e r o i s or zehrafish type; (2) Scor-

paena or scorpionfish type; and (d) Synancejn or the stonefish type. Only

the Scor paena type are found among the Brazilian scorpionfishes. Unfortun-

ately no one has described lhe venom apparatus of a Brazilian scorpionfish.

Thercfore one can only assume lhat the venom apparatus of Scorpaena brasiliensis,

S. grandicornis, and S. plumieri resemble tliat of S. guttata, the Califórnia scorpion¬

fish, which has been studied in detail. The following description is based on lhe

studies of Halstead. Chilwood, and Modglin (1955). The venom apparatus in-

cludes 12 dorsal spines, 3 anal spines, 2 pelvic spines, their associated venom

glands, and their enveloping integumentary sheaths. If lhe integumenlary sheath

is removed, a slender, elongate fusiform strand of grey or pinkish tissue can be

observed lying within the glandular grooves on either side of lhe spine. Micro-

scopic examination of cross sections of venom glands reveals a cluster a large

polygonal glandular cells with pinkish-grey, finely granular cyloplasm located in

the dermal layer within the anterolateral glandular grooves. The large venom-

producing cells have a pinnale, heart-shaped arrangement. and vary greatly in

size aml morphology.

Clinicai characteristics — Stings from scorpionfishes vary frorn one species

lo the next, hut generally the introduction of scorpaenid venom immediately

produces an intense throbbing pain. Within a few minutes lhe area about the

wound becomes ischemic and then cyanotic. The pain becomes progressively more

severe and may radiate to tbe groin or axilla. The intensity of the pain may be

comparable to that produced by renal colic and may continue for several hours.

Within a short period of time lhe affected pari becomes swollen, erythematous,

and indurated. Profuse perspiration, pallor, dyspnea, restlessness, nausea, vomil-

ing, diarrhea, loss of consciousness, and extreme tachyeardia are commonly present.

Abscesses, necrosis, and sloughing of the tissues about the wound have been re-

ported. Bayley (1940) and Colby (1943) State lhat a maculopapular or scarla-

tiniform rash over the body may occur. Cecea (1902) cites a case which resulted

in peripheral neuritis, paralysis, and muscular atrophy due to a sling by Scorpaena

nera. Secondary bacterial infections, telanus, and primary shock are frequent

complications which must be considered. According lo Blanchard (1890), Cou-

liere (1899), Scoll (1921), and Colby (1943), scorpionfish stings may result

in dcath.

T reatment — Scorpionfis

stingray envenomations.

h slina should be treated in tbe same manner as

SciELO

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Mem. Inst. Butantan

BRUCE W. HALSTEAD

Simp. Internac.

3S(1):l-26, 1966

Pharmacology — There are no reports available on the pharmacology of the

venom of Brazilian scorpionfishes.

Chemistry - There are no reports available regarding the venom of Brazilian

scorpionfishes.

Toadfishes — The BATRACHOIDIDAE, or toadfishes, are a group of

small boltom fishes which inhabit the warmer waters of the coasts of America,

Europe, África, and índia. Toadfishes are of little eommercial value and are not

generally considered as food fishes, although they are eaten in some countries.

The flesh is said to be fine-flavored, but the fishes are small in size and hony.

According to Taschenberg (1909), the liver of some of the batrachoids is poisonous

lo eal. However, the greatest interest of these fishes to biotoxicologists is their

imique and highly developed venom organs.

Family BATRACHOIDIDAE: Marcgravichthys cryptocentrus (Valenciennes). Toad-

fish (USA), niqnim-niquim, sapo (Brazil). — Brazil. Thalassophryne ama¬

zônica Steindachner. Brazilian toadfish (USA), pocomon, niquim-niquim, sapo

(Brazil). — Mouth of the rivers Negro, Amazon and Xingu (Brazil). Tha-

lassophryne branneri Starks. Toadfish (USA), niquim-niquim, sapo (Brazil).

- Brazil. Thalassophryne punctata Steindachner. Toadfish (USA), niquim-

niquim, sapo (Brazil). — Brazil.

Biology — Batrachoid fishes, wilh their broad, depressed heads and large

mouths, are somewhat repulsive in appearance. Most toadfishes are marine, but

some are estuarine or entirely fluviatile, ascending rivers for great distances.

They appear to enjoy turbid water. Regardless of the type of waler in which

they are found, batrachoids are primarily bottom fishes. They hide in crevices,

burrows, under rocks, debris, among seaweed. or lie almosl completely buried under

a few centimeters of sand or mud. Fróes (1932, 1933a) stated tliat tbe Brazilian

species of T halassophryne has the habit of covering itself witb a thin

layer of sand or mud, but witb careful observation one can usually detect the

outline and protruding eyes of lhe fish as one wades along in the clear shallow

water of sandy beaches. Toadfishes are quite hardly and are able to live for

several hours after being removed from the water. According to Goode (1884),

the bottom temperature of the water frequented hy these fishes would appear to

range from 10"C to 32°C. Toadfishes tend to migrate lo deeper water during

the winter months where they remain in a somewhat torpid condition.

They also are experts at carnouflage. Their ability to change their color to

lighter or darker shades at will and their mottled pattern make these fishes dif-

ficult to see.

Most toadfishes tend lo be somewhat sluggish in their movements, but when

after food lliey can dart out witb surprising rapidity. They are somewhat

omnivorous in their eating habits but seem to prefer among other things, crabs,

mollusks, worms, and small fishes. Toadfishes are said lo be quite vicious and

will snap at almost anything lipon the slightest provocation. Although they are

not capable of producing a severe wound, they can inflict a Lite that is not

readily forgotten. When lliey are disturbed or their dorsum touched, they im-

mediately erect their dorsal spines and flare out their opercular spines in defiance.

Toadfishes do not school, but they are gregarious and tend lo congregate together.

For additional information regarding the habits of these interesting fishes. the

excellent works of Gill (1907) and Gudger (1910) are recommended.

2 3

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VENOMOUS MARINE ANIMALS OF BRAZIL

Morphology oj the venom apparatus - The venom apparatus of toadfishes

consists of Iwo dorsal fin spines, two opercular spines, and their associaled venom

glands. In the case of Thalassophryne doici, which can be considered as typical

of the group, there are Iwo dorsal spines which are enclosed together within a

single integumentary sheath. The dorsal spines are slender and hollow, slightly

curved, and terminate in acnte lips. Al the base and tip of each spine is an

opening through which the venom passes. The base of each dorsal spine is sur-

ronnded by a glandular mass from which lhe venom is produced. Each gland

empties into lhe base of its respective spine. The operculum is also highly

specialized as a defensive organ for the introduction of venom. The horizontal

litnb of the operculum is a slender hollow bone which curves slightly, and

lerminates in an acute tip. Openings are present al each end of the spine for

the passage of venom. With the exception of the extreme distai tip, the entire

opercular spine is encased within a glistening, whitish, puriform mass. The

broad rounded porlion of this mass is situated al the base of the spine, and

tapers rapidly as the lips of the spine is approached. The pyriform mass consists

of a tough sac-like outer covering of connective tissue in which is contained a

soft. granular, gelatinousdike substance having lhe appearance of fine tapioca.

This mass is the venom gland. The gland empties into the base of the hollow

opercular spine which serves as a duct.

Microscopic examination of the venom glands shows strands of aerolar con¬

nective tissue, large distended polygonal cells filled with finely granular secretion,

and vascular channels. In some instances the polygonal cells will appear lo have

undergone complete lysis and there remain only areas of amorphous secretion.

The microscopic anatomy of lhe dorsal and opercular venom glands are essentially

lhe same.

Clinicai characteristics — The pain from toadfish wounds develops rapidly,

is radiating and intense. Some have descrihed the pain as being similar to lhat

of a scorpion sting. The pain is soou followed hy swelling, redness, and heat.

No fatalities have been recorded in the literature. Little else is known ahout the

effects of toadfish venom.

7 reatment — Ioadfish stings should he handled in a rnanner similar lo sting-

ray envenomations.

Pharmacology — The only published reports ou toadfish venom are lhose

hy Fróes (1933). Injections of the venom into guinea pigs and chicks resulted

in mydriasis, ascites, paralyses, necrosis ahout the injection site. convulsions and

death. The aulhor helieves lhat the venom of T halassophryne lias both

proteolytic and neurotoxie properties.

Chemistry — Unknown.

Miscellaneous venomous FiSHES - There does not appear to he any data

availahle on other kinds of venomous fishes in Brazilian waters.

Literature

1. Baird, W. — Description of a new species of annelide belonging to the family

AMPHINOMIDAE. Trans. Linnean Soc. London, 24:449-450, pl. 45, 1864.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 1-26, 1966

BRUCE W. HALSTEAD

2 .

6 .

8 .

10 .

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Bailey, H. H. — Injuries caused by scorpion fish. Trans. Roy. Soc. Tro-p. Med.

Hyg., 34(2) :227-230, 1 pl., 1940.

Blanchard, R. — Traité de zoologie medicale. Paris, J. B. Bailiière et Fils,

1890, 2:638-695.

Boschma, H. — The species probiem in Millepora. Zool. Verhandel. Rijk-

smus. Natuur. Hist., Leiden, (1), 115 p., 13 figs., 15 pis., 1948.

Breder, C. M., Jr. — Defensive behavior and venom in Scorpaena and

Dactylopterus. Copeia, 4:698-700. 2 figs., 1963.

Carvalho, J. P. — Peixes venenosos. Seleções agrícolas, 2(12):10-15, 1947.

Cecca, R, — • Sugli effetti tossici delle punture di alcuni pesei. CUn. Med. Ital.,

41:82-87, 1902.

Cleland, J. B. - — Injuries and diseases of man in Australia attributable to

animais (except insects). Australasian Med. Gaz., 32:269-272, 1912.

Colby, M. — Poisonous marine animais in the Gulf of México. Trans. Texas

Acad. Sei., 26:62-69, 1943.

Costa, J. F. — Intoxicação com o peixe de Fernando de Noronha. Território

Federal de Fernando de Noronha, Rio de Janeiro, Imprensa Nacional, 1958,

pp. 104-105.

Coutière, H. — Poissons vénimeux et poissons vénéneux. 221 p., Paris, The

Agrég. Écoie Supérieure de Pharmacie de Paris, Carré et Naud, 1899.

Diniz, A. — Peixes venenosos. Tese, Fac. Medicina da Bahia, Salvador, 1905,

82 p.

Earle, K. V. — Pathological effects of two West Indian echinoderms. Trans.

Roy. Soc. Trop. Med. Hyg., 33(4) :447-452, 2 figs., 1940.

Earle, K. V. — Echinoderm injuries in Nauru. Med, J. Australia, 2(10) :265-

266, 1941.

Evans, H. M. — The poison of the spiny dog-fish. Brit. Med. J., 1(3087):287-

288, 5 figs., 1920.

Evans, H. M. — The poison organs and venoms of venomous fish. Brit. Med.

J., 2(3174):690-692, 2 figs., 1921.

Evans, H. M. — The defensive spines of fishes, living and fóssil, and the

glandular strueture in connection therewith, with observations on the nature

of fish venoms. Phil. Trans. Roy. Soc. London, 212:1-33, 14 figs. 3 pis., 1923.

Evans, H. M. — Sting-fish and seafarer. 180 pp., 31 figs., London, Faber &

Faber Ltd., 1943.

Fonseca, O. — Estudos sôbre peixes venenosos do Brasil. Brasil Médico, 31

(11/12) :90-91, 97-99, 1917.

Fonseca, F. — Animais peçonhentos. Instituto Butantan, São Paulo, 1952.

B'róes, H. P. — Sur un poisson toxiphore brésilien: le “niquim” Thalassophrynu

maculosa. Rev. Sud-Amer. Méd. Cir., 3(11) :871-878, 1932.

Fróes, H. P. — Peixes toxiferos do Brasil. Bahia Médica, 4(4):69-75, 1933.

Fróes, H. P. — Studies on venomous fishes of tropical countries. J. Trop.

Med. Hyg., 36:134-135, 2 figs., 1933.

2 .

6 .

8 .

10 .

11 .

12 .

13.

14.

15.

16.

17.

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19.

20 .

21 .

22 .

23.

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VENOMOUS MARINE ANIMALS OF BRAZIL

24. GUI, T. — Life histories of toadfishes (BATRACHOIDIDA), eompared with

those of weevers ( Trachinus) and stargazers W r a n o s c o y i d s). Smith-

sonian Misc. Coll., 48(1697):388-427, 21 figs., 1907.

25. Gillet, K. & McNeil, F. — The Great Barrier Reef and adjacent isles. 209 p.,

168 pis., Sidney (Australia), Coral Press Pty. Ltd., 1962.

26. Gonsalves, A. D. — Peixes venenosos. Rio de Janeiro, 1907.

27. Goode, G. B. — The fisheries and fishery industries of the United States. 2

vols., Washington, D.C., Govt. Printing Office, 1884.

28. Gudger, E. W. — Habits and life history of the toadfish ( Opsanus tau). Buli.

U.S. Bur. Fish., 1908(28): 1071-1109, pis. 107-113, 1910.

29. Halstead, B. W. — Animal phyla known to contain poisonous marine animais.

In: E. E. Buckley & N. Porges, ed., Venoms, Washington, D.C., Am. Assoe.

Adv. Sei., 1956, p. 9-27.

30. Halstead, B. W. — Dangerous marine animais. 146 p., 88 figs., Cambridge,

Cornell Maritime Press, 1959.

31. Halstead, B. W. — Poisonotis and venomous marine animais of the world. In-

vertebrates. Washington, D.C., U.S. Government Printing Office, 1965, vol. I.

32. Halstead, B. W., & N. C. Bunker — Stingray attacks and their treatment.

Am. J. Troy. Med. Hyg., 2(1) :115-128, 12 figs., 1953.

33. Halstead, B. W., Chitwood, M. •/., & F. R. Modglin — The venom apparatus

of the Califórnia scorpionfish, Scoryaena guttata Girard. Trans. Am. Microscoy.

Soc., 74(2) :145-158, 15 figs., 1955.

34. Halstead, B. W., Kuninobu, L. S., & H. G. Hebard — Catfish stings and the

venom apparatus of the Mexican catfish, Galeichthys felis (Linnaeus). Trans.

Am. Microscoy. Soc., 2(4) :297-314, 6 figs., 1953.

35. Halstead, B. W. & Mitchell, L. R. — A review of the venomous fishes of the

Pacific area. In: H. L. Keegan & W. V. Mac Farlane, ed., Venomous and

yoisonous animais and noxious ylants of the Pacific region, New York, Pergamon

Press, 1963, p. 173-202, 16 figs.

36. Hinton, S. — Unusual defense movements in Scoryaena ylumieri mystes. Coyeia,

(4) :842, 1962.

37. Hyman, L. H. — The invertebrates: PROTOZOA through CTENOPHO-

R A. 726 p., 221 figs., New York, McGraw-Hill Book Co., 1940, vol. I.

38. Hyman, L. H. —■ The invertebrates: ECHINODERMATA. The coelo-

mate bilateria. 763 p., 280 figs., New York, McGraw-Hill Book Co., 1955, vol. IV.

39. Lagler, K. F., Bardach, J. E., & Miller, R. R. — Ichthyology. 545 p., New

York, Wiley and Sons, 1962.

40. Lane, C. E. — The toxin of P hysalia nematocysts. Ann. N.Y. Acad. Sen.,

90(3):742-750, 5 figs., 1960.

41. Le Mare, D. W. — Poisonous Malayan fish. Med. J. Malaya, 7(1): 1-8, 1 pl.

1952.

42. Levrat, E. — Les réactions cutanées vis-à-vis de certains animaux marins.

Toulouse Méd., 28(7): 194-202, 1927.

43. Mendes, E. G., Abbud, L., & S. Umiji — Cholinergic action of homogenates of

sea urchin pedicellariae. Science, 139(3553) :408-409, 1963.

2 3

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Mem. Inst. Butantan

Simp. Internae.

33(1):l-26, 1966

BRUCE W. HALSTEAD

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Mullin, C. A. — Report on some polychaetous annelids; collected by the Bar¬

bados — Antigua expedition from the University of Iowa in 1918. Univ. Iowa

Studies Nat. Hist., l(K3):39-45, pis. 5-6, 1923.

Mullins, J. F., Wilson, C. J., & W. C. Best — Cryotherapy in the treatment

of stingray wounds. Southern Med. J„ 50(4) :533-535, 1957.

Nielly, M. — Animaux et vegetaux nuisibles. In: M. Nielly, Éléments de pa-

thologie exotique. Paris, Delahaye & Lecrosnier, 1881, p. 709-710.

Paradice, W. E. — Injuries and lesions caused by the bites of animais and

insects. Med. J. Austrália, 2:650-652, 2 figs., 1924.

Parker, G. H. — Neuromuscular activities of the fishing filaments of Phy -

salia. J. Cellular Comp. Physiol., 1:53-63, 2 figs., 1932.

Phillips, C., & Brady, W. H. — Sea pests-poisonous or harmful sea life of

Florida and West Indies. 78 p., 38 figs., 7 pis., Miami, Fia., Univ. Miami

Press, 1953.

Pope, E. C. — Some sea animais that sting and bite. Austrálian Mus. Mag.,

0(5): 164-168, 5 figs., 1947.

Pope, E. C. — Some noxious marine invertebrates from Australian seas. In:

J. W. Evans, chmn., Proc. First International Convention on Life Saving

Techniques, Part III. Scientific section, suppl. Buli. Post Grad. Comm. Med.,

Univ. Sidney, 1963, p. 91-102, 2 figs.

Reed, H. D. — The structure of the poison glands of Schilbeodes gyrinus.

Proc. Am. Assoe. Adv. Sei., 49th Mtg., p. 232-233, 1900.

Reed, H. D. — Notes on the poison organs in fishes. Science, 24:293, 1906.

Reed, H. D. — The poison glands of N o t urus and Schilbeodes. Am.

Naturalist, 41:553-566, 5 figs., 1907.

Reed, H. D. — The morphology of the dermal glands in Nematognathus

fishes. Z. Morphol. Anthropol., 24:227-264, 32 figs., 1924.

Roughley, T. C. — Where nature runs riot. On Australia’s Great Barrier Reef

marine animais grow to unusual size, develop strange weapons of attack and

defense, and aequire brilliant colors. Nat. Geogr. Mag., 77 (6):823-850, 33 figs.,

1940.

Roughley, T. C. — Wonders of the Great Barrier Reef. 282 p., New York,

Charles Scribner’s Sons, 1947.

Russell, F. E. — Stingray injuries: a review and discussion of their treatment.

Am. J. Med. Sei., 226:611-622, 3 figs., 1953.

Russell, F. E. — Stingray injuries. Public Health Repts., 74(10) :855-859, 1959.

Russell, F. E. — Marine toxins and venomous and poisonous marine animais.

Adv. Marine Biol., 3:255-383, 1965.

Russell, F. E., Barritt, W. C., & M. D. Fairchild — Electrocardiographic pat-

terns evoked by venom of the stingray. Proc. Soc. Exp. Biol. Med., 96:634-

635, 1957.

Russell, F. E., & V. C. Bohr — Intraventricular injection of venom. Toxicol.

Appl. Pharmacol., 4:165-173, 1962.

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of the stingray. Med, Arts Sei., 12(21:78-86, 3 figs., 1958.

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VENOMOUS MARINE ANIMALS OF BRAZIL

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for stingray injuries. Venoms. Am. Assoe. Adv. Sei., (44):43-53, 3 figs. 1956.

65. Russell, F. E., & Long, T. E. — Effects of venoms on neuromuscular trans-

mission. In: H. R. Viets, ed., Miasthenia gravis, Springfield, Charles C. Thomas,

1960, p. 101-116, 3 figs.

66. Russell, F. E., Panos, T. C., Kang, L. W., Warner, W. M., & T. C. Colket —

Studies on the mechanism of death from stingray venom. A report of two

fatal cases. Am. J. Med. Sei., 235:566-584, 1958.

67. Russell, F. E., & A. van Harreveld — Cardiovascular effects of the venom

of the round stingray, Urobatis halleri. Arch. Internat. Physiol., 62(31:322-

333, 6 figs., 1954.

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Rio de Janeiro, F. Briquiet & Cia., 1952.

69. Scott, H. H. — Vegetal and fish poisoning in the tropies. In: W. Byam &

R. G. Archibald, ed., The practice of medicine in the trerpies, London, Henry

Frowde and Hodder & Stoughton, 1921, 1:790-798, 2 figs.

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SciELO

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Mem. Inst. Butantan

Simp. Internac.

33(1): 27-30, 1966

DIVA DINIZ CORRÊA

2. TAX0N0M1A DE ANTIIOZOA (COELENTERATA) BRASILEIROS:

DISTRIBUIÇÃO E FREQUÊNCIA EM AGUAS BRASILEIRAS

DIVA DINIZ CORRÊA

Departamento de Zoologia, Universidade de São Paulo, São Paulo, Brasil

A classe ANTIIOZOA, a maior classe do filo COELENTERATA. é

composta de duas subclasses, ALCYONARIA (OCTOCORALLIA), que

abrange seis ordens e ZOANTHARIA (HEXACORALLIA), com cinco

ordens.

Entre as ordens maiores as melhores conhecidas para o Brasil são a ordem

GORGONACEA (subclasse ALCYONAKIA ) com 17 espécies, a ordem ACTI-

NIARIA (subclasse ZOANTHARIA ) com 14 espécies e a ordem 3IADREPO-

RARIA (subclasse ZOANTHARIA ) com 14 espécies. Entre as ordens menores,

que são ainda menos conhecidas, posso mencionar apenas uma espécie da ordem

TEEESTACEA (subclasse ALCYONARIA ), T ele st o risei , uma espécie da or¬

dem PENNATULACEA (subclasse ALCYONARIA)', Renilla mülleri e uma es¬

pécie da ordem CERIANTHARIA (subclasse ZOANTHARIA ), Ceriantheomor-

phe brasiliensis. Isto significa um conhecimento ainda muito pequeno para a to¬

talidade do litoral brasileiro. São no total 18 espécies numa classe que contém

cêrca de 6.000 espécies.

De acordo com meu conhecimento, os livros mais modernos sôhre a sistemá¬

tica da classe ANTIIOZOA, nos quais material brasileiro foi considerado são

o de Frederick M. Bayer (1961), ''The shallow-water OCTOCORALLLIA of

lhe West Indian region”, e o de F. G. Walton Smith (1948), “Atlantic reef corais

- a handbook of the common reef and shallow-water corais of Bermuda, West

Indies and BraziU.

A inclusão de animais brasileiros nos dois livros é baseada em razões zoo-

geográficas. Faunisticamente extensões das índias Ocidentais penetram no Gôlfo

do México e ao longo da costa leste da Flórida, para o sul, ao longo da costa

nordeste da América do Sul até os recifes do Brasil. É impossível traçar um

limite rígido entre a fauna do Gôlfo do México e da costa sudeste dos Estados

Unidos com aquela das índias Ocidentais, a qual inclui também quase lôda a

informação disponível da fauna de ANTIIOZOA do Brasil.

Com exceção de alguns relatórios fornecidos por algumas investigações pes¬

queiras recentes, a costa da América do Sul. ao sul e a leste de Trinidad. constitui

uma lacuna nos nossos conhecimentos faunísticos neste grupo de animais. Algu¬

mas coleções mais antigas desta área foram relatadas, muito inadequadamente, por

Stiasny (1951) e ainda mais antigo é o estudo de Verril (1912) sôhre GORGO¬

NACEA da costa brasileira. Coleta completa nesta região está ainda grandemente

necessitada.

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TAXONOMIA DE ANTHOZOA (COELENTERATA ) BRASILEIROS: DISTRIBUIÇÃO

E FREQUÊNCIA EM AGUAS BRASILEIRAS

De particular interesse é o material de (JOKGONACEA obtido, durante as

explorações pesqueiras mencionadas, na costa das Guianas e do llrasil pelo navio

pesqueiro ‘Coquetle’ em 1957 e pelo ‘Oregon’ em 1957 e 1958. A presença

nestas regiões de algumas espécies como Iciligorgia schrammi, Diodogorgia noduli-

jera, Elisella barbadensis e A7t.sc//a elongata mostra que a fauna fora da praia,

mas em águas relativamenle rasas, apresenta decididamente um sabor das índias

Ocidentais e as regiões citadas seriam apenas uma extensão faunística antilhana.

Em profundidades maiores, de cêrca de 90 a 200 melros, a ocorrência de gêneros

corno Thesea, Muricea, Eli se ll a e Callogorgia indica que as

afinidades faunísticas com as índias Ocidentais ainda persistem nesta indicação

batimétrica.

A fauna de recife do Brasil contém elementos endêmicos como as espécies

Phyllogorgia dilatata e 1'lexaurella grandijlora e elementos não endêmicos, do Atlân¬

tico Ocidental, como Leplogorgia virgulata e Lophogorgia hebes, os quais se es¬

tendem em direção ao norte até às vizinhanças do Cabo Hatteras na costa norte-

americana. Os limites geográficos dos componentes endêmicos não são conhecidos.

Aproximadamente as mesmas afirmações podem ser tomadas em consideração para

os corais verdadeiros, ordem MADREPORARIA.

Com exceção das 14 espécies da ordem ACTINIARIA, as quais foram estu¬

dadas por especialista brasileiro, tôdas as outras espécies de ANTHOZOA men¬

cionarias foram estudadas fora do llrasil por especialistas estrangeiros.

A fim de melhorar o conhecimento desta grande classe de animais marinhos

foi iniciado reeenlemenle pelo Departamento de Zoologia da Universidade de São

Paulo, um levantamento das espécies ocorrentes na costa do Estado de São Paulo.

Este levantamento abrange no momento o estudo de três ordens apenas, a ordem

(JORGONACEA, as gorgônias, a ordem ACTINIARIA, as anémonas do mar e a

ordem ZOANTHIDAE, cujos representantes são chamados popularmente de paliloas,

de acordo com o seu gênero maior e melhor conhecido, o gênero Palythoa.

O estudo das anémonas do mar está mais adiantado que o das outras ordens,

mas ainda encontra-se em sua fase inicial. Por enquanto estamos realizando um

levantamento faunístico, sistemático, a fim de conhecermos os animais e podermos

com isso fornecer material classificado para interessados em estudos de fisiologia,

ecologia, genética, microscopia eletrônica e bioquímica. Como disse Cadet Hand

(1961), estudioso americano dos Celenterados, o conhecimento dêste importante

filo do reino animal ainda está na sua infância e dificilmente outro campo da

Zoologia pode apresentar tantos problemas como o dos nematocistos, cápsulas ur-

ticantes, característica do filo.

A área escolhida situa-se entre Itanhaém e Ubatuba, abrangendo, portanto,

uma parte do litoral sul de São Paulo, lodo o litoral centro e lodo o litoral norte.

As espécies estudadas, com exceção de uma que é comensal com eremitas ou ca¬

ranguejos, ocorrem na zona das marés, usando rochas, areia, lôdo e algas como

substrato. Enumeradas em ordem sistemática, essas espécies são: Actinia bermu-

densis (McMurrich, 1889). Ane.monia sargassensis Hargitl, 1908, Runodosoma cais-

saram Corrêa, 1961, Bunodosoma cangicum Corrêa, 1964, Anthopleura cascaia

Corrêa, 1964, Phyllactis conchilega? (Dueb. & Mich.. 1860). Phynianthus canoas

Corrêa, 1961, Paranthus rapiformis (Lesueur, 1817). Calliactis tricolor (Lesueur.

1817) e Aiptasia pallida ( Verrill. 1864).

Pouco pode ser dito a respeito da distribuição e frequência destas espécies

para a totalidade do litoral brasileiro. A espécie comensal. Calliactis tricolor, ocor¬

re desde o Ceará até o extremo sul do Estado de São Paulo, liba do Bom Abrigo,

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Mem. Inst. Butantan

DIVA DINIZ CORRÊA

Slmp. Internac.

K3UI:27-30, 1966

Cananéia. sendo relativamente comum em profundidades de cêrca de 5 metros.

Sete espécies são conhecidas desde Ubatuba até Itanhaém e apresentam, algumas

delas, intensa freqiiência em certas localidades como a Enseada do Flamengo e

a Praia do Segredo, no litoral norte, a Ilha das Palmas, na Baía de Santos e a

Praia do Sonho, em Itanhaém. Duas espécies apresentaram até agora uma ocor¬

rência muito restrita em ambientes muito especiais e freqiiência bastante baixa.

São o Phymanthus canous de praia lodosa do Saco da Ribeira e o Paranlhus

rapiformis de areia da Praia de Baraqueçaba, ambos os locais situados no litoral

norte do Estado de São Paulo.

Tendo estudado também 16 espécies de anémonas do mar da Ilha de Curaçau,

Antilhas Holandesas, sugeri a hipótese que algumas espécies que ocorrem nesta

ilha em praias tipicamente coralinas, ricas em corais vivos, deveriam também ser

encontradas em ambientes coralinos do litoral brasileiro. Quatro confirmações já

foram obtidas. Lebrunia coralligens (Wilson, 1890) e Condylactis gigantea (We-

inland, 1860) ocorrem no Arquipélago dos Abrolhos, sul da Bahia. Lebrunia

danae (Duch. & Midi.. 1860) e Stoichactis helicmthus (Ellis, 1767) foram verifi¬

cadas em uma praia de Recife, Pernambuco.

Nenhum caso de queimadura ou de envenenamento é do meu conhecimento

nas regiões onde tenho trabalhado, a Ilha de Curaçau e a costa de São Paulo.

Afora a capacidade intensa de adesão tentacular que algumas espécies apresentam,

não tive até agora nenhuma sensação de queimadura após o contato direto com

as 23 espécies que estudei.

Consultando o livro de Keegan e MacFarlane. referente ao Simpósio sóbre

animais venenosos e plantas nocivas da região do Pacífico (1963), no artigo de

R. V. Soulhcott, sôbre Celenterados de importância médica, verifiquei que os 30

casos de envenenamento conhecidos ocorreram entre o norte da Austrália e o con¬

tinente asiático até o norte das Filipinas. São mencionados casos de injúrias cau¬

sadas pelo contato com anémonas do mar, com corais verdadeiros e com corais

falsos que pertencem a outra classe dos Celenterados.

A anémona do mar Sagartia elegans (Dalyell. 1848), de acordo com Halslead

(1959), é a causadora da “sponge diver’s disease” ou "maladie des plongeurs"

no Mediterrâneo. Fstas anémonas utilizam-se das esponjas como seu principal

substrato. Os velhos pescadores de esponjas possuem numerosas cicatrizes nas

mãos causadas pelas injúrias. Outras espécies como Actinodendron plumosum,

Actinodendron arboreum, Actinia equina, Adamsia palliata e Anemonia sulcata

são mencionadas como causadoras de severas queimaduras. Corais causam úlceras

na região indo-pacífica assim como Acropora palrnata das índias Ocidentais.

I ma espécie conhecida como “matamalu"' em Samoa, Rhodactis houesii, se¬

melhante a uma anémona do mar verdadeira, quando cozida, faz parte da alimen¬

tação dos nativos. São conhecidos casos de envenenamento após ingestão acidental

ou intencional destas anémonas cruas.

SuMMARY

The present “mise au point” aboul the State of research on ANTIIOZOA

(COELENTERATA) from the Brazilian coast, including taxonomy, distribu-

lion, and frequency, shows that very little is known up to now of this large Class of

rnarine invertebrates. Of the about 6,100 species in the group, only 17 GORGO-

NACEA, 14 ACTINIA RIA, 14 MA DREPORARIA, 1 TELESTACEA, 1 PENNATU-

LACEA and 1 CERIANTHARIA, were already described from Brazilian waters.

2 3

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11 12 13 14 15

30 TAXONOMIA DE ANTHOZOA ( COELENTERATA ) BRASILEIROS: DISTRIBUIÇÃO

E FREQUÊNCIA EM AGUAS BRASILEIRAS

The Department of Zoology, University of São Paulo, Brazil, has receutly

started a survey of tho species of two larger Orclers, the GORGONACEA and the

ACTINIARIA, occurring on lhe coast of São Paulo. The chosen area extends

from Itanhaém, on the Southern, lo Uhatulia, on the Northern part. Ten species

of sea-anemones were already described and a few more are in process of study

together with several species of GORGONACEA.

Some of lhe ten above mentioned species of sea-anemones have a large dis-

tribution and frequency in the Western Atlantic Ocean. A few others are as yct

known only for the coast of São Paulo, one or 2 of which have a low frequency.

No severe injure to man, caused by these animais, has heen noticed among

the Brazilian species, as it is known for some sea-anemones and corais occurring

in the others parts of lhe world.

Literatura

1. Buyer, F. M. — The shallow-water OCTOCORAEEIA of the West Indian

Region. A manual for marine biologists. Haia, Hol.. Martinus Nijhoff, 1961.

2. Corrêa, D. D. — CORALLIIVIORPHARIA e ACTINIARIA do Atlântico Oeste

Tropical. Serv. Docum. R.U.S.P., pp. 1-139, São Paulo, Brasil, 1964.

3. Halstead, B. W. — Dangerous Marine Animais. Cambridge, Maryland, Cornell

Maritime Press (citado de Keegan & Mac Farlane, 1963), 1959.

4. Hand, C. — Present State of Nematocyst Research: Types, Structure and

Function. The biology of Hydra. Miami, Lenhoff, H. M. & Loomis, W. F.,

1961, pp. 187-202, f. 1 a-b.

5. Smith, F. G. W. — Atlantic reef corais — a handbook of the common reef and

shallow-water corais of Bermuda, West Indies and Brasil. Miami, 1948, pp.

1 - 112 .

6. Southcott, R. V. — Coelenterates of medicai importance. In: Venomous and

Poisonous and noxious plants of the Pacific Region. New York, Keegan, H. L.

& Mac Farlane, W. V., 1963, pp. 41-65.

7. Stiasny. G. — Alcyonids et Gorgonides des collections du Muséum d’Histoire

Naturelle (II). Mem. Mus. Nat. Hist., Sér. A, Zool., 3(l):l-80, 1951.

8. Verril, A. E. —• The Gorgonians of the Brazilian Coast. Journ. Acad. Nat. Sei.

Philad., 205) :393-404, 1912.

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Mem. Inst. Butantan

Simp. Internac.

33(1):31-34, 1966

PAULO SAWAYA

3. TOXIC MARÍNE INVERTEBRATES — VENOMOUS AND NOXIOUS

FISHES OF FRESH WATER

PAULO SAWAYA

Departamento de Fisiologia Geral e Animal, Universidade de São Paulo,

São Paulo, Brasil

Toxic MARÍNE AN1MALS — Research on toxic inarine animais has heen done

in Brazil by Faria (1914, p. 29) on PROTOZOA. Pteridinium tochoideum.

which is associated willi mass mortality of marine organisms, and Prorocenlrum

sp. were described. As it is well known, lhe first one is very common on the

Brazilian eoasts, hui their appearance in mass is, fortunately, nol very frequent.

In the sound of São Sebastião, where the laboratory of the Marine Institute of

Biology is located, the red lide has heen observed in 10 years only once.

Marine invertebrates — PO RI FERA — De Laubenfels (1932, p. 85)

says that Tedania toxicalis is toxic and its toxicity is one of its striking characters.

The spicules are long and provoke a very strong irritation on the hands. Even

other animais are sensitive lo lhose spicules. According to De Laubenfels (l.c.)

if a speeimen of this sponge be placed in a bueket with other living sea animais,

as for example, fish, molluscs, crabs, and worms, in an hour or less they are

observed lo die. while in Controls lacking the sponge they survive.

It seems that not all species are sensitive to the toxin because the sponges

live in very dense community with other animais, chiefly, Echinoderms (Ophiu-

roids), small crustaceans (Amphipods, Polychaetes), etc.

The zonation of these animais is very interesting. Their habitat is restricted

to the middle and inferior littoral.

In the same place, on the beach of the São Sebastião sound, there are a

number of Coelenterates. For example, Palylhoa sp. are very common. The.

abundant mucus secreled by these animais, injected into the vascular System of

some mammals, such as the Rat, seems to be toxic. The mechanism of secretion

of this mucus, and the toxicity is unknown. Some experiments liave heen run at

the laboratory of the Institute of Marine Biology al São Sebastião, 230 km to the

north of São Paulo, and we expect lo confirm or not its toxicity.

Echinoderms — It is known that several Echinoids are venomous. In the

Institute of Marine Biology, Mendes, Abbud and Umiji (1963, p. 408) have

studied a substance produced by the pedieellariae of the Sea Urchin Lytechinus

variegatus. It has an acetylcholine-like behavior, according lo the residts obtained

on the responses of lhe guinea pig illeum. rat uterus, blood pressure in the dog,

heart-beating of toad, longitudinal muscle of holothurian, and the protractor muscle

of the sea urchin lantern.

2 3

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TOXIC MARINE INVERTEBRATES — VENOMOUS AND NOXIOUS FISHES

OF FRESH WATER

The famous venomous sea-urchin gen. Diadema does not occur on the

Brazilian coast. The Sea Urchins common here are Lytechinus variegatus, Echi-

nometra lucunter and Arbacia lixula. Echinometra lucunter is lhe niost common

sea-urchin on the Brazilian seashores. The spines are straight and pointed. lt

is not known if lhe spines of Echinometra have lhe so-called poison glands.

They are horne in a tubercule of the thecal plate. When the spines of Echi¬

nometra perfurate the skin llie wound is painful, and sometimes an inflam-

mation occurs.

Diadema has been captured in the hay of Acapulco, México. Some spines

ean exceed 50 cm in lenglh, and injure the skin when touched. I know lliis by

my own experience when I visited the Acapulco hay. Russell (1965, p. 285)

says: “There does not appear to be any biochemical or toxicological evidence, at

the present lime, to indicate that these structures do indeed contain a poison”.

All 1 k now is that when the spines are touched the skin shows an irritation and

is painfull.

Holothurians — On the Brazilian sea-shore Holothuria grisea is very

common. They have some toxin in lhe Cuvierian tubules. Some investigators

think that the toxin secreted by the Cuverian tubules is related lo the fact of

evisceration, shown by this animal, when oul of water, or when exposed lo excessive

changes of temperature, pH and oxygen tension. Nigrelli (1952, p. 89) named

lhe substance of the Bahamian sea cucumber, Actinopyra agassizi, as holothurin.

lt is composed of 60% glycosides and 30% pigment salts, polypeptides and free

aminoacids, 5 lo 10% insoluble protein and 1% cholesterol. Holothurin has

deleterious effects on Hydra, P lano r bis, and Tubifex.

Worms — Among the Polychaetes several species are toxie. On the Brazilian

coasts the AMPHINOMIDAE are very frequent. Hartmann (1951, p. 21) indicates

that the common name, “fire-worm” allndes lo the stinging sensation caused from

handling specimens. The injury is mechanical, resulting from the penetration

into the skin of numerous, fine, glass-like, harpoon-shaped setae that are difficult

to remove. Inflammation and considerable discomfort result. but there is no per-

manent injury.

I have observed these worms under the stones in the intertidal zone, and the

collectors must be careful, because they can irritate the hands. Several cases,

that I observed indicate that the sensitiveness of people is different in degree,

probably related to some allergic reaction.

The same Polychaetes (AMPHINOMIDAE) were considered long time ago as

venomous, according lo Baird (1864, p. 450) who says: “The specimens of this

worm Amphinome didymobranchiata carne from lhe Island of Ascención where

they are collected by lhe boatmen and sold as curiosities. They pretend that they

are of venomous nature, and are able to inflict serious wound upon lhose who

incautiusly handle them. This idea no doubt takes its origin from numerous

setae with which their feet are clothed, but which (to judge from their appear-

ance...) in realily powerful weapons for offense and defense against ibese animais

whicb ])rey upon or are filted for food for them are in fact powerless for harm

to human beings”. Arndt (1930, p. 292) refers lo noxious Polychaetes such as

Hermodice corunculata, which is very toxic.

According to Kussell (1964, p. 480) the composilion of the venoms of marine

animais varies considerable. Among the Coelenterates there are the following

composilion: several quaternary ammonium compounds, lhe most toxic of which

1, | SciELO

Mem. Inst. Butantan

PAULO SAWAYA

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33(1): 31-34, 1966

being the tetramelhylammonium hydroxide our ''letramine”, 5-hydroxytriptamine,

histamine and histamine releasers, several proteins whose composilion has nol yet

been determined.

Venomous and noxious FHESHWATEK EISUES — South American freshwatera

are populated by several species of venomous fishes.

The so-called black rays or Arraia arara and Arraia pintada (Trygon stron-

gylopterus, Potamotrygon brachyurus or Par trygon motoro ), are small rays, found

in lhe rivers Amazon and Paraguai. One or Hvo stings are located in lhe tail.

They are known also by the common name of stingray. The sting is bilaterally

serrated. The venom and also the venom apparatus of Brazilian stingray have

nol been studied. Russell (l.c., p. 339) descrihe the marine stingray, Urolophus

halleri, and says that the venom is known to exert a deleterious effect on the mam-

malian cardiovascular system. It causes bradycardia and increase the PR inlerval

of the ECC.

Observations of some cases of attacks by the Partrygon from the Araguaia

river, in the north of Brazil, indicates that the sting delivers a large quantity of

mucus, and the venomous substances probably exist in it.

Another venomous fish is Potamotrygon brachyurus from Guyana, Venezuela

and the Amazon region. Some information on lhe biology of this ray has been

quoted by Dr. Hermann von Ihering. The fishermen say that this fish when

attacked keep the fry in the vagina.

SILURIDAE — In Brazilian rivers several fishes of this family are very

common. Most of them have the firsl ray of the pectoral and dorsal fins ser¬

rated. When caught tliey attack the men witli this serrated ray. In the begin-

ning the vvound provokes only a strong pain, but afterwards as a result of the

mucus left inside the skin, inflammation occurs, followed by headache and in

some cases vomiling. ll has nol been demonstrated up to now if this serrated

fin ray is venomous or not in spite of the information from Lagler, Bardach and

Miller (1962, p. 132) who indicate that catfishes ( Ciarias and others) possess

spines of dorsal and pectoral fins vvitb glands beneath the skin, opening through

poses at bases of spines.

The same authors (l.c., p. 184) inform that in the North American fresh-

water catfishes ( ICTALURIDAEl , the sharp. hardened ray of the leading edge

of the pectoral fin has a locking structure, which enables the catfish to erect and

hold it erect, presumably as an instrument of combat. In the madtonus ( N o t u -

r ii s ) this spinous ray has a special gland at its base. The secretion of the

gland, injected by the spine, has a stinging. paralyzing effect on man.

In this family several fishes are edible and the fishermen eut out the first

ray of the pectoral and dorsal fins lo maintain lhe fishes in the hand. In the

creeks and rivers of the outskirts of São Paulo, fishes of the family PIMELODIDAE

(Pimelodus sp., and Ciarias sp.) and others can be easily found. When caught

the fish spread out the rays of the dorsal and pectoral fins for defense. If the

fisherman is wounded by the serrated firsl ray and inflammation usually follows.

Russell (1965, p. 480) informs that fish venoms are composed of 3 to 10

proteins and have liltle or no enzymatic activity.

Other fishes — The famous piranha (sufi fam. Serrasai.moninae) from

tropical waters can be here included. These fishes usually damage ealtle, other

2 3

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11 12 13 14 15

TOXIC MARINE INVERTEBRATES — VENOMOUS AND NOXIOUS FISHES

OF FRESH WATER

fishes and mcn, The fishermen say lhat lhe piranhas smell blood, and by lliis

attack the victim. Cannibalism also occurs.

Another curious fisb noxious to man is the so-called Candini (Vandellia cir-

rhosa or Hemicetopsis candiru). People say that tliis small fish of 3-5 cm in length

penetrates lhe urethra of man and the vagina of woman when they are taking a

batli. When the fish penetrates lhe urethra there is an copious bleeding. Eingen-

mann and Allen (1942, p. 142) give a long report on the “candiru” and say (p.

146-147) that “there is little clear-cut evidence by which we can definitely sort

out cases of urinotropism from parasitism”.

Santos (1962, p. 114) also describes this curious fish. but by the figure and

the size of lhe animal il does not seem probable that it can enter into a man’s

urethra.

Within lhe TETRAODONTIDAE there is a species, Colomesus psitlacus, named

“baiacu” or “mamaiacu”, which similar to its relative from the sea water, is be-

lieved to be venomous (Couto Magalhães 1931, p. 95). It lives in the rivcrs of

lhe North of Ilrazil. The flesh is edible but the liver is very toxic. The fish is

small (18 cm in length) and usually can be kepl in aquarium as an ornamental fish.

The South American electrical — Electrophorus clectricus — could be listed

here. This fish is neither toxic nor venomous, but is noxious to the human being

and other animais.

Literature

1. Arnd, W. — Polychaeten ais Gesund-Heitschãdlinge des Menschen. Sitz. d.

Gessell. naturforschenden Freunde, 1930.

2. Baird, W. ■ — Description of a new species of ANNELIDA belonging to the

Family AMPHINOMIDAE. Trans. Linn. Soc., 24:449-450, 1864.

3. De Laubenfels, M. W. — The Marine and Fresh-Water Sponges of Califórnia.

Proc. U.8. Nat. Mus., 81:1-140, 1932.

4. Ergennann, G. H. & Allen, W. R. —- Fishes of Western South America. XIII +

494 pp., The Univ. Kentucky, Lexington, Kent., 1942.

5. Faria, J. G. de — Um ensaio sôbre o plankton seguido de observações sôbre

ocorrência de plankton monótono, causando mortalidade entre peixes na baia

do Rio de Janeiro. 48 pp., Rio de Janeiro, 1914.

6. Hartman, O. — The Litoral Marine Annelids of the Gulf of México. Publ.

Inst. Marine Science, 2(11:7-124, 1951.

7. Lagler, K. F., Bardach, J. E. & Miller, R. R. —• lchthyology. 545 pp., The

Univ. Michigan. Ann Arbor, Michigan, 1962.

8. Magalhães, A. C. de — Monografia Brasileira de Peixes Fluviais. 260 pp., Sec.

Agric. Ind. Com. Est. S. Paulo, São Paulo, 1931.

9. Mendes, E. G., Abbud, L. & Umiji, S. — Cholinergic Action of Homogenates

of Sea Urchin Pedicelliariae. Science, 139(35531:408-409, 1963.

10. Nigrelli, R. F. — The effects of Holothurin on Fish and Mice with Sarcoma

180. Zoologica N. York, New York, 37(11:89-90, 1952.

11. Russell, F. E. — Marine Toxins and Venomous and Poisonous Marine Animais.

In: Russell, F. S., Advances in Marine Biology, London & New York, 3:255-

384, 1965.

12. Russell, F. E. — Venomous animais and their toxins. Ann. Rey. Smithsonian

Inst., Washington, pp. 477-487, 2 t, 1965.

13. Santos, E. — Peixes d’água doce. 2" ed., 278 pp. Rio de Janeiro, F. Briguiet

& Cia., ed., 1962.

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Mem. Inst. Butantan

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33(1): 35-44, 1966

J. VELLARD

4. LA FONCTION VENIMEUSE CHEZ LES ARAIGNÉES

J. VELLARD

Instituto Boliviano de Biologia de Altura, La Pag, Bolivia

Morphologie des glandes — Deux familles à caracteres archaiques bien

marques, les FILISTATIDAE et les SCYTODIDAE s’éloignent de loutes los autres

araignées par la morphologie des glandes de leurs chélicères.

Celles des FILISTATIDAE sont multilobées et occupent une grande partie du

céphalothorax.

Dans le genre Scytodes , des glandes volumineuses donnent à la région

thoracique sa forme bombée, caractéristique du genre. Les travaux de Millot, et

avant lui ceux de Monlerosso, ont rnontré que ces glandes se divísent en deux

parties histologiquement differentiées, séparées par mi repli interne: une partie an-

térieure, souvent réduite, correspondant aux glandes venimeuses habituelles; et une

partie postérieure beaucoup plus développée, à sécretion basophile, visqueuse, très

voisine de celle des glandes sérigènes et servant à ces araignées à engluer leurs

proiés. Cetlo double fonction rappelle celle des glandes des chélicères des pseudo-

scorpions.

Dans iin second genre de SCYTODIDAE, le genre Sicarius (nous avons

surtout étudié S. peruviensis) , les glandes volumineuses, pouvant atteindre 6 mm

de longueur, sont divisées extérieurement en nombreux lobes granuliformes, à paroi

très mince.

Un troisième genre, Loxosceles ( L. laeta et autres espèces sud-américai-

nes) marque le lerme de 1’évolution de cette famille. A 1’extérieur, les glandes

sont divisées du côté interne par de profondes incisions correspondant a des cloi-

sons intérieures séparant incomplètement la lumière de la glande en plusieurs lobes.

Des glandes en jjartie lobulées existent encore cbez les PALPIMANIDAE: Ani-

saedus stridulans de la côte du Pérou.

Le type classique, en forme de sac, lisse extérieurment, sub-cylindrique ou

plus ou moins arqué ou eoudé, se trouve dans toutes les autres familles étudiées.

Les glandes des Mygalomorphes, sauf rares exceptions, sont logées dans la

convexité des chélicères.

Cbez les aranéomorphes, elles sont entièrement thoraciques ou partiellement

engagés à la base des chélicères ( CLUBIONIDAE, DRASSIDAE, SALTICIDAE,

TETRAGNATHIDAE ). Les glandes des PALPIMANIDAE très réduites, sont si-

tuées dans les chélicères (Anisaedus).

Histologie des glandes — Chez loutes les araignées les glandes sont cons-

tituées par une fine adventice conjonctive externe, qui parfois disparait sur les

coupes; une tunique musculaire d épaisseur variable, formée de fibres striées dis-

posées en spirale et une basale sur laquelle repose 1’épithelium glandulaire.

SciELO

10 11 12 13 14 15

LA FONCTION VENIMEUSE CHEZ LAS ARAIGNÉES

L adventice conjonctive envoie de. fins prolongements à Iravers les filires

musculaires jusqu’à la basale.

La tunique musculaire, bien développée chez les Mygalomorphes el ehez beau-

conp d’Aranémorphes, peut être parfois très mince, comme chez Ctcnus medius et

les Dysdéridés; chez d’autres araignées elle alteinl au contraire un développement

considérahle (Nephila cruentata).

Sur les coupes 1’aspect de la glande varie suivant la phase de sécrétion

(tu venin.

Au débul, les cellules épithéliales, formées d’éléments prismatiques, à pelit

noyau basal, re])osenl sur la basale el sur les franges internes très développées

formant un réseau remplissant la Iumière de la glande.

A une période plus avancée, les cellules augmentent de volume de la base

au sommet de ces franges qui disparaissent en grande partie; les cellules se rem-

plisscnt de granulations acidophiles comprimant le noyau vers la périphèrie; puis

elles se rompent et les noyaiix déjà peu visibles sont mis en liberte au milieu de

la sécrétion acidophile remplissant le centre de la glande.

En fin de sécrétion, les franges internes ont à peu près dispam. La séeré-

tion acidophile, presque sans vesliges de noyaux, occupe toute la Iumière de la

glande, comprimant contre la basale les cellules épithéliales. La glande 11 ’étant

plus soutenue par le réseau des mailles internes se déforme facilement à la coupe.

Dès que la glande a vidé son contenu, les cellules épithéliales entrent en pro-

Iifération active.

Le temps nécessaire pour remplir les glandes de venin dépend de nombreux

facteurs et varie d une espèce à Lautre: 5 ou 6 jours au moin pour Phoneu-

tria fera.

Rôle du venin. Mécanisme d inocueation — Les chélicères des araignées

ont conservé leur rôle primilif d’organes de préhension. LVxistence de la glande

venimeuse accroil leur valeur d’armes de chasse.

L’injection du venin est volontaire, non subordonnée au mouvement des ché¬

licères, et sous la dépendance de la tunique musculaire striée propre des glandes.

L’action mécanique des chélicères suffit souvent pour immobiliser et tuer les pe-

tites proies, sans intervention du venin. Tenue avec une pince 1’araignée peut

tnordre les animaux qui lui sont présentés sans innoculer de venin, faussant ainsi

le résultat de beaucoup d’expériences. Le cas est fréquent avec des mygales à

venin très actif, telle que Trechona venosa, dont 1’action de la morsure ne peut

être étudiée par à procédé.

Le venin des araignées est dépourvu d’action protéolytique et n’intervient

pas dans la digestion; ce rôle est réservé aux glandes des maxilles dont 1’activilé

digestive est très élevée.

Variation du venin — L’activité et les proprietés du venin varient de mode

considérahle d’un groupe à l autre et il est possible de caraetèriser des types de

genres ou de familles, tels que Ie venin des DIPLURIDAE, celui des Aviculaires

ou ceux de Latrodectus ou de Phoneutria. Les propriétés du venin

s’ajoutenl aux élements morphologiques pour identifier certains phylums.

Dans une famille ou dans un genre, quelques es[)èces peuvent se distinguer

par une acti vi té particulière, exaltalion des propriétés communes à tout le groupe:

parmi les SCYTODIDAE, les Loxosceles possèdent un venin de même nature

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Mem. Inst. Butantan

Simp. Internac.

33(1): 35-44, 1966

J. VELLARD

que celui des Si cari a.s, mais beaucoup plus aetif. Seuls les Latrodectes noirs

et rouges sont dangereux pour lhonime: le venin des Lalrocdetes fauves ou gris

(/.. geornetricus, par exemple), montre des propriétés identiques, mais bien moins

accentuées, propriétés qui se retrouvent à im degré moindre ehez de nombreux

THERIDIIDAE.

Ces variations du venin, d’origine génètique, peuvent s’observer à l’intérieur

d’une espèce, associées ou non à celles d’autres caractères morphologiques ou

éthologiques peu apparents; certains auteurs ont conclu ainsi à 1’existence d'es-

pèces physiologiques ou cryptiques: le cas s’est produit ]>our Latrodectus mactans.

de Santiago dei Estero. Le venin de cette espèce offre d’ailleurs de grandes

variations régionales dans son aire très vaste de dispersion.

Les différences individuelles ont beaucoup moins d’importance et il est tou-

jours possible d’établir des moyennes d’activité pour une espèce dans une région

ou dans des conditions saisonnières ou climatiques données.

LMnfluence du climat et de la température sont en effet considérables sur

les araignées. Dans mes premiers travaux au Rrésil j'avais déjà noté de varia¬

tions sensibles de 1’activité du venin de divers Ctenides des environs de Rio de

Janeiro (Ph. fera, Cl. medias , Ct. ornatus ) suivant 1'époque de 1’année, la tem¬

pérature et le degré hygrométrique. Le maximum de toxicité s’observait par

lemps chaud el humicle. II m‘avait même été possible en plaçant des araignées

vivantes dans des chambres à températures différenles (+15° et +30“C) de con-

trôler expérimentalement ces observalions. Les modifications n’affectent le venin

uue pendant Ia période de séerétion et non le venin déjà élaboré remplissant la

glande. En même temps que la toxicité augmente, le pH du venin s’abaisse.

J'ai eu 1’occasion d’observer des faits identiques avec Loxosceles lacta. Le

venin des exemplaires des environs de Lima est beaucoup plus toxique que celui

fourni par des exemplaires du Chili.

Exemplaires de Lima: avec 0,25 glande, 100% de mortalité pour le cobaye; avec

0,10 glande, 50 à 70% de mortalité.

Exemplaires de Santiago: avec 0,40 glande, 50 à 70% de mortalité; beaucoup

d’animaux résistent à 1’injection d’une demie glande.

11 est par contre plus difficile d’obtenir chez le cobaye des lésions de nécrose

avec le venin de Lima: la dose limite mortelle étant très proche de la dose né-

crosante, ces deux seuils sont plus largement séparés avec le venin de Chili.

Des exemplaires recueillis non pas sur la côte, mais au-dessus de Lima. entre

2.000 et 2.500 mètres dallitude, en climat plus froid, ont donné des résullals

identiques à ceux du venin chilien.

Spécialisation du venin — La toxicité plus marquée du venin pour les

proies habituelles est un fait banal chez beaucoup d’animaux venimeux. II se

relrouve chez les araignées et pour juger de 1’activité réelle de leur venin, il est

nécessaire de Tétudier sur une série étendue d’animaux d’experience.

Tons ces venins sont généralement très actifs pour les insectes, mais un bon

nombre d’entre eux présentent aussi une toxicité élevée pour des groupes très

differents.

Le venin des Latrodectus possède une aetivité particulière ])our les

scorpions, mais est également dangereux pour les vertébrés et pour l’homme.

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LA FONCTION VENIMEUSE CIIEZ LAS ARAIGNÉES

Le venin de T r e c li a I e a et cTautres PISAURIDAE aquatiques possède une

action marquée sur les têtarcls et les petits poissons.

Les grands Enoploctenus qui chassent la nuit à Penlrée des groltcs et

sur les parois de rochers ont un venin beaucoup plus actií que celui des autres

C t e n a s ])our les geckos qui abondent aux mêmes endroits.

Le venin de nombreuses THERAPHOSIDAE se inontre curarisant pour les

vertébrés; beaucoup de ces grosses mygales capturent des lézards, des petits ron-

geurs, ou même de jeunes oiseaux.

Un des cas les plus remarquables est celui de grandes G r a m m o s t o l a dont

le venin est particulièrement actif pour des batraciens ou des petits reptiles, per-

mettant à ces araignées de lues de jeunes serpents.

Je n’ai pu vérifier 1’action du venins des ARCHEIDAE aniéricaines sur les

autres araignées, mais j’ai Irouvé à plusieurs reprises Mecysmauchenius segmen-

tatus, qui vit sons les écorces humides des Notojagus de la Terre de Feu,

dévorant des araignées beaucoup plus grosses quYlle: Anyphaena et Ho¬

me o m m a .

Caracteres généraux du venin ■—• II existe trop peu de travaux sur 1'ana-

lyse chromatographique du venin et de Fhémolymphe des araignéees pour en tirer

des conclusions valables sur leur structure dans tout l’ordre.

Nous avons, par contre, de nombreuses études sur les propriétés pharmaco-

logiques de ces venins dans les diverses familles d’araignées.

Ces venins, bien plus simples que ccux des serpents, n’ont pas 1’action com-

plcxe de ces derniers.

Les venins d’araignées peuvent se ranger dans deux grandes catégories: les

venins d’action neuro-musculaire et les venins cytotoxiques à propriétés protéasi-

ques prépondérantes.

Les venins neuro-musculaires sont des curarisants, tels ceux de la plupart des

THERAPHOSIDAE; ou des venins élevant le tonus musculaire et provoquant des

contractures de la musculature striée et lisse et des convulsions de type tonique

(Diplures, C t e n u s ) ou clonique ( Latrodectus ).

Certains venins cytotoxiques limitant leur action au derme sont respon-

sables de lésions culanées plus ou moins étendues, sans répercussion sur 1’état gé-

néral (Lycoses). D’autres déterminent la mise en liberte d’histamine, se tradui-

sant par des lésions locales et un état de choe avec son cortège habituei de ma-

nifeslations: hypotension, stases viscérales intenses, cbüte du nombre des globules

rouges, bémolyse, diminution des protéines sanguines (aranéisme cutanéo-hémo-

lytique), suivies généralement de lésions hépatiques et rénales tardives; avec d’au-

tres venins enfin |)redominent les altéralions du foie et des reins.

Aucun des venins étudiées, même pas ceux responsables de Paranéisme cuta-

néo-hémolytique (Loxosceles), n’ont montré d’action coagulante ni (Paction

lytiquc in vitro sur les globules rouges, seulcs ou en présence de lécilhine ou de

seriirn normal, soulignant le mécanisme indirect de leur puissante action liémo-

lytique dans Porganisme. Leur action ia vitro protéolytique, anti-coagulante ou

sur le complément est Irès peu marquée et ne s’exerce, qidavec des doses élevées

de venin.

Résistance des araignées à leur propre venin — Le problèmc de Pexis-

tence d une véritable immunité et de son mécanisme chez les invertébrés, a été

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Mem. Inst. Butantan

Simp. Internac.

33(1): 35-44, 1966

J. VELLARD

SOUVent ]>osé sans recevoir de solulioii définitive. Cetle immunité est générale-

ment considérée comme différente de celle des vertébrés, et serait liée aux bé-

mocytes, sans intervention de sensibilisalrice ni de complément.

La résistance des invertébrés venimeux à leur propre venin est im cas par-

ticulier de ce problème général. Son étude se heurte à de nombreuses difficultés

tecbniques: sensibilité très grande de la plupart de ces animaux, des araignées

en particulier, aux injections et aux hémorragies; toxicité de leur bémolymphe

pour les animaux habitueis de laboratoire, ne permettant pas le plus souvent d’em-

ployer des doses suffisantes pour étudier son pouvoir protecteur ou son action

neutralisante sur le venin.

Les travaux de Metchnikof, de Calouillard et de Marie Phisalix ont eepen-

dant bien établi que les scorpions offrent une résistance élevée à leur propre

venin et que leur hémolymphe possède contre ce venin un pouvoir neutralisateur

et même préventif.

Peu de choses ont été publiées sur les araignées. R. Lévy, en 1916, a mon-

tré le premier que 1 bémolymphe des Tégénaires et de quelques autres araignées

exerce une légère action préventive contre le venin homologue.

Toute les araignées sonl loin de se prêter à ces recherches.

La toxicité élevée pour le pigeon de 1’hémolymphe de Phoneutria fera et de

Trechona venosa ( DIPLURIDAE ) ne m’avait permis dans mes premières recher-

ches que des résultats incomplets: 1’injection endoveineuse d’une dose inframortelle

(1’hémolymphe (0.1 à 0,2 ml) additionée d’une dose mortelle de venin, avail

évité la mort, mais non les symptômes graves avec la première espèce, et atténué

les ])hénomènes convulsifs sans survie appréciable des animaux avec la seconde.

Ces recherches onl été reprises dans des conditions bien plus favorables avec

une mygale de taille moyenne de la côte du Pérou, Hapabpus Umensis. Son

venin curarisant est très actif pour les vertébrés, de la souris au cbien; souris,

lapin et cobaye supportent sans accident 1 injection inlramusculaire, intrapérito-

néale ou endoveineuse de doses élevées, 1 à 2 ml d hémolymphe. La curarisation

ehez le lapin el le cobaye se produit en 10 à 15 minutes avec des doses de venin

voisines de la minima mortelle.

0.50 ml d’hémolymphe injeclés par voie intramusculaire en même temps que

le venin protègenl complètement le cobaye contre une dose mortelle de venin

(0,5 gl.) ; 1,0 ml protege contre deux doses mortelles.

0,25 ml ont protégé 50% des animaux contre une dose mortelle de venin;

les autres ont succombé en une vingtaine d beures; mort des témoins en moins

d une heure.

L’hémolymphe a montré une action préventive nette: 0,5 ml ont protégé le

cobaye contre 1’injection postérieure, 12 à 30 minutes, d’une dose mortelle de

venin; témoins curarrisés en 20 minutes.

La même dose, dans des conditions idéntiques, a retarde considérablement

la mort avec 2 doses mortelles de venin: curarisation en 80 minutes, mort en 12

heures; controle, curarisation en 3 minutes, mort en 17 minutes.

L’hémolymphe d'une grande mygale du Haut-Amazone, Paniphobetens ntgri-

color, a montré une action analogue vis à vis de son propre venin:

1,0 ml d’hémolymphe, en injection mixte par voie veineuse au lapin. a neu-

tralisé complètement le venin d une demie-glande; curarisation du controle en 15

minutes, mort en 18 heures.

SciELO

10 11 12 13 14 15

LA FONCTION VENIMEUSE CIIEZ LAS ARAIGNÉES

Par contre, avec une grande mygale du nord de 1’Argentine, Acanthoscurria

chucoana, les résullats ont été négalifs. Le mélange d’hémolymphe et de venin

injecté par voie veineuse au lapin a accentué l'effet de choc de ce venin. Le

neu d’exemplaires dont nous disposions n’a pas permis de poursuivre ces expé-

rienees par dautres voies:

1 ml d’hémolymphe plus le contenu d’une glande ont provoque une chute

immédiate de la pression arlérielle, tuanl 1’animal en 3 minutes. Des controles

recevant 1 ml d’hémolymphe ou le venin d’une glande ont montré seulement une

ehúte passagère de la pression.

L hémolymphe des araignées est donc capalile, de proteger contre le venin

homologue et possède même im pouvoir protecteur contre 1’injection postérieure

du venin; mais celte action neulralisante est souvent marquée par 1’effel toxique

ou hypotenseur de l’hémolymphe pour les vertébrés supérieurs.

Sérothérapie contre i.es venins i/arasgnées: La toxicité de Phémolym-

phe ne garde aucun rapporte avec celle du venin et un serum préparé avec I hé¬

molymphe ou avec une macération totale du corps de 1’araignée n’a aucine action

sur le venin.

Les premières tentatives pour ohtenir un serum actif contre le venin d"a-

raignés remontent au début du siècle et sonl dues à des auteurs russes (Sehtscher-

hina et Konstausoff) qui utilisèrent le chameau pour préparer un serum contre

le venin du Latrodecte russe, le Karakurt.

En 1928, avec Vital Brazil, nous avons préparé les premiers serums théra-

peutiques contre le venin de Lycos a, contre celui de divers Ctenus et un

polyvalent anti-ctenolycosique, avec comine animal productcur le mouton, afin

d’économiser Fantigène. Le choix nétail pas très bon. Le mouton es un mé-

diocre productcur d anticorps et le rendement en serum est faible. Cet exemple

a eependant été suivi aux Elats LInis (d’Amor, 1939; Smith Dorms, 1929) pour

ohtenir un serum anti-Latrodecte, et en Afrique du Sud (Finlayson, 1937) pour

un polyvalent anti-Latrodectes.

En 1939, Maxianovich a utilisé, pour Ia première fois et en Russie, le cheval

avec le venin du Karakurt et en 1942, à Buenos Aires, Pirosky et ses collabora-

teurs ont obtenu avec le cheval un serum très actif contre Latrodectus mactans.

En 1953, à Lima, avec 1’âne et le cheval, j’ai préparé des serums très actifs

contre le venin de Loxosceles lacta, loujours utilisés depuis au Pérou. Ces tra-

vaux ont été continués à Butantan, en 1961, par Reynaldo Schwindl Furlanetto,

qui a préparé des serums actifs contre les venins de Phoneutria, Lycos a,

Loxosceles rufipes et rufescens et de scorpions.

Les venins d araignées sonl des bons antigènes et la préparation de ces serums

n offre aucune difficulté.

L action rclativement simple de ces venins permel aussi de combattre les ac-

cidents avec une medication symptomatique, qui peut donner d’assez bons résul¬

lats. mais inférieurs à la sérothérapie spéeifique.

Le glyconate de chaux a parfois une action spéctaculaire mais inconstante

dans les accidents par Latrodectus. La cortisonne, la néo-stigmine, la chlo-

ropromazine ont donné quelques résultats favorables avec les Latrodectus.

Les anti-histaminiques sont indiqués en cas de piqüre de Loxosceles.

L’association du traitement symptomatique avec la sérothérapie spéeifique

constitue Ia méthode de choix.

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):35-44, 1966

J. VELLARD

Le venin des prixcipales famili.es d araignes

Yous ne pouvons ici entrer dans de longs details sur les propriétés du venin

dans les principales familles daraignées et nous nous limiterons à quelques con¬

siderai ions générales.

MYGALOMOBPHES: Les propriétés du venin sont três voisines chez

les ACTINOPODIDAE, les CTENIZIDAE et les DIPLURIDAE. Ce sont des venins

neurotropes provoquant des tremblements, des contractures toniques et dans les

cas graves des convulsions et la paralysie, avec exagération des sécrétions, sans

réaction locale. Celui des deux premières familles est surtout aetif pour les in-

sectes et parfois pour les petits réptiles et Imtraciens. Aucune espèce n est dan-

gereuse pour 1’homme.

La toxicité du venin des DIPLURIDAE est plus élevée. La géante de la

famille, Trechona venosa peut tuer des petits mammifères et des oiseaux; 1/100

de glande suffit pour un pigeon. Vivant dans de profondes terriers, elle n’offrc

aucun danger pour l’homme. Un venin aussi aetif, mais en faible quantité se

trouve chez des petites espèces de cette famille, Ischnothele, Diplura mon-

ticolens. En Australie, deux représentanls du genre Atrax peuvent être causes

daccidents graves.

Le venin des THERAPHOSIDAE et BARYCHELIDAE est de type curarisanl.

Les espèces étudiées d' AVICULARIIDAE et d’IsCHNOCOLlNAE tuent rapidement des

petits mammifères de la taille d’une souris au d un cobaye et même des lapins

ou de jeunes chiens; au-dessus d une dizaine de kilos leur piqüre est sans cffel.

II en est de même pour les Grammostolinae; quelques unes de celles-ci pré-

sentent, nous 1 avons vu, une activité particulière pour les reptiles et les batraciens.

Le venin de nombreuses Theraphosinae. principalement des Acanthos-

c u r r i a, P a m p hobo et e u s, P h o r m ic to p u s et formes voisines possède

en plus une action cytotorique marquée, provoquant une lésion locale pouvant

aboutir à l’escharre, un état de choc plus ou moins accusé e! des lésions hépati-

ques et rénales tardives, les rendant dangereuses pour Ihomme.

ABANEOMOKPHES : Nous avons étudié des représentanls de presque

toutcs les famill(‘s américaines de ce groupe,

Seules les THERIDIIDAE, les CTEN1DAE, les HETEROPODIDAE, les LYCO-

SIDAE et les SCYTODIDAE comptent des espèces ayant une réelle importance

pratique.

BeauCoup d autres araignées possèdent des venins très intéressants, mais dont

nous ne pouvons nous occuper ici. Leur étude permet de comprendre que les

espèces dangereuses ne représentent que 1'exaltation d’un caractere existant dans

lout un genre ou loute une famille.

THERIDIIDAE: Dans toutes les régions tropicales et tempérées. Les L u -

t r o i e c tu s sont redoutés et causes d’accidents graves. Ce sont des araignées

uhiquistes, s’adaptant aux eonditions les plus diverses, apparaissanl certaines an-

néeS en grande abondance; elles peuvent alors occasioner de nombreux accidents.

de véritables épidémies, comme en 1947 en Italie Centrale et en Yougoslavie.

Leur venin neurotoxique provoque des doideurs intenses, irradiantes, une by-

perexcitabilité généralisée, des contractures musculaires cloniques, des convulsions.

une élévation notable de la PA; certains cas peuvent faire penser à un abdômen

SciELO

10 11 12 13 14 15

LA FONCTION VENIMEUSE CHEZ LES ARAIGNÉES

aigu. Leur adiou parail sVxercer direclement Fiir le système nerveux central et

le système nerveux végélalif.

Parmi los animaus d’expérience lo cobaye ost particulièrement sensible et

succombe à un oedème aigu du poumon.

L’espèce amérioaino, L. mactans, s’etend dos Etats Unis à 1’Argentine ot au

Chili. Dans cette vaste aire do dispersion, ollc présente de nombreuses variations

de colorées. Lactivité do sou venin offre également dune région à 1’autre de

sensibles différences, soil pour los conditions olimatiques, soit pour los différentes

inodalités d’aceidonts, soit onfin pour des variations génétiques.

Los propriétés du venin do L. mactans se retrouvent, avoc quelques diffe-

rences, dans toutes los espèces du genro, parfois Irès accusées avec uno action

plus marquée sur la fibre musculaire lise (/,. indistinctus ). parfois Irès alténuées

( L. geometricus).

Ces mêmes propriétés oxistent dans le venin de nombreuses autres THER1-

DIIDAE, mais beaucoup moins accentuées; les glandes de plusieurs exomplaires

sont nécessaires pour produire dos symptômes analogues. Nous avons beaucoup

étudié, par exemple, le venin de diverses espèces do Lithyph antes du Férou,

/.. andinas, L. nigrofemoratus et quelques autres. Tous ont un venin analogue

à celui dos Latrodeclus, mais bien moins aolif. Nous avons également vé-

rifié que le venin des espèces do la côte péruvienne est 3 ou 4 fois plus toxiques

que celui dos mêmes espèces vivant à 3.000 mèlres dans los Andes.

CTENIDAE: Nous nous sommes longuement étendus dans dos travaux an-

térieurs sur lo venin des C t e n u s . Ce sont dos venins neurotropes. provoquant

des contractures et des convulsions toniques, et une douleur intense avec élévation

de la FA, et dos altérations profondes du rythme cardiaquo. La mort avoc les

grandes espèces du sous-genre Phoneutria I fera, nigriventer du Brésil; ru-

fibarbis, d’Argentine; reidyi ot andrewsi, do 1’Amazone; boliviensis , do Bolivie),

peut survenir choz 1 hommo en 2 ou 3 honres.

Dos propriétés identiques so retrouvent dans lo venin dos araignées do taille

moyenne de cette famille. ( C. medias, ornatus, curvipes et autres) qid no possè-

dent pas une dose suffisanto do venin pour être dangereuses pour Phomme. Le

venin de grosses espèces voisines, les C a p i e n n i u s est on général bien moins

toxique; cependant uno espèce do ce genro, non déterminée du Mato Grosso —

les exemplaires ont été perdus au cours d'un voyage aceidenlé — a montré un

venin aussi aclif que celui dos P h o n r u t ria, et Irès redouté des indiens

Nambikwaras qui lui imputont des accidents mortels.

Les mêmes propriétés oxistent à un faible degré chez les espèces amoindries

de cette famille, du genro O do, qui ne disposent que do quantités réduites

de venin.

LYCOSIDAE: Los Lycoses possèdenl un venin d’action nécrosanto limitée,

sans action générale sur los vortébrés. II est facile de reproduire ces lésions

chez le cobaye ou par injection intradermique dans roroille du lapin.

Quelques grandes espèces, Lycosa raptoria, L. erythrognatha, du Brésil, oc-

casionnent dos accidents íocaux, mais sérieux chez 1’hommo. I) autres espèces du

Honduras, du Férou, du Chili no m’ont donné que dos résultats insignificants.

II en est do mêmo d une grosso espèce do Bolivie, L. rujimanoides. des onvirons

de La Faz.

Des araignées d’un genre voisin de la mêmo famille, los Por rima (P. di¬

versa, du Brésil; P. harknessi, du Pérou). provoquenl égalomont dos pelitos lé¬

sions localos chez les animaux d’oxpérionco.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 35-44, 1966

J. VELLARD

HETEROPODIDAE: LVspèce lype, la grande lleteropoda venatoria, est nne

des araignées domieiliaires les plus communes dans loutes les régions tropicales

et tempérées chaudes.

Son venin, assez pen actif, de ty|)e histaminique, provoque un cedème local

parfois assez important, accompagné de vésicules et suivi d’une petite escharre

superficielle. Dans certains cas on observe une éruption scarlatifornie généralisée.

Ces accidents n ont aucune gravite, mais onl pu êlre confondus avec des manifes-

tations allergiques.

Une espèce voisine, //. meticulosa, du Haut-Amazone, de |)lns petite taille,

possède un venin du même type mais un peu plus actif. La morsure, ou l’in-

jection du contenu de deux glandes, peiit tuer le cobaye; 1'autopsie montre, en

dehors de l’o'dème local, une stase viscérale généralisée.

D’autres espèces de la même famille, entre autres le gros Polybetes macula-

tas d’Argentine, ont un venin analogue, mais três peu actif.

SCYTODIDAE: Dans cette famille se trouvent les venins cytotoxiques, d’action

histaminique, les plus typiques.

Les diverses espèces de Loxosceles que nous avons étudiées ( laeta , yura,

taeniopalpus, rufipes) présentent peu de différences dans 1'activité de leur venin.

La première est repasable de nombreux cas d aranéisme classe sons le nom de

cutanéo-ictéro-hémolytique et longtemps attribués à diverses araignées domieiliai¬

res, Fi li st ata, Dysdera et autres, avant que la véritable responsable ne

fiil déterminée en 1935, au Chili, par Escudero.

Les autres Loxosceles étudiés, bien que possèdant un venin aussi actif,

étant moins domieiliaires, ne causent pas d accidents.

Nous avons déjà indiqué que le venin des S içarias est du même type,

avec une aclivité bien moindre.

Les FILISTATIDAE ( Filistata hibernalis, du Brésil; /'. brevipes, du Pérou),

n'offrent aucun danger, leur venin étant peu actif, mais cependant du même type

que celui des Loxosceles.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):45-54, 1966

ROBERTO GAJARDO-TOBAR

EL ARANEISMO EN EL MUNDO TROPICAL Y SUBTROPICAL

ROBERTO GAJARDO-TOBAR

Chile

Hace muchos anos, siendo médico rural, presencié un hecho que me impre-

sionó vivameute y que nunca podré olvidar.

Regresaba, en un mediodía de ardiente verano, al hospital dei modesto pueblo

donde ejercía mi profesión, cuando unos gritos despavoridos comenzaron a oirse

desde una cuadra de distancia, proferidos por un mozo joven que transportaban

dos camilleros sobre una improvisada parihuela. Consternaba escuchar la cre-

ciente intensidad de los quejidos a medida que el pequeno cortejo se acercaba.

Un atleta de 18 aííos, convertido en una miséria, sudoroso, se retorcia gritando

desesperado por terribles dolores en todo el cuerpo y por convulsiones que le

hacían presagiar una muerte inminente. jHabía sido picado por una araria, en

un trigal! La araria dei trigo o viuda negra, jEl Latrodectus mactansl

Desde entonces comenzó mi inlerés por las aranas, sus costumbres y la ac-

ción de su veneno.

Desde tiempo inmemorial estos extranos seres han ocupado la atención dei

hombre. Unos les temieron, otros ensalzaron sus cualidades curativas, muchos les

despreciaron y algunas tribus indígenas ban empleado la maceración de sus cuer-

pos para envenenar sus armas defensivas. Han transcurrido siglos antes de que

se les estudiara y les fuese asignada una justa posición médico-zoológica y un

lugar entre los agentes determinantes de emponzonamientos.

Aristóteles cita en sus escritos aranas venenosas y Plinio describe la ‘‘phalan-

gia’’ y recomienda como tratamiento para su picadura el uso dei cuerpo macerado

de la araria.

Durante la Edad Media son empleadas las aranas en la composición de

drogas y filtros misteriosos y su tela se convierte en el remedio más eficaz contra

las hemorragias.

Surge después la historia fantástica de la tarântula, una araíía de Tarento

(Lycosa tarcntula) a la que se atribuía una curiosa enfermedad, de carácter

epidêmico y de la que se dice se exlendió desde Italia Meridional hasta Europa

Central.

El tarantulismo describiose en las formas más extranas y la gente llegó a

temerle más que a Satanás. jEl picado de arana caía presa de una agitación

espantosa, reía y lloraba alternativamente, moviéndose desesperado, de un lado

para otro, gritando y gesticulando, saltando y haciendo cosas absurdas! jEslaba

picado de arana!

De este raro mal sobrevenían epidemias y muchas personas enfermaban a

un mismo tiempo. x

> _

O / O A

SciELO

10 11 12 13 14 15

EL ARANEISMO EN EL MUNDO TROPICAL Y SUBTROPICAL

En vista de las dolências y de los sintonias, pensaron los contemporâneos

que lo más vialile seria somelerles a tratamiento colectivo y como habían obser¬

vado que mejoraban mejor y más luego los que transpiraban más abundante-

mente, les animaron a danzar con aires musicales de movimientos muy vivos,

horas de horas, hasta que caían al suelo totalmente agotados, banados en sudor

y se dormían. I)e ese sueno despertaban sanos. jLa tarantela les había me-

j ora do!

Aliora no hay epidemias. Se ha comprolmdo que lo que se llamó tarantu-

lismo corresponde al envenenamiento producido por la picadura de otra araria:

el Lalrodeclus tredecimguttatus. Sin embargo, en Italia siguieron por bastante

tieni|)o, en rnuchas juirtes, asignando a la tarântula los menlados accidentes. Por

oiro lado, algunos taimados se hacían pasar por "picados de araria" para come¬

ter sus picardias!

En mi país, a aquellos mozos jóvenes muy enamoradizos, de corazón ardien-

te, de ideas estrambóticas y de eonducta extrana, se les tilda de “picados de

araria”.

Antes de la Conquista, ya los aborígenes y sobre todo los araucanos cono-

cían muy bien los accidentes producidos por el Latrodectus mactans, al que 11a-

maban “guina y “pallú”, araiía cuya picadura experimentaron también los es-

panoles y a la que llegaron a temer tanto como a la viruela (Padre Valdivia.

1606. Padre Febres, 1765).

No corresponde ahora tratar de la anatomia, de la fisiologia ni de la siste¬

mática de los arácnidos. El tema sólo comprende los accidentes producidos, en

el hombre, por el veneno.

Exlranos habitantes de la tierra, las ararias disponen de un arma poderosa,

los quelíceros, para coger y matar a las bestezuelas conque se alimentan o para

defenderse cuando son agredidas.

Los quelíceros son apêndices quitinosos situados en la parte frontal dei céfalo-

tórax, por encima de la boca, de la que están separados por el rostrum. Son

dos, uno al lado dei otro. Cada uno consta de dos segmentos: uno basal, el tallo,

de mayor volumen, cilindroídeo, rígido, muy firme y, otro terminal, la garra o

gancho, móvil, puntudo, en la extremidad libre dei tallo.

El quelícero es el órgano destinado a inyectar el veneno que preparan dos

glândulas, alojadas dentro dei tallo o en el interior dei céfalo-tórax. La ponzona

sale de la glândula por un pequeno conducto que va a desembocar cerca dei ex¬

tremo puntudo dei gancho.

Los quelíceros son antenas modificadas, homólogos con el segundo par de

antenas de los crustáceos, pero no con las antenas de los insectos.

El gancho o garra de los quelíceros es móvil, pero sólo en un plano. En

Ias ararias Mygalomorphas se rnueven paralelamente uno al otro en sentido ver¬

tical, mientras que en las Arachnomorphas se eruzan yendo de fuera a adentro.

LI veneno de las ararias es un líquido claro, que deja después de ser sorne-

lido a la desecación un polvo amarillento. Presenta los caracteres de los proteí-

dos, pero de su íntima composición no es mucho lo que se sabe. La cantidad

de veneno que son capaces de producir las glândulas no guarda relación con el

tamano de la araria. El total de veneno seco, por glândula oscila entre 0.05 mg. y

6.00 mg., segun la especie. La ponzona es alcalina en tiempo caluroso y se aci¬

difica en la estación fria. Es más activa en su condición alcalina. El polv

disuelto en concentraciones adecuadas en suero fisiológico tiene igual

el veneno fresco.

polvo,

aceión que

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):45-54, 1966

ROBERTO GAJARDO-TOBAR

Las ararias tienen olras toxinas en su cuerpo, especialmente a nivel de los

ovários en el período dei ceio. Ya rnuchos anos atrás Kobert y después Walbum

dieron cuenta de ello, pero no intervienen en las actividades ofensivas ni defen¬

sivas de los arácnidos.

Los venenos de las glândulas ponzonosas de las ararias están destinados a

actuar sobre los seres que constituyen el alimento de ellas y naturalmente se en-

cuentran acondicionados para tal evento. Son diferentes en su acción segiin cada

espécie, aun cuando hay algunas muy vecinas con efeclos parecidos.

Las aranas pican al hombre cuando éste las irrita, apretándolas o lastimán-

dolas intencional o accidentalmente. Jamás atacan en forma deliberada.

Aún cuando todas las aranas, con excepción dei pequeno grupo de los ULO-

BORIDAE, poseen aparato venenoso, sólo algunas especies disponen de veneno

suficientemente activo y en cantidad adecuada como para producir accidentes

graves o mortales para el hombre.

Fuera dei género Latrodectus, de una amplia repartición por el mun¬

do. la rnayor parte de las especies peligrosas son sudamericanas.

Los accidentes de araneísmo son más o menos frecuentes. Hasta antes de

las pruebas experimentales hubo entre los hombres de ciência incredulidad, en

tanto que en el público existia exuberância imaginativa.

Han actuado en forma negativa en la exacta determinación de los agentes

causantes de los emponzonamientos vários hechos: Por una parte, ante una pi-

cadura, el afectado no siempre veia la arana, o si la advertia no era capaz de

eogerla, y si la atrapaba la destruía, de manera que resultaba bien difícil poder

juntar el efecto con la causa. Luego, la experimentación, cuando pudo hacerse,

por errores de clasificación de las aranas o por falias de técnica, muehas veces

fracasó en sus resultados.

En otras circunstancias, los pacientes aseguraban haber sido picados por ara¬

nas cuando se trataba de insectos o atr buían a aranas forúnculos y abscesos sin

ninguna relación con ellas.

Por fortuna, no siempre ha sido así y gracias a que en un huen número de

veces fue posible pillar a la arana picando, la causa etiológica de la rnayor parte

de los emponzonamientos se ha logrado establecer y más tarde con la reproduc-

ción experimental dei accidente se ha prohado.

En cualesquier caso, en matéria de emponzonamiento por veneno de aranas

tienen importância algunos faetores: 1." — Durante el verano las aranas están

nuk vivaces y el calor, alcalinizando las ponzofias, las hace más activas. 2." —

Una arana bien nutrida está fisiológicamente en mejores condiciones para actuar.

— Si no ha usado de su veneno durante un tiempo, tendrá más ponzona con

que defenderse. 4. u — La piei fina o las regiones muy irrigadas son las que

van a determinar los cuadros clínicos más sérios. 5." — El camino seguido por

el veneno y su ubicación, en la piei, el paso al torrente circulatório o la invasión

dei sistema nervioso eondicionarán el proceso. 6." — La resistência humana y

la edad de los afectados influirán a su vez. Los ninos son los que hacen los

más graves casos de araneísmo. 7.° — Einalmente también importarán las va-

riaciones de venenosidad que ocurren durante el período dei ceio.

Los accidentes causados por la picadura de las aranas, aracnidismo o ara¬

neísmo, que se acostumbra a denominarlos según el nombre dei género al cual

pertenece la arana, determinan cuadros clínicos bien característicos, que pueden

SciELO

10 11 12 13 14 15

EL ARANEISMO EN EL MUNDO TROPICAL Y SUBTROPICAL

agruparse en Ires tipos: nerviosos, cutâneos y cutáneo-viscerales, dependientes, na-

luralmente, dei caracter de la ponzona, dei tropismo de cila y de los tejidos sobre

los que actúa.

Los accidenles más espectaculares son los producidos por venenos neurotóxi-

cos, es decir aquellos que actúan fundamentalmente sobre el sistema nervioso.

Las ararias, más importantes, que los provocan pertenecen a los géneros Ctenus

o P ho n eutria, Latrodect u s y THERAPHOSIDAE.

El emponzonamiento desencadenado por la toxina de los C t e n u s o P h o -

neutria es de desarrollo rapidísimo y dramático.

Sobreviene el pinchazo de los ganchos de los quelíceros, en cualesquier parte

de la piei. más frecuentemente en las partes descubiertas. El dolor es agudo,

localizado en el sitio afeetado al principio, irradiado más tarde y generalizado

por fin. Sobrevienen calambres en las extremidades y contracturas musculares

violentas. Surgen vértigos y transtornos de la visión. Hay rigidez toráxica y

abdominal, dolores precordiales, angustia, malestar general, escalofríos y grandes

sudores.

A este cortejo sintomatológico se suman formidable temblor permanente, hi-

perestesia marcada y convulsiones tónicas. Luego aumento sensacional de las

secreeiones salivales, nasales y bronquiales. Le acompanan también taquicardia,

muchas veces arritmia, mientras el pulso se hace incontable y filiforme.

Cuando el proceso se agrava más se imponen la hipotermia, el aumento de¬

las contracturas musculares llegando hasta la rigidez general en o])istótono y con

crisis convulsivas de tal manera imponentes que hacen pensar en tétanos.

No hay lesiones locales en el sitio de la picadura.

Hay a veces retención de orina. No se ha comprobado albuminúria ni he¬

matúria. En general se produce estrenimiento rebelde.

En los casos mortales ha sobrevenido la muerte en las primeras cinco horas.

En los casos de evolución favorable, el [)ulso se va regularizando lentamente,

la temperatura se normaliza, los dolores y calambres se aplacan y, cosa curiosa,

son reemplazados por adormecimiento local, paresia y anestesia de las zonas cir-

cunvecinas al lugar picado. Esto acontece más o menos en una semana.

El período más agudo, de sintomatologia más violenta y más grave dura

entre seis y doce horas.

La casuística más importante y mejor estudiada corresponde a las regiones

rurales de São Paulo, hacia el final de los veranos, siendo con mayor frecuencia

responsable de los accidenles Phoneutria nigriventer. En cambio, en los aledanos

de Rio de Janeiro se deben, de preferencia, a Phoneutria jera. En Argentina

se inculpa a Phoneutria rujibarbis.

Las Phoneutrias son ararias bien conformadas, de 4 a 5 cm, de 8 ojos. de

patas robustas, peludas, de color gris amarillento, de caracter muy agresivo, noc¬

turnas y errantes.

Son ararias tropicales, pero llegan con los cargamentos de bananas a los

países subtropicales, donde también han producido accidenles, menos sérios que

en su tierra de origen.

Oiro tipo de araneísmo nervioso es el producido por el veneno dei L a t ro¬

dee tus, que en América está representado por Latrodectus mactans y habita

desde Califórnia hasta Chile, Argentina y l ruguay. Araria muy temida, cono-

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internae.

33(1) : 45-54, 1968

ROBERTO GAJARDO-TOBAR

cida bajo diversos noinbres, como ser arafia dei trigo, dei lino, rastrojera, araíía

brava, guina, pallú, mico-mico, lucacha y black widow, según la zona donde viva.

Es una araíía mediana, negra con el abdômen globuloso con manchas rojas,

patas largas y firmes, vive en las grietas dei terreno, en la base de las plantas,

sobre todo en los trigales; hace su tela irregular y deposita una decena de ca-

pullos amarillentos.

El color y la disposición de las manchas coloradas dei abdômen han inducido

a muchos autores a crear especies que en vcrdad deben ser sólo variedades de

Latrodectus mactans.

El veneno de Latrodectus mactans, esencialmente nenrotropo, determina un

emponzonamiento violento, de rápida y patética evolución, grave y a veces mortal.

A siete dias de grandes padecimientos siguen profunda astenia y severa fatiga

intelectual.

Al lancetazo de la picadura de la araíía sigue un período mudo de una vein-

tena de minutos. Después aparece dolor local que acrece, quema e irradia a

todo el cuerpo, más marcado en la cintura y extremidades. Con angustia y te¬

mor, aniquílanse las fuerzas y abátese el espírilu. Contracciones musculares, tem-

blores y convulsiones estremecen cl cuerpo. Las paredes toráxicas y abdominales

se ponen rígidas. Malestar, dolores, opresión al pecho y abdômen condicionan

la impresión de muerte inminente.

Sudores extenuantes, sialorrea. lagrimeo, hipersensibilidad dérmica, exagera-

ción de los reflejos, disnea y superficialización de los movimientos respiratórios,

taquicardia que va a bradicardia después, a veces arritmia (con alteraciones

electrocardiográficas), fuerte crisis hipertensiva, parálisis vesical e intestinal, di-

suria, enuresis y anuria, priapismo, ocasionalmente polucioncs, y |ior oiro lado

hiperglicemia fugaz, uremia marcada y albuminúria conforman los cuadros clí¬

nicos típicos.

Contrastando con el espectacular compromiso dei sistema nervioso, las lesio¬

nes locales se reducen a una manchita rosada y a los pequenos orifícios dejados

por los quelíceros de la araíía en la piei afectada.

Con exacerbaeiones y atcnuaciones el proceso evoluciona bacia la mejoría,

en la mayor parte de los casos.

Cuando la gravedad es extrema, la muerte se produce por edema agudo dcl

pulmón, entre las 30 y las 50 horas después dei accidente.

En un centenar de casos, hemos tenido 4% de mortalidad.

El veneno ejerce su máxima actividad sobre los núcleos centrales dcl sistema

nervioso vegetativo, en la médula, bulbo, protuberância y cerebro.

La anatomia patológica cnsena intenso edema pulmonar con acentuada liipe-

rcrnia de las bases, hiperemia de la pia medular y cerebral, hiperemia y edema

dcl cérebro, hiperemia dcl hígado y rinones, gastritis catarral, dilatación gastro¬

intestinal y vesical.

11 ay un bonito estúdio, muy reciente, de los Drs. Lebez, Maretic y Kristan

de Pula, en el que describcn sus experimentos destinados a determinar la distri-

bución dei veneno dei Latrodectus tredecimguttatus, marcado con P32 en coneji-

llos de índia emponzoíiados. Mediante un proccdimiento muy sencillo, dejando

a las ararias sin agua una semana, les ofrecían después agua conteniendo 660 u.

por ml. de Na2HP3204. Cuatro dias después hacían picar a los cobayos. Sa¬

crificados éslos entre 3 v 120 minutos después de picados y algunos 2 y 3 horas

SciELO

10 11 12 13 14 15

EL ARANEISMO EN EL MUNDO TROPICAL Y SUBTROPICAL

más larde, ensenaron grandes cantidades de P32 en el sistema nervioso central

v en los nervios periféricos y pequenas cantidades en el hígado, bazo, pulmones,

corazón, rinones, suprarenales, músculos y sangre.

Cabe recordar aqui un hecho histórico que pone de. relieve el brutal efeelo

dei veneno de Lalrudectus. En la víspera de la batalla de Loncomilla (8-

XI1-1851), numerosos batallones aguardaban en una semcntera, ocultos y listos

para dar una sorpresa, cuando al anochecer, las ararias picaron a muchos solda¬

dos de tal rnanera que los gritos y las lamentaciones de los infelices habrían

comprometido la posición dei ejéreilo de no haber mediado la medida extrema

de lener que cloroformizarlos.

Por último, dentro de los accidentes de tipo nervioso, están los causados por

las ararias MYGALOMORPHAE, que algunos médicos consideran básicamente

como narcóticos y oiros como eurarizantes.

La picadura de algunas de estas ararias, causa, como las olras, el dolor de

una elavadura. Luego sobreviene adormecimienlo local, con anestesia y posterior-

mente paresia y parálisis de los músculos vecinos al sitio afectado.

En general son casos benignos. Otras veces producen lesiones loeales, y en

algunos comprometeu cl estado general.

Se ba baldado y lran sido publicados trabajos sobre accidentes producidos

por la picadura de algunas de las grandes ararias “pollito”, senalando que la pi¬

cadura es poco dolorosa, que apareceu edema, fliclenas y eritema, acompanados

de fiebre, que a las 24 a 48 horas babría icterícia, oligúria y albuminúria, e in¬

cluso hematúria. Otras veces se ha publicado de grandes uleeraeiones. También

se citan casos mortales entre el 4.° y el 5." día. Se seíiala que este tipo de ac¬

cidentes seria produeido por los venenos de A c anthoscurria, P li o r -

m i c I o p u s y P <i m p h o b e t e u s .

Experimentalmente no siempre se ha podido probar. lloy existe la impre-

sión que en Sud-América no hay ararias “pollito” (Mygalas) peligrosas. El ve¬

neno de ellas ha demostrado ser anestésico relajante de la musculatura.

Está bien probado que los pelos finos que cubren el cuerpo de estas ararias

(Theraphoslnae) producen, en muchas personas, fuerte prurito cutâneo y erup-

ción urlicariforme.

Totalmente diferentes son los emponzofiamienlos producidos por la picadura

de ararias cuyo veneno tiene acción necrosante. Entre estos hay jronzofias que

sólo actúan sobre la piei y, otros que obran sobre la piei pero, también, y, a

veces en forma mortal, sobre sangre y vísceras.

El veneno de las Lycosas produce dolor, más o menos acentuado, en el punto

de la picadura. más larde engendra una pápula blanquecina, con una zona sin

sensibilidad. rodeada de una guarda rojiza, congestiva, dolorosa. Paulatinamenle

el rubor se va extendiendo a su alrededor, a veces con erupción generalizada.

Sobrevienen edema voluminoso, manchas equimótícas y flictenas. Se produce la

necrosis entre el 4." y 5." dias, después de formarse una escara seca, que delimi¬

tada. cae al 15.” día, dejando una úlcera irregular que llega hasta las aponeu-

rosis de los músculos.

La cicatrización es lenta y defecluosa. No hay compromiso dei estado ge¬

neral. Muchas veces los afei tados se agravan por infecciones secundarias. Ly-

cosu raptoria ha sido inculpada en São Paulo y otras partes.

En el Perú, Escomel ba descrito una acción local parecida en casos de pi¬

cadura de Glyptocranium gastheracunthuidvx pero, en los que babría además com¬

promiso dei estado general.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Stmp. Internar.

38(l):45-54, 1966

ROBERTO GAJARDO-TOBAR

Por último, por muchos anos, ya desde 1852. vienen dando cuenta los mé¬

dicos de Chile, de la existência de una enfermedad llamada "la mancha gangre¬

nosa” que, siendo cn la mayor parte de los casos un proceso local, había oportu¬

nidades en que, comprometiendo gravemente el estado general, llevaha a la muerte

en pocas horas.

Habiendo sido este accidente muy hien descrito y muchísimas veces obser¬

vado y estudiado, sólo en 1934, con los hallazgos en Antofagasta y la experimen-

tación hechos por A. Macchiavello, pudo determinarse al agente causal: Loxos-

celes laeta (que al decir de otros investigadores debe llevar el nombre de rufipes).

Loxosceles produce dos cuadros clínicos diferentes, según como actúc

su veneno, en razón al camino seguido en el organismo: En uno hay acción local

de la ponzona, con extensa o limitada acción gangrenosa de la piei, con nulo o

escaso eompromiso dei estado general. En el oiro, con fulminante y grave alte-

raeión dei rinón y dei hígado y una violenta hemólisis, acusadas por fiebre,

postraeión, ictericia, hematúria y hemoglobinuria.

En la forma cutânea, al dolor de la clavadura de los quelíceros de la arana

sigue un período sin sintomas de minutos a horas, al cabo dei cual irrumpe fuerte

dolor local intensamenle quemante, a veces con un poeo de prurito. La violência

dei dolor crea impotência, causa insomnio y desesperación creciente. Una man¬

cha rojo-vinosa senala el sitio de la picadura. A la 24 horas se torna violácea

y después negra. Se forma allí una placa de 1 a 30 y más centímetros de diâ¬

metro, muy característica, con zonas pálidas, blancas y moradas, como vetas irre¬

gulares, rodeada de un halo intensamente rojo. Es la lesion que nosotros hemos

llamado “la placa marmórea".

Sobreviene edema que se extiende muchísimo y una gran infiltración dura

bajo la placa. En la parle negra y en la placa marmórea en general desaparece

la sensibilidad al dolor y térmica, mientras que en Ia periférica se exageram

Surgen desde el comienzo, sobre la placa en formación, grandes flictenas sero-

hemáticas.

Al 6.° día se deslinda una escara apergaminada, seca, brillante y negra. A

la 3“ semana se desprende y cesa el dolor. A este proceso es al que se llama,

en Chile, desde más de cien aiios, “la mancha gangrenosa".

La curación es muy lenta y a veces requiere de injertos. La cicatriz es

irregular y azuleja.

El estado general no se compromete o lo hace en poea monta, con calofríos,

fiebre, desasosiego, insomnio, etc. Con esto o sin ello, el dolor quemante es tan

terrible, a veces, que algunos pacientes desesperados han deseado que les fuese

amputado el miembro afeclado para librarse de él. o han llegado hasta intentar

suicidarse en su aflicción.

Citando el veneno pasa la barrera de la piei y ya sea por la cantidad intro-

ducida o por su pasaje directo al torrente circulatório e invasión de todo el or¬

ganismo, se produce la forma cutáneo-visceral, en que la lesión dérmica se insinua

y no alcanza a desarrollarse porque el eompromiso dei estado general mata antes

que aquella evolucione.

Se extiende el dolor local, surgen escalofríos, cefaleas, decaímienlo y desa¬

sosiego. A las doce horas hay vómitos, a veces con sangre, luego ictericia, más

tarde hematúria y hemoglobinuria. El proceso avanza con insomnio, taquicardia.

hipertermia, hipotensión, disnea, cianosis y anemia. Por último vienen conges-

tión y edema pulmonar.

SciELO

10 11 12 13 14 15

EI- ARANEISMO EN EI- MUNDO TROPICAL Y SUBTROPICAL

La anemia es hemolítica, con grau destrucción fie los glóbulos rojos, hemo-

globinemia, con brusca leucocitosis de tipo leucemoíde, trombocitopenia variable

y sedimentación alta.

Manifiéstanse hematúria, hemoglohinuria y cilindruria. Les acompanan grau

alza de la uremia, hiperglicemia fugaz y luego hipoglicemia e hiperbilirubinemia.

La muerte acaece entre las 30 y las 40 horas después dei accidente. Hay

lesiones de neerosis incipiente de la piei, hiperemia y edema polivisceral, grau

hemólisis, neerosis focal hepática, nefrosis hemoglobinúrica y hemorragias múlti-

ples. Nosotros, en 200 casos de loxoscelismo hemos tenido 22 casos de grave

forma cutáneo-visccral con 7 muertes, cs decir 2.5% de los 200 y 31.81% de

los 22 de tipo cutâneo-visceral.

La casuística humana de araneísmo ha dependido, naturalmente, de la ex-

tensión dei habitat de las especies peligrosas y, hoy dia, se descrihen accidentes

de loxoscelismo desde los Estados Unidos ( Loxosceles reclusa), Chile, Perú. Bo-

livia. Argentina, hasta Uruguay (Loxosceles laeta o rufipes) ; y de latrodectismo,

de muy antigna ohservación hay casos de casi todo el mundo, a saber, en Jlalia,

Yugoslavia, Espana {Latrodectus tredecimguttatus) , en Rusia dei Snr, Turquestán,

los países dei Mar Negro y dei Mar Cáspio y Asia Menor (Latrodectus erebus

o Karacurl), en Australia y Nueva Zelanda I Latrodectus hasselli o Katipo), en

Madagascar (Latrodectus menavodi ) y en África dei Norte (Latrodectus trede¬

cimguttatus), etc., etc.

Muchas otras ararias han sido inculpadas de producir accidentes importantes

humanos de emponzonamiento pero no hay al respecto demostración experimental

irredarguible. Con lodo. lenemos nosotros también accidentes por el veneno de

otras ararias, de lo que daremos cuenta en otra oportunidad.

Con relaeión al diagnóstico, el antecedente de haber sorprendido a la arana

picando cs categórico pero. muchas veces los afectados no ven a la hechora y es

entonces cuando hay que proceder con ingenio, paciência y agudeza a analizar

el cuadro clínico para hacer el diagnóstico.

Ya sabemos de la acción específica de muchos venenos, luego nos ayudarán

la procedência dei enfermo, el tipo de su trabajo, la presencia de arácnidos en

el lugar y por sobre todo el cuadro clínico.

Luego, según el lij>o de la lesión dehemos tener en cuenta todas las afeccio-

nes parecidas dei país donde el accidente ha sucedido. Con cierta experiencia

no es problema muy difícil.

En euanto al tratamienlo, es interesante recordar que ya los indios usaban

muchas yerbas, agua de estiercol de guanaco e incluso un remedio que pasó al

uso popular, la celebremente repugnante ulpada (deposieiones humanas diluídas

con agua) empleadas para combater la parálisis intestinal dei latrodectismo.

De todas maneras, para impedir la difusión dei veneno no parece servir nin-

gún procedimiento.

1,1 ideal, en eualesquier araneísmo, es la terapêutica específica o sea el em-

pleo de sueros específicos.

Las aranas tienen venenos bien definidos para cada espeeie y los sueros de-

ben ser entonces específicos. Sin embargo, las especies de un mismo género

poseen ponzonas muy afines, de manera que Ia preparación de sueros contra una

espécie dei género dará un producto que puede ser usado y resultar bien para

2 3

5 6

10 11 12 13 14 15

Mem. Tnst. Butantan

Simp. Internac.

3311): 45-5 4, 1ÍJG6

ROBERTO GAJARDO-TOBAR

el cmponzoíiamiento producido por otra dei mismo género, como lo demostramos

usando suero contra el veneno dei Latrodectus tredecimguttatus en casos de latro-

dectismo por Latrodectus mactans, con excelente resultado.

Hemos empleado el tratamiento con sueros específicos, en latrodectismo y

loxoscelismo, en los casos que estimamos más graves, en nuestro país, siempre

con muy buenos resultados. El suero utilizado lo obtuvimos gracias a la genti¬

leza de los l)rs. Pirosky, Sampayo y colaboradores dei Instituto Malbrán de Bue¬

nos Aires, en casos de latrodectismo y de los Drs. Stanic y Maretic dei instituto

de Higiene de Pula, también para el latrodectismo. En loxoscelismo usamos suero

específico dei Instituto Butantan que nos proporciono el Dr. Bücherl y otro pre¬

parado por el Dr. Vellard.

Hoy dia el Instituto Butantan prepara excelentes sueros contra el veneno de

Phoneutria fera, Lycos a y Loxosceles. El Instituto Malbrán obtiene suero

contra el veneno dei Latrodectus, pero en diversas partes dei mundo se

producen también, sobre Iodos en los Estados tinidos y en Yugoslavia.

Otra arma efectiva en el araneísmo, sobre todo en la casuística visceral, está

constituída por los corticosteroídes, empleados tempranamente y con generosidad.

También debemos seguir recurriendo al tratamiento sintomático, destinado pri-

mero que a nada a calmar las manifestaciones fundamentales. En cualesquier

araneísmo el dolor predomina, luego las crisis nerviosas, las convulsiones, los trans¬

tornos de las secreciones, las alteraciones digestivas, la deshidratación, la anemia

y la postración deben juiciosa y rápidamente ser atendidas. Sueros glucosalinos,

gluconato de cálcio, sedantes, opiáceos, cardiotónicos, etc., encuentran su aplica-

ción en el araneísmo.

No creemos, a pesar de lodo lo dicho, que habría que liquidar o tratar de

hacer desaparecer a las ararias. jPor ningún motivo! Elias mantienen el equi-

1 ibrio biológico en la Naturaleza y destruyen un gran número de especies daninas

para la agricultura y para el hombre. Pero como, por otro lado, liemos visto

de lo que son eapaces mediante el empleo de su veneno, como arma defensiva,

estimamos que frente a las arailas no hay que tener temor, pero tampoco ser con¬

fiados. Corremos el riesgo de que nos digan, como en nuestra tierra se acostum-

bra, con mucha picardia, que somos unos "‘picados de araria”!

SciELO

10 11 12 13 14 15

KL ARANEISMO EN EL MUNDO TROPICAL Y SUBTROPICAL

LOXOSCELISMO CUTÂNEO

edema.

Fig.

1 —

Fig.

2 —

Loxosceles sp. Fig. 3

mancha gangrenosa.

flictenas.

Fig.

4 —

SciELO

Mem. Inst. Butantan

Simp. Internac.

33(1):55-59, 1966

BERTHA LUTZ

6. BIOLOGICAL SIGNIFICANCE OF CUTANEOUS SECRETIONS l\ TOADS

AND FROGS

BERTHA LUTZ

Museu Nacional, Universidade Federal, Rio de Janeiro, Brasil

The skin plays a very important role in the biology of lhe AMPHIIIIA

ANURA, i.e., Toads and Frogs. lt suhserves lhe respiralory function. since

pulmonary respiration is nol quite adequate for their needs. It is a naked skin

unprovided with scales. lt is kept moist thus permitting gaseous exchanges

through the superficial capillaries.

The skin of the ANURA receives a special blood-supply over a large area

of the hody through cutaneous arteries and returns it through museulocutaneous

veins. The total respiratory capillary lengtli of the skin varies from 20.5 to 65.1

in the anurans studied for this factor. It is greater in thin-skinned and in aquatic

forms llian in terrestrial toads with a glandular skin and better developed lungs.

Classic and rnodern studies of certain species show that carbon dioxide is mostly

excreted hy the skin and varies with physiological condition and activity wliere

as the intake of oxygen hy the lungs, being dependent on outside tension, is more

constant.

Two main types of mullicellular glands occur in the skin of adult Anurans:

mucous glands and granular glands. The mucous glands produce a rather fluid

secretion which luhricates the skin. The granular glands produce a creamy

granular secretion which contains poisonous substances. The skin glands may

he disseminated over the hody but they tend to become localized and form masses

at certain points, especially the granular glands. Some frogs, such as lhe neo¬

tropical genus Cyclorhamphus and a number of small kinds of P alu¬

dicola, have gelatinous glandular disks on the flanks. In the genus Bufo.

which comprises the true toads, lhe granular glands form large masses in lhe post-

ocular region; they are called paratoids, from a false analogy with the parotoid

glands of mammals. Some other frogs also have parotoids, f.i. the large species

of neotropical tree-frogs belonging to the genera Pliyllo medusa and Pi-

thecopus, but their parotoids are long and thin and continue along the dorso-

lateral edges of the hody. The skin glands of Anurans, especially the glandular

glands of toads, are of interest to students of Venomous Animais on account of

the poisonous substances contained in their secretions but they are well-known in

oídy a few genera and species.

From a biological point of view the skin glands of the ANURA are a me-

chanism of defense which aids survival of the individual.

The slimy secretion of lhe mucous glands is greatly increased the moment a

frog is seized and makes it difficult to maintain the hold, the more so as lubrica-

tion is accompanied hy intense wriggling to get away. The secretion frequently

SciELO

10 11 12 13 14 15

BIOLOGICAL SIGNIFICANCE OF CUTANEOUS SECRETIONS IN

TOADS AND FROGS

has a strong odor, for inslance of pepper, garlic, crushed leaves, or musk. It is

often sternutatory and sometimes induces tears. Some frogs, like lhe garlic toad.

or “Knoblauchkroete”, Pelobates, cause the deatli of other frogs put into

lhe same Container with them. Another European frog, Bom bina, can be

kept alive in a vivarium full of lurtles because it is not attacked by them. One

of the mosl interesting cutaneous secretions is thal of the. large, glandular spccies

of tree-frog of lhe neotropical Ilyla venulosa gronp. The secretion dries lo a rub-

hery consistence. Often, hui not invariably, it provokes a reaction after hand-

ling the frogs.

These substances are poorly known. Their virulence may vary from one

form to another but possibly also in lhe same form at different seasons or in

different physiological conditions. My former assistant Miss Kloss ha d a pro-

longed and violent headache on inadvertently rubbing her eyes soon after hand-

liiig the Amazonian form of Ilyla venulosa. Other specimens, from Belém do

Pará, very much handled by me, caused no symptoms at all. A painful rash,

lasting a few hours carne out on my hand and arm after catching a Ilyla imi-

tatrix, of the same group, dropped by a bird seuffling with it. The other frogs

put with it arrived dead and glued together. Another specimen of the same

form left a painless weal, whieh lasted many days, on my hand by laying a leg

across it. The bromealiad collectors Mr. and Mrs. Racine and Mulford Foster

called the Chaco form, Ilyla hebes Cope, the "india rubber frog”, because the

secretion was ahundant enough to permit them to make small pellets with it.

Neither tliey nol I fclt any effects from it, even when I rubbed it on the inside

of my lip. Professor Mertens mentions a burning sensation on scizing the un-

related Ilyla vasta from Sanlo Domingo.

The secretions of the parotoid glands of lhe true loads are easier lo obtain

and have been studicd more consistently. A number of poisonous substances have

been extracted from them and named. There seem to be specific differences.

However, lhese glands also constitute a mechanism of defense. Thcir secretion is

seldom released spontaneously and even llien after a greal deal of provocation

has been endured. The author only saw it spurt out once, in a toad lliat hit

the ground after being dropped from a height. Nor can the toads introduce their

venom inlo the body of their enemies. For it to lake effect, the glands have

generally got to be bitten into. Small dogs have worried toads may die or be-

come very ill as a result of their indiscretion but lhe toad is a passive element.

Experiments with toad venom by injection may be of biochemical interest but

biologically they are artificial and disregard natural conditions.

Cutaneous secretions are supplemented by a few olher simple defense me-

chanisms. The first is derived from lhe integument and is the coloring, especially

of the permanently visible dorsal aspect. There are two main types of protective

coloration, procrypt and apósematic. In procrypt animais lhe color is concealing

either by resemblance to the background, or by a disruptive pattern whieh con-

veys a false outline and breaks up the visual irnage of the surface. The Iree-

frogs 1’hyllomedusa and Pithecopus are protected by similarily to lhe

background. They live on the vegetation and in day-time the dorsal aspect is

uniformely green. The color is brusquely out off at the edges of lhe permanently

visible surfaces and thus separates lhem abruptly from lhose concealed in repose,

whieh may have bright spots of apósematic color on them. At night, when they

move about, the large species become very dark, purple or chocolate-colored, al-

most black. The true toads, li u j o, may have a uniform or mottled dorsal

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1) : 55-59, 1966

BERTIIA LUTZ

surface, witli a sexual clichroism in some species; whieh is very unusual in Anu-

rans. The color may blend inlo the background or the surface he disrupted l>y

rnottling. Bufo typhonius and other forest-forms may look like leaves from above;

some of them, like Bufo gultatus, are so dark beneath that tliey seem flat. In

aposematic frogs, lhe hright coloring is seldom general, ihough il is uniform in

a few, f.i.. lhe minute pumpkin-eolored Brachycephalus ephippium. As a rule,

the hright coiors eilher serve to disrupt lhe visible surface or lhey are flash-

eolors, often on lhe thighs, whieh are only seen momentarily when the (rog moves.

An additional means of defense derives from increase of ossification, in di-

rect opposition lo lhe evolutionary reduction of lhe skeleton from ancient lo recent

amphibians. Some have a hony shield on lhe back, such as the tiny Brachyce-

phalus ephippium just mentioned and the huge horned toads. More often the

increased ossification is on the !op of the head, the pari of lhe body most vulner-

able to altack. Many species of Bufo have hony crests whieh stand out; lhey

vary from species to species and are used in taxonomy. Their greatest develop-

ment is attained in Bufo lyphonius, old and large specimens of whieh may have

veritable wings lo lhe sides of the head. Olher loads develop a more or less

complete skull-cap or helmet; these are burrowing fornis. The function of tliese

adaptative struelures evidently is to preveni seizure and crushing of the head.

Habits and patterns of hehaviour also provide means of defense. Nocturna]

life is lhe rule in frogs and toads. One way of avoiding their enemies is by hid-

ing and sleeping in day-time in boles and burrows, bolh natural or artificial

(ground-dwellers), in bromeliads or among leaves (tree-frogs). Only the most

agile running-water frogs, f.i., E l o s i a and M e galo si a , can afford the

htxury of diurnal life.

Some anurans have evolved altitudes of defense. The besl known is probably

lhe “l iikenreflex”, f rom lhe German popular name “Unke” for the genus B o m -

bina. lt consists in arching the body witli the back uppermost and curving

the limbs up over it and the head, bringing the flash-colors of the ventral aspect

into evidence. Many neotropical tree-frogs “play possum”, lying quietly on the

ground as if dead, while they await the chance lo turn rapidly and leap away.

Toads inflate their lungs to the utmost. Some rear up on their hind legs and

butt with their heads. Others lean to one side and present a lateral view to the

predator. This greatly increases the surface that has to be bitten into or swall-

owed. Holaden bradei, a minute toad from the Itatiaia, whieh lays terrestrial

eggs and guards its nest, also rises on its legs and leans forward, hissing, when

its spawn is threatened.

The simple mechanisms enumerated above exhaust lhe modest arsenal of

defense of the toads and frogs, leaving them often at the mercy of their enemies.

Some are better proteeted lhan others, f.i. the Antillean, phragmolic, casque-headed,

toads whieh live in the ground and plug the openning of lhe burrow with their

heads. Small toads with insignificant crests, like B. granulosas, hiding in hollows

in dunes are however sometimes yanked out of their mobile homes by the head

by snakes, as observed by Gliesch in Rio Grande do Sul. The British naturalist

Loveridge once saw a rat rip lhe skin off a toad down the middle of the back

and start lo devour its flesh. He also observed other small mammals avoid the

parotoids by tumbling over the toad and attacking il from the belly. In nature

the predalor-pray relationship is apt lo be less favorable to anurans lhan to their

enemies and loss may have to be compensated for by excessive spawning or by

protection of eggs and larvae; this is done in many ways, beginning with the

unpalatable and perhaps poisonous eggs of toads. They are seldom interfered with.

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HÍOI.OGICAL SIGNIFICANCE OF CUTANEOUS SECRETIONS IN

TOADS AND FROGS

Among their enemies the A M P H I li IA must reckon human beings.

Toads liave been barbarously used in witchcraft and primitive medicine; to

lliis day many are flayed alive for lheir skins and lei loose to die or be devoured

by fire ants. The Tucana Indians imprison the large Phyllomedusa bicolor in

little cages like birds. After bouts of drinking and gorging they scarify the

skin at the temples and wrists and rub the paratoids of the frog on lhe scratches

to bring on vomiling and catharsis. Many beautiful little frogs of the genus

Dendrobnt.es are caught by Amazonian Indians and impaled alive on sticks

to roasl over the fire; the agonie gouts of poison gained are used on arrows for

killing monkeys and other small game. Can one really consider Venomous Ani¬

mais those that never allack and can barely defend themselves? In lhe interests

of Science one must study them hut the study should be carried ont in a logic

and in a humane manner.

References

1. Lutz, A. — Ueber die Drepaaanidien der Schtangen. Ein Beitrag zur Kentniss

der Haemosporidien. Zbl. Bctlct., 29(91:390-398, 1901.

2. Lutz & Mello — Descripção de um novo genero e de duas novas especies de co-

lubrideos aglyphos. A Fôlha Médica, 3(13) :97, 1922.

3. Lutz & Mello —• Elaps ezeguieli e Rhinostoma bimaculatum, cobras novas do

Estado de Minas Geraes. Mem. Inst. O. Cruz, 15(1):235-9, 1922.

4. Lutz & Mello — Duas especies de colubrideos brasileiros (Nota previa). A Fôllin

Médica, 4(1): 2-3, 1923.

5. Miranda Ribeiro, A. — Jararaca de Santa Maria no Rio Corrente, Estado da

Bahia. Lachesis lutzi. Mem. Inst. O. Cruz, 7:50, 1915.

Discussion

H. Edery: “During the last years Prof. Erspamer and his collaborators have

found in the skin of a number of frogs and toads extremely active peptides, some

them related to bradykinin. Have you any information how they are formed?

Are they originated in precursors proteins? The second question: which are the

natural enemies of these frogs and toads you mentioned? I suppose they should

be particular sensitive to the venom. Have you any information on this point?”

B. Lutz: “First question: I am afraid that I do not know the answer, but 1

refer you to Dr. J. M. Cei of the University at Mendoza, Argentina, who will answer

your question.

Second question: The main enemies are snakes, arboreal (tree-frogs), terrestrial

and aquatic. They seize the frogs by the head. Those which are casgne-headed

are better protected, especially if they are phragmotic, taking refuge in a cavity

and plugging the lienem with a snake. I once observed a Bothrops jararaca try-

ing to get one out of a bromealiad, unsuccessfully. Those with less perfect helmet,

like small Bufo granulus with crests only, are less well protected. They live in

holes in the dunes, are janked out of their mobile burrow by the head, as seen by

Prof. Rudolph Gliesch in Rio Grande do Sul.

Tree frogs, both niale and female, seized by snakes, give a loud cry of distress.

Mammals also sometimes eat frogs and toads. I presume that some of the predators

are insensitive to the venom or avoid biting directly into the parotid glands. Dr.

Freiberg of Argentina saw a very large chulean frog, Calyptocephalella goeji,

swallow toads.

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

-Simp. Internac.

:55-59, 196fi

BERTHA LUTZ

A British naturalist, Loveridge, saw a rat attack a toacl in the middle of the

back, behind the glands. He also saw small mammals tumble over the toad and

attack it on the belly. I mentioned a birth that released, in mid-air, a Hyla imi-

tatrix with irritant secretion. Aquatic frogs may be attacked by leeches. Many

neotropical ones, especially bromeliad-dwellers, have larval mites in the skin. Some

mosquitoes bite frogs. They also have worms, f.i. Trematodes. Dr. Adolpho Lutz

íound an oligochaete worm Schomardaella lutzi Michaelson in the bladder of some

tree-frogs.”

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DORIS M. COCHRAN

7. TAXONOMY AND DISTRIBUTION OF ARROW-POISON FROGS

IN COLOMBIA

DORIS M. COCHRAN

Di vision of Reptiles and Amphibians, U.S. National Museum,

Washington, D.C., U.S.A.

Some of the dendrobatid frogs are known to secrete from their skin a

substance used by native tribes of Colombia for poisoning lheir arrows. The

genus Phyllobates, to which the highly toxic ‘"kokoi” belongs, is re-

presented by 14 known forms in Colombia, while Dendrobates, also poi-

sonous, has 11 kinds within tbe borders of that country.

These frogs are all rather small, lhe largest reaching a head-an-body length

of less than two inches. They are often brilliantly colored with yellow or orange

spots or stripes on a dark background in a certain pattern characteristic of earh

form. Unfortunately, these bright hues disappear in preservative, and the specimen

becomes gray or bluish, sometimes with darker areas.

Arrow-poison frogs of lhe genera Phyllobates and Dendrobates

are told apart by the presente or absence of teeth on the maxilla, members of

Phyllobates possessing teeth that may be felt with the point of a pin

along lhe inner maxillary horder, while in Dendrobates this area is com-

pletely smooth and devoid of snch teeth.

lt was formerly believed that lhe presenee or absence of a web between the

toes of the hind foot further divided the genus Phyllobates , the frogs

lacking such webs being placed in tbe genus Prostherapis. Tbe variation

in degree of webbing in some of lhe species is so great. however. that no valid

reliance can be placed on il to justify such a separation on that characteristic,

therefore since Phyllobates is the older name, all frogs since referred to

as Prosthera p i s have been placed under Phyllobates.

While mucli collecting remains to be done in Colombia before we can have a

complete picture of the distribution of tbe 25 forms there, at present the largest

number of kinds is recorded from Antioquia and Chocó, each of these depart-

ments having fonr Dendrobates and five Phyllobates. Undoubtedly

more intensive searching in olher favourable regions will turn uj) as many or

more for some of the other departments.

Other Central and Soulb American countries also have a good population

of arrow-poison frogs, although these at present appear to be more numerous in

Colombia than in any of the adjoining regions.

These little frogs are terrestrial as a rule, living among the vegetation on

the floor of the forest, or in heavy grass near some stream or pool. Their dappled

golden spots or lines render them nearly invisible in such siluations. They give

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TAXONOMY AND DISTRIBUTION OF ARROW-POISON FROGS

IN COLOMBIA

a gentle, whislling call, and this hetrays lheir hiding place, altliougli they are not

readily seen even then. Presumably lliey feed upoii small insects and other

minute invertebrates. Very little lias been recorded as to their actual feeding

habits in the wild slale.

Breeding occurs during the spring and summer, When the eggs have hatched,

a inale will present himself among the very young tadpoles, and they instinctively

attach themselves to the skin of liis back hy their suction organs. He then car-

ries thern safely for several weeks, until they have grown suffieiently to fend for

themselves. While the male is tinis “incubatiríg” lhe tadpoles, he frequently

visits a shallow stream or a tree-hole containing rainwater, in which he immerses

himself and lhe tadpoles for some lime, thus keeping them from becoming too dry.

When using slivers of bamboo as blow-gun darts, the Indians had to emplov

a very quick-acting killing or paralyzing substance that would drop the bird or

mammal they had hit hefore il could escape into the dense underbrush heside

lhe jungle trail and so he lost to them as an item of food. For this purpose

kokoi poison was in use long hefore the coming of the white man. and was

singularly quick and effective once it had entered the bloodstream of the animal

shot. According to Dr. Bernard Wilkop of the National Inslitute of Health,

whose analysis of it will he presented to you later in this symposium. it is one

of the most jiowerful animal poisons yet known.

As a taxonomist, my chief interest in these frogs has been the Identification

of the Colombian forms, together wilh their aparent relationships. There follows

a shorl summary of some of their characteristics, taken from a forthcoming paper

on the frogs of Colombia hy Dr. Coleman J. Goin and myself.

Both D endro bates and Phyllobates are now considered to belong

10 the Suhfamily DendrObatinae of the family HANIDAE. As I have already

mcntioned, members of the genus Dendrobates have no teeth on the maxil-

lary hone, while Phyllobates possess such teeth, not readily visible except

under a high-power lens, hut easily felt hy drawing a pin or other pointed ohject

along the inside of the maxilla. Both genera have a longitudinal furrow along

lhe top of each digit, forming two distinct platelets, unlike the single undivided

disks found in most other frogs having enlarged toe and finger tijis.

I shall consider first the 11 forms of Dendrobates now known from

Colombia. In these the relative lengths of the first and second fingers are usually

diagnoslic, also the position reached hy the adpressed heel in relation to the eye

or tympanum. The color patlern is usually highlv characteristie of the species.

Due lo lhe considerable amount of variation in the individuais of each species,

the following statements as to the finger proportion. leg length and color pattern

are only generally applicahle, however, and each frog should he compared wilh

the best availahle description and figure.

The largest Colombian Dendrobates known al present is trivittatus,

reaching a head-and-hody length of 46 mm. In alcohol it is mostly slatecolor,

wilh a lighter stripe down each side of the back, and a lighter unspotted venter.

11 is one of the two species in Colombia in which lhe first finger is distinctly

longer than lhe second. It is known from Brazil, British Guiana, Ecuador and

Peru, as well as from Amazonas in Colombia.

The other D e n d r o b a t e s wilh the first finger longer than the second

represents a new species found in Caquetá, Colombia. Until its description has

been published, I am not at liberty to discuss it furlher.

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 61-65, 1966

DORIS M. COCHRAN

Two well-known Dendrobates willi lhe first finger nearly as long as

lhe second and wilh lhe heel reaching to lhe center of lhe eye when extended

forward are hahneli and lugubris. A pale axillary spot, lighl narrow dorsolateral

Iines and reticulations on lhe posterior half of lhe helly distinguish hahneli, a

small species reaching 23 mm in length and known from Mela and Putumayo

in Colomhia, as well as Peru. Lacking the axillary spot, having wider dorso¬

lateral lines, and with light spots or reticulations scaltered on lhe helly, lugubris

in alcohol is a slate-colored frog of 34 mm., perhaps much brighter in life; it

is widely distributed in Colomhia and is known also from Panama.

The next large division of Colombian Dendrobates consists of those

having the first finger much shorter than the second. The first subgroup contains

very small frogs, nol longer, than 18 mm. In opisthomelas and minutus ventri

maculatus there is a large light spot on the under side of the upper arm reach¬

ing from the axilla nearly to the elbow. In the former the heel reaches the

anterior border of the eye, and there are no definite dorsal stripes; il is known from

Antioquia in Colomhia. The latter, first described from Ecuador, has a shorter leg,

with heel reaching only to the posterior corner of the eye; usually there is a

wide middorsal and a pair of dorsolateral light stripes. It is found in Caldas

and Caquetá, Colomhia.

The nominate form of the last-named has no conspicuous light spot below

the upper arm. It is known at presenl from Panama and from Antioquia in

Colomhia.

The last subgroup under those frogs with short first fingers are lhe “clown-

frogs”, so-called because of their brilliant colors and patterns. The subspecific

name of one of lhese is histrionicus, meaning an “actor”. This one is hlack above

with a large light spot (yellow or orange in life) on lhe snout and another be-

tween the shoulders. IIs lower surface is also dark, wilh a rectangular light s]>ot

covering throat and chest, and another on the posterior pari of the helly. Its

maximum known size is 38 mm., and it occurs in Antioquia, Caldas and Chocó

in Colomhia.

A second forni of tinctorius is wittei, about the size but with many small

rounded silvery-while (orange in life) spots on the anterior part of the baek,

lhese becoming much larger on lhe sacrum. lhe lower surface is pearl gray in

alcohol, with an irregular hlack patch on lhe center of the throat. Il is known

only from Chocó in Colomhia.

A third subspecies is chocoensis, light above with several irregular dark spots

on the back and dark brown below. Possible intergrades between chocoensis and

histrionicus have been taken at Playa de Oro on Rio San Juan, Chocó. This

most interesting mountainous region gives rise to streams draining two watersheds,

one pouring into the Caribhean, the other into the Pacific. Perhaps the early

geological history of this arca explains the occurrence liere within a short distanec

of each other of the three subspecies of tinctorius just mentioned.

A fòurth subspecies of tinctorius, conjluens, occurs in Cauca and Narino.

Colomhia. in alcohol it is olive-gray with many small irregular hlack spots, but

was said to be scarlet in life. A great deal more remains lo be done both in

eollecting and in analysing the characters of these lillle frogs before the final

word as to their identity can be said.

The genus P hyllob ate s is as variable in some of its characteristics as

is the foregoing Dendrobates. Some authorities have recognized Cope's

genus Prost hera pis for frogs with maxillary teeth having webbed toes.

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TAXONOMY AND DISTRIBUTION OF ARROW-POISON FROGS

IN COI.OMBIA

reserving Phyllo bates for those withoul webbing on the feet. I finei lliat

lho variation in regard lo clegree of webbing is so great in somo Colombian

spocies lhat lliis distinclion can no longer be applied, as some frogs may have

no perceptible web, wh ilt* olhers of lho samo spocies from the same locality may

have a small but distinct vostigal web. A separation on the eharacter of skin,

— whether il tears easily or seems resistant to abrasion — couplod with large

si/e, lioavy build and laek of distinct light striped may yet be possible. Such

a distinclion using only preserved material lias not proved feasihle. Al any rate,

the 14 Colombian forms to be discussed are now considered lo belong to lhe

genus P h y ll o b a t e s .

In the first section of these, in which the toes are free or nearly so, and

the first finger is usually longer than the second, we find fonr spocies. In bi¬

color (the poisonous “kokoi” of which you will bear later), the baek and posterior

pari of belly are uniform pearl-gray or slate color, although these paris are known

to be bright red-orange in lifo, while lhe upper surfaces of lhe limbs, now gray,

were once yelowish in tone. The anterior half of the belly, as well as the lliroat

and chest, are cinereous in alcohol, but in life were straw yellow to cream color.

A Peruvian example had numerous blaek s|iots on the tbroat and chest. with a

dark triangle on the center of the belly. The "kokoi” is about 42 mm. in length

when adult. In addition to Peru, this frog has heen collected in Antioquia,

Caldas, Cauca, Chocó and Valle.

A light dorsolateral st ripe, sometimes faint, occurs in the remaining three

species having lhe toes free. The first finger is longer than the second, and the

hcel reaches between the anterior and posterior corners of the eye in boulengeri

and femoralis. The former has coarse dark reticulations on lhe belly which con¬

tinue on the tbroat and forni a pair of shorl parallel dark stripes on the chin.

It is a small frog measuring only 22 mm. and is found in abundance on Gorgona

Island, and rarely on the adjoing mainland in Nariho and Valle. Its close relative

is femoralis, with lhe chin and anterior part of belly blaek, lhe poslerior half of

belly being marhled dark and light. It is not known to exceed 26 mm in length.

We know it from British Guiana and Peru, and from Amazonas, Caldas, Caquetá,

Mala, Putumayo and Valle in Colomhia. A fourth species in this section is mer-

tensi, with toes that may be barely to almost 1/8 webbed. Its legs are short, the

heel reaching between the tympanum and lhe posterior corner of the eye. Its

belly is drab with heavy dark reticulations, and it reaches a length of some

80 mm. At present it is known only from Cauca.

In the remaining Colombian Phyllobates, the toes are webbed at the

base up to 2/3 webbed. Some have the first finger shorter than the second.

Among this, nominate subpunctatus has lhe toes webbed only at lhe base, while

its heel reaches the posterior corner of the tympanum. Its belly is pinkish buff

often small scattered dark spots, and its maximum size is about 22 mm. ll ap-

pears lo be restricled to Cundinamarca. The next three have more extensive

webs. In vergeli the hcel reaches lhe tip of lhe snout, lhe belly is immaculate,

and the size is about 22.5 mm. It likewise is unknown outside of Cundinamarca.

In lhe two remaining in this section, both have dark spots or reticulations on

the belly; in chocoensis the toes are 1/2 webbed, the maximum size is about

27.5 mm., and the range is Chocó and Antioquia. A new species not yet

puhlishcd falis near the three preceding.

In lhe next section lhe first and second fingers are subequal. Palmatus

has the toes 1/3 to 1/2 webbed; the belly is immaculate drab, and the size is

up lo 36.5 mm. This is one of the species most notable for its easily abraded

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Mem. Inst. Butantan

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DORIS M. COCHRAN

skin. In lhe rather large number of preservcd specimens examined, I found

almost none in which lhe skin was not ragged and lorn, even when obvions carc

had been taken in collecting and packing lhe specimens. In spite of its suscep-

tibility to abrasion, it is one of lhe commonest Phyllobates in Colombia,

being recorded from over half lhe States. A new subspecies of subpunctatus from

Boyacá is Iikewise placed with frogs having the first and second fingers subequal.

The final species have the first finger longer than the second and possess

distinct webs. In brunneus the heel reaches the anterior corner of the eye, the

belly may be immaculate or finely spotted, while ils size does not exceed 22.5 mm.

It is found over a wide range, from Panama, Ecuador and li raz.il. and including

most of the States of Colombia. Two species have lhe heel reaching lhe center

of the eye; one of these, pratti, lias the belly immaculate and is very small, measur-

ing only 18 mm. in length. It is known at present only from Chocó. The other,

latinasus, has the lower surface immaculate except for a dark line around the

lower jaw and a patch of dark dots below the shoulder. Its size may be up to

26 mm., and its range is Panama, Ecuador and through much of Colombia. The

final species, inguinaUs, has the toes 1/4 webbed, the heel reaching to the center

of the eye, and the belly drab with a few darker spots below the femur and

tibia. It attains a length of 29 mm., and occurs in Panama and in northern

and western Colombia.

Needless to add, a great deal more collecting and comparison of species is

needed before we can say the final word on the taxonomy and variation of these

remarkable genera of arrow-poison frogs.

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Mem. Inst. Butantan

Simp. Intcrnac.

33(1):67-72, 1966

.TOSEPII R. BAILEY

MODES OF EVOLUTION IN. NEW WORLD OPISTHOGLYPH SNAKES

JOSEPH R. BAILEY

Department of Zoology, Duke University, Durham, U.S.A.

The area I have chosen lo discuss this afternoon is the general phenomenon

of opisthoglyphy and ils evolutionary implications wilh emphasis on New World

forms. Opisthoglyphy is the condition in colubrid snakes marked by the presence

of grooved teeth on the posterior expanded portion of the maxillary bone im-

mediately helow its articulation with the ectopterygoid. These teeth are usually

in pairs on either side, with one of each pair shed alternately, so that at least

one is functional on a side at any given time. The grooves are on the anterior

or outer face and serve as channels for the flow of venom by ducts from glands

in the temporal region of the head. These glands, lheir histology, homology

and biochemistry are currently being studied by my colleague, Aaron Tauh, at

Pennsylvania State University.

A half century ago the possession of grooved posterior maxillary teeth by a

snake was thought to indicate suhfamilial status among the COLUBRIDAE; i.e., all

snakes with such dentition were considered to have been derived from a common

ancestor. Since that time there has been a growing consensus among ophio-

logists that this opisthoglyph dentition has arisen many times in lhe course of

snake evolution, and today systematists consider the grooved rear teeth to be use-

ful taxonomically only at the generic and specific leveis, and at times to vary

even within a species. While being discredited, or at least demoted, as a nsefnl

character in classification, the morphological condition has, at the same time, been

grossly neglected as a biological phenomenon. The fact that a considerable

number of species of opisthoglyphs on three continents are known to be dangerous

to man, and at times even fatal, indicates that the rear fangs as functional

mechanisms are worlhy subjeets of investigation in their own right.

This paper examines opisthoglyphy from evolutionary, phylogenetic, geo-

graphical and ecological aspecls in an attempt lo discover any generalizations

which might pertain to the condition, and which indicate promising avenues for

further research.

In some species the grooves become subjective since their degree of develop-

ment is sufficiently variahle that one specimen may show it and another may

not. This has been shown by Stickel (1943) in Sonora, Eryth rolamprus

rnimus by Dunn and Bailey (1939) and it occurs in Rhadinaea guntheri accord-

ing to verbal information from Charles Meyers who is making an intensive study

of that genus and its relatives. This latter species currently resides in the lite-

rature under two names in two different genera.

Apparently any teeth may show a grooved condition. 1 have examined a

skull of Oxyrhopus jorrnosus which hears distinct grooves on the outer faces of

the mandibular teeth. The type of Calamodontophis has them on the

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MODES OF EVOLUTION IN NEW WORLD OPISTHOGHYPH SNAKES

anterior maxillary teelli as well as lhe posterior, and a sknll of Tomodon dor-

satus has heen examined in which the posterior fangs are deeply and douhly

grooved. These rear fangs vary considerahly frorn speeies lo species as to their

ahsolute lenglh and as to their length relative to lhe more anterior teeth. In

many these are scareely differentiated. The rnost extreme eondition I have ob-

scrved is in Tomodon dorsalus in which the length of the fang may he nearly

seventy per eent of lhe length of lhe maxillary bone.

what is probably the extreme of evolution towards

the fangs, since in some individuais the maxillary

or may he representei! by one or Iwo teeth only. I

This species also illnstrates

functional dependenee ii|)on

teeth are missing altogelher

n these individuais lhe denti-

gerous blade of lhe maxillary bone is thinned lo mueh less than lhe basal diameter

of a tooth. If only a single tooth is present the bone is thickened al that point

only sufficiently to aeeommodate il, as if in development the tooth primordium

had induced the deposition of the necessary bony base. Curiously and admittedly

on an insufficiency of data, it appears that the fangs are relatively shorter when

lhe anterior maxillary teeth are lacking. It is my belief that the anterior maxil¬

lary teeth are in the process of being lost and lhe fangs will take over as the

whole maxillary dentition. This has perhaps reached the quantum slage (Simp-

son, 1953:389) and its evohitionary progression is eurrently very rapid. We

know litlle of the functional aspects of this problem. No one lo my knowledge

has even described the feeding behavior of Tomodon, and until a careful

analysis of the mechanical aspects of feeding in this species has been made I

think it premature to speculate further.

How many different limes the opisthoglyph eondition has been derived in

lhe eourse of evolution may never be determined. However its multiplc origin

would indicate that under certain ecological situations there is an adaptive ad-

vantage conferred on its possessors just as there is in lhe case of wehbed feet

in aquatic birds or mammals.

I am

p a n o i d t

convinced from a long time intensive study of lhe relatives of Dre

s that its flaltened sabre-like posterior maxillary teeth, lacking grooves,

have evolved from an opistoglyph Cie lia -like ancestor. The only food record

I have for it is reptile eggs which hardly require lhe injection of venom for

subjugation.

Where, geographieally, do we find opisthoglyphy? Table I makes it clear

that the opisthoglyph eondition is increasingly more prevalent as we move out-

ward from a north polar center toward the tips of lhe three peninsulae of Austral-

Asia, África and South America. Whether hased on genera or species the per-

centages range from zero in the north to approximately fifly per cenl in Australia,

South Afriea and Argentina. Fifty years ago it might have been tempting to

invoke Mathew’s (1915) then recently proposed lhesis that the more primitive

opisthoglyphous forms were forced oul of more northern centers of origin by more

Progressive aglyphous forms. However, this inlerpretalion would run counter lo

classical, and to my belicfs, of the Irtie phylogenies. I think the answer is to

be found in the ecological-behavioral area, possibly combined with recent invasions

of northern geographical elements.

Habitudinally, without going into details and citing long lists of examples,

opisthoglyps in lhe New World are well representei! in arboreal, fossorial. and

terrestrial habitais, and have only failed to establish thcmselves in the aquatic zone.

In the Old World however, the opisthoglyph HomolopsiNae are highly aquatic.

A quantitativo analysis of New World Snakes by habitats at this time is imprudent

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JOSEPH R. BAILEY

<lue lo lhe dearth of reliable fielcl knowledge of lhe snakes preferences. Many

genera include species of diverse hahitat selection, as for instance Philodryus,

an opistlioglyph, and Thammophis, an aglyph. Tabulation then becomes

neeessary on a specific basis which is, of course, well beyond lhe scope of loday’s

discussion.

Granted the supposition lhat an elliptical pupil indicates greater noclurnal

activily, as inlerpreted from histological evidence by Gordon Walls (1982), we

find a geographic analysis on this morphological hasis revealing in the New

World fauna. In Talile 2 we see that among lhe aglyphous genera the round

pupil greatly predominates in North and South America. Among ophisthogly-

phous forms lhe elliptical pupil predominates when lhe conlinents are combined

and lhey form 60% of the ophisthoglyph coluhrid fauna of South America. The

prevalence of noclurnal forms in the tropics is not all surprising among poekilo-

iherms since cool nights of temperate regions are inhospitable to requisite nocturnal

activity, hui the hospitable Iropic evening invites exploitalion. The opislhoglyphs

have apparently heen better able to exploit nocturnal niches efficiently lhan the

aglyphous groups. Whelher ihis is due to the presence of a venom apparatus

may be open lo question. However, lhe more specialized dangerously venomous

families are predominately night adapted. On the other hand the American

opistlioglyph genera with elliptical pupils may be traced to not more than four

phylogenetic stocks and Ivvo of these, comprising the relatives of Pseudoboa

and those of Tachymenis contribute two thirds of lhe genera so that the

success of these two lines in South America lips lhe balance in their favour.

The phylogenetic stocks comprising the round pupil ophisthoglyph element are far

more diverse.

In both groups the greater exploitalion of the nocturnal environment in the

tropics goes far to explain the greater diversity of colubrids in low latitudes.

Turning lo the question of comparative food habits we are again confronted

with a dearth of reliahle knowledge of the animais in nature. Throughout my

systemalic studies of snake collections 1 have kept careful notes of food items,

but these accumulate slowly, detailed literature reporls are few and scattered, and

the general ones are moslly repetilions of previous sketchy data.

Again, without going into detail, I suggesl that on the average, and í

emphasize average, the diet of opislhoglyphs is more narrowly circumseribed than

that of aglyphous species. Perhaps it would be more accurate to say the diet

of nocturnal forms is more restricted lhan that of diurnal species, because within

my limited knowledge the nocturnal aglyphous species have as limited dietary

selection as do lhe opislhoglyphs. I do not know any opistlioglyph which has

the broad spectrum of food selection which is exhibited by Coluber coiistrictor,

the elaphes, Agkistrodon mokeson and A. piscivorous. The mollusc eating Dipsa-

dinak are tropical, aglyphous, and nocturnal with very narrow dietary restriction.

Most opistlioglyph species which are adequately known fil this pattern. For

instance from my own work I find that adults of Clelia feed on other snakes

and usually good sized venomous ones, perhaps, not because lhey are venomous

but because lhey are more sedentary and available. Erythrolamprus is a

snake feeder. Adults of Oxyrhopus and Pseudoboa are mammal eaters

whereas R a chidelus is reputed lo eal birds. P h i m o p h i s and S i p h lo •

phis are lizard feeders as are lhe juveniles of Clelia, Oxyrhopus, and

Pseudoboa. T antilla, with its curious short blunt maxillary teeth, is

lhe only gcnus I know to specialize in centipedes, as indicated by the scattered

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MODES OF EVOLUTION IN NEW WORLD OPISTHOGHYPH SNAKES

half dozen or so records 1 ha ve plus literature reports (Hamilton, 1956; Force,

1935). Our knowledge of this area of snake liiology is too embryonic to justify

conclusion at this time. Not only do we lack information on which food items

are taken, but just as important, we do not know to what extent choice enters

the picture. Are items taken simply because they are available at the lime and

place of snake activily and are of a suitable size, or does real choice and bio-

logical selection influence the residis?

In conclusion I would like to briefly summarize the points suggested above;

and then to point out other areas of insufficient knowledge:

1. Grooves may appear irregularly on teeth in several parts of the mouth.

2. Opisthoglyphy is polyphyletic in origin.

3. In Tomodon dorsatus the anterior maxillary teeth are in the process of being

lost entirely and the large fangs will remain as the oniy maxillary dentition.

4. Aglyphy may be secondarily derived from an opisthoglyph condition, as in

Drepanoides.

5. Opisthoglyphy is absent in the most northern colubrid faunas but is at least

equally numerous with the aglyphous in the most Southern faunas.

6. Opisthoglyphy is found in all habitats (except the aquatic in the New World).

7. Opisthoglyph genera predominate among nocturnal colubrids.

8. Opisthoglyph snakes take a wide variety of foods, but the diet of each species

(or genera) is rather narrowly circumscribed.

Areas of future research:

1. Little has been done of an experimental nature on the activity patterns of

snake species; at what hours are they active, at what temperatures, light in-

tensities, humidities, etc.... This subject could best be approached on snakes

in captivity.

2. We know far too little of the detailed food habits of all snakes in nature.

I suggest these be kept and recorded in the literature in detailed and quantitative

fashion including the size of the snake. Records in captivity should be noted

as such, and food items rejected noted along with those accepted.

3. We need careful observation and analysis of the feeding behaviour and mechanics

of nearly all snakes. Such analysis should utilize high speed photography when

possible.

4. Virtually nothing is known of the pharmacology, immunology and biochemistry

of opisthoglyph venoms. Will these disciplines help us in understanding opistho¬

glyph phylogenies or explain restricted food habits?

Literature

1. Dun, E. R. and Bailey, J. R. — Snakes from the uplands of the Canal Zone

and Darien. BuU. Mus. Comp. Zool., 8G(l):l-22, 1939.

2. Force, Edith R. ■— A local study of the opisthoglyph snake, Tantilla gracilis

Baird and Girard. Papers Midi. Acad. Sei., Arts and Letters, 20:645-659, 1935.

3. Hamilton, William J. — The food of some colubrid snakes from Fort Benning,

Geórgia. Ecology, 37(3) :519-526, 1956.

4. Mathew, William D. — Climate and evolution. Ann. N. Y. Acad. Sei., 24:171-

318, 1915.

5. Simpson, George G. — The Major Features of Evolution. 434 pp. New York,

1953.

í, | SciELO

Mem. Inst. Butantan

Simp. Internac.

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JOSEPH R. BAILEY

6. Stickel, William H. — The Mexican snakes of the genus Sonora and Chio-

nactis with notes on the status of other colubrid genera. Proc. Biol. Soc.

Wash., 56:109-128, 1943.

7. Walls , Gordon —■ Pupil shapes in reptilian eyes. 'Buli. Antivenin Inst. Amer.,

5(3): 68-70, 1932.

TABLE 1 — LATITUDINAL TRENDS IN OPISTHOGLYPIIY IN THE COLUBRID AE

New World

Genera

Percent

Opisthoglyph

Species

Percent

Opisthoglyph

Canada .

North Carolina .

5.0

2.7

México .

22.7

264

31.4

Costa Riea .

28.9

115

27.8

Ecuador .

35.7

117

27.4

Argentina .

50.0

47.1

Europe- África

Genera

Percent

Opisthoglyph

Species

Percent

Opisthoglyph

Englaml .

Europe .

37.5

14.3

África .

45.2

South África .

53.8

46.3

Asia-Australia

Genera

Percent

Opisthoglyph

Species

Percent

Opisthoglyph

Japan .

26.6

14.0

China .

22.2

11.1

Thailand .

33.3

30.0

Malaya .

40.0

34.0

Australia .

55.5

46.7

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L. D. BRONGERSMA

9. POISONOUS SNAKES OF SURINAM

L. D. BRONGERSMA

Rijksmuseum van Natuurlijke Histoire, Leiden, Hullund

Relatively little lias been published alioul the poisonous snakes o[ Surinam.

Scattered notes on isolated specimens or on small collections liave lieen published

in various journals. Moreover, some Information may be obtained from compre-

bensive Works, like SchlegeEs (1837) “Essai”, BoulengeEs (1896) catalogue, Ama¬

rais (1929, 1931) check lists, KlemmeEs (1963) lisl of the poisonous snakes of

the world, and as far as ELAPIDAE are concerned from Schmidts (1936)

preliminary account of South American coral snakes. ll seems lhat in the last

hundred years only two authors (Kappler, Van Lidth de Jeude) have dealt with

the Surinam snake fauna as such. Van Lidth de Jeude (1914-1916) dealt with

poisonous snakes in a series of articles in an encyclopaedia; he did not aim at

completeness, and his notes have only a very limited value for our purpose. From

1842 lo 1846 Kappler made it his business lo collect zoological specimens in Su¬

rinam, and in two books he published notes on lhe snakes (Kappler, 1881:137-139,

166-167; 1887:128-137). Of most interest is the list of species (Kappler, 1881:

166-167) of which he senl specimens to the Stuttgart Museum; il is not knovvn

lo me who was responsible for lhe identifications. The following poisonous snakes

were recorded by Kappler (1881:166-167): Elaps surinamensis, E. hemprichii, E.

lemniscatus, E. collaris, Crotalus horridus, Lachesis nuiliis (on pp. 33, 138, named:

Trigonocephalus rhombeatus ), Bothrops bilincatus, 11. atrox.

Since 1881 only three. species bave been added to Ibis list, viz., Micrurus

psyches, M. averyi, and Bothrops neglecta.

Taking into account laxonomic and nomenclatorial changes, to-day a list of

lhe poisonous snakes of Surinam reads as follows:

ELAPIDAE

Leptomicrurus collaris ( Schlegel, 1837),

Micrurus averyi Schmidt, 1939,

Micrurus hemprichii hemprichii (Jan, 1858),

Micrurus lemniscatus lemniscatus (Linnaeus, 1758),

Micrurus psyches , ( Daudin, 1802-1803),

Micrurus surinamensis surinamensis (Cuvier, 18 17)

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POISONOUS SNAKES OF SURINAM

CROTALIDAE

Bothrops atrox ( Linnaeus, 1758),

Bothrops bilineatus ( Wied, 1825),

Bothrops neglecta Amaral, 1923,

Crotalus durissus terrijicus ( Laurenti, 1768),

I, ache sis mulas miitus (Linnaeus, 1766).

Leptomicrurus collaris (Schlegel)

The history of Elaps collaris Schlegel was discussed l>y Schmidt (1937; 1939:

45, note 1), hui as his survey is incomplete, and erroneous as regards some

details. a more complete account will he puhlished hy me elsewhere. For long

years ihis species has heen included in surveys in lhe Philippine fauna as Hemi-

bungarus collaris. Schmidt (1937:361) believed lhat lhe specimen from (British)

Guiana was lhe first lhat proved in which part of lhe world lhe species is to he

found. This is incorrect, however. Both Schmidt (1937) and Thompson (1913)

overlooked lhat the species had heen recorded from Surinam already hy Kappler

(1881:167). This record is suhstantiated hy a specimen, which Kappler sent in

1844 to the Slutlgart Museum (now: Staatliches Museum fiir Naturkunde, Lud-

wigsburg). Anolher specimen from Surinam, collected more than hundred years

ago, was discovered recently in the collections of the Zoologisch Museum. Amster-

dam. At one time il helonged lo lhe Vrolik collection *, part of which was

acquired hy lhe Anatomieal Institute of Amsterdam Iniversily; in 1943 il was

passed on lo Zoological Museum. Sehmidl made Elaps collaris Schlegel the type

of his new genus L e p I o rn i c r u r u s .

Leptomicrurus collaris is one of the Coral Snakes that lacks an annulate

pattern. Except for a whitish collar. an indication of a whitish liar across the

snout, and large whitish spols on the ventrals reaching laterally on to scales of

lhe first two rows, il is uniformly dark hrown.

Mirraras lemniscatus Icniniscatus (L.)

The Surinam specimens examined hy me agree with the typical subspecies

hoth in the number of ventrals and in coloration. A specimen from Macasseema.

(British) Guiana (BM 87.1.22.14), with 256 ventrals also agrees with lliis sub-

species. A male from Cayenne (French Guiana) (ML 1122) has only 223

venlrals, and in (his respect il would come within the range of variation of

M. I. diutius Burger (1955), which, according to the original description, occurs

in Trinidad, Venezuela, and part of Guiana; in coloration this Cayenne specimen

agrees with the typical subspecies.

« The Vrolik collection belonged to Proí. G. Vrolik (25-ÍV-1775 — 10-xi-1859), and later

to his son Prof. W. Vrolik (29-iv-1801 — 22-xii-1863) (Engel, 1939:329-330).

2 3

5 6

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Mem. Inst. Butantan

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33 ( 1 ): 73 - 79 , 1966

L. D. BRONGERSMA

Micrurus surinamensis surinamensis (Cuv.)

Although it is usually stated that one of llie characters of Elapid snakes is

the absence of a loreal, llie (posterior) nasal being in contact with the preocular,

and llius separating the prefrontal from the npper labiais, there are some abnormal

specimens of M. s. surinamensis in which a scale is present between lhe nasal

and lhe preocular, and which hence show a loreal. In one Surinam specimen

(ML no. 1398) such a loreal is present on either side; another Surinam specimen

(ML no. 1417) has a loreal on lhe right side, and in a further specimen (ML

no. 1419) two loreals, one hehind the other, are present on the left side; in a

specimen from (British) Guiana (BM, purchased of Mr. Leadbeater) a loreal is

present on the left side.

Bothrops atrox (L.)

Bothrops atrox is a species with a very wide range of distribution in South-

America, and it is not to be wondered that it is foimd also in Surinam. 1 have

not used trinomials in this case, because 1 believe that more research is necessary

on the variation of this species hefore one can safely divide lhe species into sub-

species. Should the occasion arise, that Guiana specimens have to be recognized

as a distinct subspecies, it must be borne in mind that at least three names are

available, viz., Bothrops subscutatus Gray (1842:47), Bothrops sabinii Gray

(1842:47), and Bothrops affinis Gray (1849:7).

Bothrops atrox is fairly common in the low coastal area, but it also occitrs

farther into the interior. In the Nassau Mountains it was found in a river valley

at 464 m above sea levei. As far as our information goes at present, it seems

that it has a preference for damp areas near water.

Bothrops neglecta Amaral

This species was described by Amaral (1923:100-101) from two specimens,

both males. The holotype carne from Bahia, Brazil, the paratype from (British)

Guiana. At the time, Amaral (1923:101) suggested that the localily record for

the paratype might be erroneous. Amaral (1929:237; 1931:100, reprint: 8),

mentions the species from Bahia only; Klemmer (1963:408) mentions it from

Bahia and Venezuela. However, it seems to be unlikely that the paratype, which

the British Museum (Natural History) received from the Demerara Miiseum carne

from anywhere cise than Guiana. Moreover, Parker (1935:525) mentions four

other Guiana specimens received by the British Museum (Natural History), and

since that time slill another specimen was added to the London collection. Lrom

Surinam 1 have examined eight specimens. The range of variation in the num-

bers of ventrals and subcaudals is small, and as far as the small number of spe¬

cimens allows, of any tentative conclusions, tliere seems to be very litlle difference

between the sexes; in eight males the number of ventrals varies from 156-162,

that of subcaudals (pairs + one) from 45-52; three females show 156-162 ventrals,

and 43-46 subcaudals.

A specimen, taken on the Upper Tapanohoni river, in the mountains on lhe

Surinam-Brazil border. was referred by floge (1964:63) to Bothrops brazili Hoge

(1953). After having examined this specimen, I identified it as being Bothrops

neglecta, for the following reasons. The specimen, a male, has 159 ventrals, and

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POISONOUS SNAKES OF SURINAM

the subcaudals 8/7 + 4 + 26/26 + 1 + 6/6 + 1 + 0/1 + 1 (47 in all). Wilh these

counts it comes within the range of variation of 11 . neglecta, but it remains below

thc counts of the two type specimens ol B. brazili, which liave 175 and 179

ventrals, and 55 and 60 subcaudals respectively. Jt must be remembered ibat

Hoge (1953:15) pointed out that there was a strong resemblanee in colour pal-

tern between lí. brazili and lí. neglecta (as well as with lí. pirajai Amaral). The

colour pattern of lhe Tapanahoni specimen agrees very well with that of lhe

other Surinam and Guiana specimens of li. neglecta, but with reference lo Hoge’s

(1953:15) remarks, ihis need not be decisive. However, li. brazili is stated lo

lack a nasal pore (Hoge, 1953:15), whilst in B. neglecta sucli a pore is present.

Afler careful examination of lhe Tapanahoni specimen, 1 arrived al lhe conclu-

sion lhat a nasal pore is present, and that it is of the sarne shape as that of

lí. neglecta. Taking all these features (ventral and subcaudal counts, colour pat¬

tern, nasal pore) into account I feel convinced lhat the specimen mitsl be refcrred

to Bothrops neglecta Amaral.

Whether lhe Guiana specimens (including lhe paratype) and the Surinam

specimens of B. neglecta are conspecific with the holotype, which carne from

Bahia, is a question lhat can only be sellled by direct comparison.

As far as our [)resent knowldege goes, lí. neglecta is not found in lhe Coastal

area of Surinam, but only on higher grounds more in the interior. It seems

thal it does not have lhe preference of 11. atrox for lhe vicinily of water, but

that it occurs in the forest on higher ground. The only specimen with a definite

record of lhe altitude was taken in lhe Nassau Mountains at 406 m above sea

levei in the forest on the slope of a hill.

Bothrops bilineatus (Wied)

This species lias been recorded from Surinam already by Kappler (1881:167;

1887:137); Schlegel (1837, 11:540, Trigonocephalus bilineatus ) mentioned its

occurrence in Cayenne (Frencli Guiana); Quelch (1899:407, Lachesis bilineatus),

and Parker (1935:525, 529) recorded it from (British) Guiana. Thcrefore, it

is rather aslonishing that the occurrence of lliis species in thc three Guianas is

not mentioned in comprehensive works, like Klemmer’s (1963:404) list.

Crotalus durissus terrijicus (Laur.)

Allen & Neill (1957) have pointèd to the possible exístence of two ecological

forms of Crotalus durissus terrijicus in (British) Guiana. In Surinam too it is

said lhat there are two different forms of rattlesnake, which differ in coloration,

and which occur in different habitais. The material available to me is too small

to form a definite opinion.

Gomparing the snake fauna of the three Guianas, there seems to be no dif-

ference, al least as regards the poisonous snakes. It is true that Bothrops ne¬

glecta has not yet been recorded from French Guiana, but this will be only a

matter of time. Micrurus averyi is known from a single specimen only, but 1

do no doubt that it will be found lo occur in all three Guianas.

Very liltle is known about the distribution of snakes within Surinam. In

the old times “Surinam” as a locality record was considered lo be sufficient, and

in any case most collecting was done fairly close to the coast. Gradually some

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L. D. BRONGERSMA

information is coming from the interior, as more oollectors are penelrating farther

lo the soul li. Bolhrops neglecta apparenlly is a species that prefers the higher

parts of lhe country, and ihis may explain why it has been reported from Su-

rinam only fairly recenlly. Coral snakes (genera Micrurus and Leptomi-

c r u r u s) are not very often represented iti collections, but íbis need not mean

that lhey are exceedingly rare. More probahly it is a matter of not knowing

the habitats preferred by these species. M. s. surinamensis and M. I. lemniscatus

are more often represented in collections than the other species, and this may also

poinl to their heing lowland s])ecies.

lf little is known about the distribution of snakes within Surinam, still less

is known about the frequency of snake bite. Recently, Kabaart (1962) reviewed

the situation. Allhough military personnel often goes on patrol into the jungle,

the data colleeled by Kabaart show that in lhe period 1925-1958 not a single

case of snake bite by a poisonotts snake occurred. In 1958 two civilians died

from snake bite, but the species of snake is not mentioned. There have been

a few cases of snake bite, apparently by non-poisonous snakes, no effects of poi-

soning being apparent. Earlier authors (reviewed by Kabaart, 1962:220-221, re-

print: 3-4) also State that snake bite is very rare in Surinam.

Of course one does not know bow many cases of snake bile occur in the

interior, because these are not reported lo lhe medicai aulhorities.

As an inheritance of their African ancestors, the negro population of Suri¬

nam (and many other people as well) put great faith in “sneki koti”, which may

be used for inoculation, or as an antidote after snake bite has occurred. Its

composition is not completely known, excepl that the main ingredient is the head

of a poisonous snake, roasled and ground into powder. Opinions differ slightly

as to what is added, but usually it is stated that roasted and ground leaves of

various plants are added. Those, who know bow to prepare “sneki-koti” are not

allowed to tell what the ingredients are, because then lhe antidote would lose

its power. Moreover, inoculated persons have to refrain from eating some kinds

of food, e.g., deer or turtle, etc. Although it has repeatedly been shown that

“sneki koti” is of no value at all, it is very difficult to eradicate this superstition.

Only very rarely it is known which species of snake was responsible in a case

of snake bite. “Sneki koti” will be applied to biles of harmless snakes too, and

if the patients after this treatment do not show any signs of poisoning, this is

ascrihcd lo the effect of “sneki koti”. If the patients dies, it is assumed that

he has eaten of forbidden food.

References

1. Allen, E. R. & Neill, W. T. — Some interesting Rattlesnakes from Southern

British Guiana. Herpetologica, 13:67-74, 2 fig., 1957.

2. Amaral, A. do, — New Genera and Species of Snakes. Proc. New Engl. Zoõl.

CL, 8:85-105, 1923.

3. Amaral, A. do, — Estudos sôbre Ophidios neolrópicos. XVIII. Lista remissiva

dos ophidios da região neotrópica. Mem. Inst. Butantan, 4(2):i-iv, 129-271,

1929.

4. Amaral, A. cio, — Serpentes Venenosas Sul-Americanas. Arch. Socied. Biol.

Montevideo, Supl., 1, 1930, 93-107 (repr.:l-15), 1931.

5. Boulenger, G. A. — Catalogue of the Snakes in the British Museum (Nat.

Hist). London, 3:xiv + 721, 37 text. figs., 25 pis., 1896.

SciELO

10 11 12 13 14 15

POISONOUS SNAKES OF SURINAM

6. Burger, W. L. — A new subspecies of the Coral Snake Micrurus lemniscatus,

from Venezuela, British Guiana and Trinidad; and a key for the Identification

of associated species of coral snakes. Boi. Mus. Cienc. Nat., Caracas, 1(2) :1-

18, 1955.

7. Engel, H. —■ Alphabetical List of Dutch Zoological Cabinets and Menageries.

Bijdr. Díerk., 27:247-346, 1939.

8. Cray, J. E. — Synopsis of the species of Rattle-Snakes or Family of CROTA-

LIDAE. Zool. Miscellany, 47-51, 1842.

9. Gray, J. E. — Catalogue of the specimens of Snakes in the collection of the

British Museum. London, XV+ 125, 1489.

10. Hoge, A. R. — A new Bothrops from Brazil: Bothrops brazili, sp. nov.

Mem. Inst. Butantan, 25:15-22, 7 figs., 1953.

11. Hoge, A. R. — Serpentes da Fundação “Surinam Museum”. Mem. Inst. Bu¬

tantan, 30:51-64, map, 1960-1962.

12. Kabaart, J. — Slangebeten. Ned. Milit. Geneesk. Tijdschr., 15:218-240 (repr.:

1-23), 9 figs., 1962.

13. Kappler, A. — Hollãndisch-Guianu. Erlebnisse und Erfahrüngen wãhrend eines

J/3 jãhrigen Aufenthalts in der Kolonie Surinam. x + 495 p., Stuttgart, W.

Kohlhammer, 1881.

14. Kappler, A. — Surinam; sem Land, seine Natur, Bevôlkerung und seine Kultur-

Verhãltnisse mit Bezug auf Kolonisation. Stuttgart, J. G. Cotta, 1887.

15. Klemmer, K. — Liste der rezenten Giftschlangen. In: Die Giftschlangen der

Erde. Behringwerk-Mitteilungen, Sonderband, 255-464, 36 col. pis., 1963.

16. Lidth de Jeude, Th. W. van, — Various articles on snakes. In: Encyclopaedie

van Nederlandsch West-Indiê, ed. H. D. Benjamins en J. F. Snelleman.

's-Gravenhage, M. Nijhoff and Leiden, E. J. Brill, pp. 245, 280 (1914), p. 439

(1915), pp. 517, 535-538, 1916.

17. Parker, H. W. — The Frogs, Lizards and Snakes of British Guiana. Proc.

Zool. Soc. London, pp. 505-530, 1935.

18. Quelch , J. J. — The Poisonous Snakes of British Guiana. Ann. Mag. Nat.

Hist., 7(3):402-409 (reprinted from Timehri, 12, 1, n.s., 1898, 26-36), 1899.

19 Schlegel, H. — Essai sur la Physionomie des Serpents. Leiden, Arnz & Co.,

part. I, xxviii + 251; part 11(2)+606 + xvi; atlas: 21 pis., 3 maps, 1837.

20. Schmidt, K. P. — Preliminary Account of Coral Snakes of South America.

Zool. Ser. Field Mus. Nat. Hist., 20(19): 189-203, 1936.

21. Schmidt, K. P. — The History of Elaps collaris Schlegel 1837-1937. Zool.

Ser. Field Mus. Nat. Hist., 20(261:361-364, 1937.

22. Schmidt, K. P. — A new Coral Snake from British Guiana. Zool. Ser. Field

Mus. Nat. Hist., 24(6):45-47, fig. 5, 1939.

23. Thompson, J. C. — The correct status of Elaps collaris Schlegel. Notes

Leyden Mus., 35:171-175, 1913.

Discussion

A. do Amaral: "The generic name Bothrops being of feminine gender oblige

us to say Bothrops neglecta and not neglectus. Wilh regard to the variabilities of

the markings and changes that occur during growth I have written a paper, in

1925, about the variations in colour pattern in other species.”

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33U):73-79, 1966

L. D. BRONGERSMA

B. Lutz: “The Guiana-specimens of Bothrops bilineata belong to the same sub-

species as those occurring further lo the south of Bahia?”

L. D. Brongersma: “Perhaps Dr. Hoge may answer the last question because

he has recently distinguished between two subspecies of B. bilineata.”

A. R. Hoge: “I have only seen a few specimen from the Guianas and although

very similar to B. bilineata. There are siight differences in colour and pattern

but more material is needed to arrive to a conclusion.”

After the meeting A. do Amaral, A. R. Hoge and L. D. Brongersma have

examined and discussed the specimens of Bothrops neglecta, B. pirajai and B. bra-

zili in the collection of the Instituto Butantan. It became clear to all that the

Guiana- and Surinam-specimens, referred to B. neglecta by Brongersma, must be

placed with B. brazili, of which this probably represents a subspecies. Brongersma

and Hoge agree that B. neglecta is a synonym of B. pirajai; Amaral does not

agree with this synonymy, in as much as the former comes from the subxerophytic

section (N.E.) of Bahia and the latter from the S. wooded area. In addition A. R.

Hoge informs: “The information about the origin of Bothrops neglecta type speci¬

men is from Amaral, who never published it and there is no information in the

snake-register of the Instituto Butantan.”

AmaraVs additional remarks: In view of the profound divergence existing

among ophiologists concerning the real systematic status and nomenclalural situa-

tion of the various populations of Bothrops atrox and «trox-like forms (in their

mutual relations as well as in their relation to B. jararacussu: megaera, lanceolata,

aspera, neglecta , pirajai, brazili and others) as scattered from S. México, Central

America, some Antilles and S. America to N.C. Argentina, it seems to be high

time for a thorough (preferably cooperative) revision to be undertaken of that

complex group of serpents.

That revision should take into consideration, besides other possible bases of

comparison, the following points: geographic, topographic (altitudinal, clinal and

climatic) distribution; ontogenetic evolution of body markings; general pholidosis;

body and head shape and relative size; head scutellation; nasal pore; hemi-penis

formation; number and character of vertebrae; scale keel type, etc.

Whenever possible, that study should also include comparative observations of

living specimens (behaviour and striking position; average number of young in a

brood and venom characteristics: toxico-pharmacological, biochemical and physico-

chemical peculiarities of active components; venom-antivenom reactions).

— Through the same scientific approach it would be advisable to try to clear

the status of the various populations (morphologically too closely allied) of the

Neotropic rattler, gen. C r o tal u s .

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

88U): 81-83, 1966

I. S. DAREVSKY

10. ECOLOGY OF ROCK-VIPER (VI PERA XANTH1NA RADDE1

BOETTGER) IN THE NATURAL SURROUNDINGS OF ARMÊNIA

I. S. DAREVSKY

Z oologicul Institute, Science Academy, Leningrad, U.S.S.R.

l he rock-viper (Vipera xaiithina raddei Boettger ) is one of the rare prelty

poisonous snakes belonging lo lhe Soviet Union Fauna; sparse reporls concerning

its hiology and hahits are lo he found in literature. The following data ou the

hiology of this snake have heen eollected hy lhe aulhor between 1951-1901 vvhile

doing lield-work in lhe Easl Transcaucasus, particularly in the woods of mountain-

ous Armênia.

Habits — The most eharacteristic hahitats of the rock-viper in this country

are the roeky, at times steepy and scraggy slopes within lhe belt at 1500-1800 m

ahove sea levei, overgrown with thin oak forests, where they live particularly

abundant among dry rocks, thin oaks and bushes, and in piles of rock fragments

at the wooded slopes. Less frequently they rnay he met among lhe thinned out

xerophite vegetation upon hill-sides, among lhe sparse grovvth of juniper and in

the wide open, roeky and xerophite steppe. In some places the viper spreads

out into cultivated fields where it keeps lo slone heaps along the bounderies.

They shelter in cavilies under stones, rock crannies, between roots or in holes

of rodents. Their winter retreats are deep fissures of rocks, almosl too narrow

for entrance. Numbers of them, up to 20 mature specimens seek their winter

retreat in the same hole. If lhose holes really are in connection one with an-

other in lhe middle oí lhe rocks, we may indeed he right in coneluding that the

vipers hibernate in great numher logether.

Erequency — Vipers varies in accordance with lhe different seasons of the

year. After hibernation, between the end of April and the middle of May, they

remain in the vicinity of their winter quarters and lie coiled up logether en

masse. In May 1953 the aulhor could count up lo 50-60 mature vipers, in

groups of 4-6 specimens at an area less than 1 hectare, in the forested roeky

surroundings of the Antharul village (Armênia). After coupling, ahout the end

of June, higher up in the mountains even in its firsl week, the vipers disperse

over adjacent areas, reassemhling near winter quarters late in Oeloher, gradually

increasing in numher, not reaching however the amount of spring-time while

lhe firsl comers already retire into lheir deus. There may he found perhaps

20-50 specimens per hectare in those areas.

MiGRATlON — Migration from the area near winter quarters takes plaee in

daytime: the single speeimen may he seen crawling from one tree to another to

disappear finally in the thick underwood. The direction is indicated hy pieces

of shedded skin, which are seen all over the bushes, bordering the winter retreat.

SciELO

10 11 12 13 14 15

ECOLOGY OF ROCK-VIPER (VI PERA XANTHINÁ RADDUl

ROETTGER) IN THE NATURAL SURROUNDINGS OF ARMÊNIA

from the middle of June on. whereas the snakcs themselves are nowhere to lie

founcl. In orcler lo elucidate lhe believes that the snakcs really return lo the

sarnc placo for hibernating, lhe author caught and marked 63 vipers in May 1952

on the Southern side of the Aragatz rnountain (Armênia). On the end of October

of lhe same year, 7 of thosc were caught within a radius of 100-200 in from

the place of the firsl capture. Tinis, it is confirmed that at least a part of suakes

return to lhe same winler quarters.

Seasonal and DAILY ACTiviTiES Awakeniiig from hihernation depends ou

the altitude above sea levei of the rei reais and takes place in the middle of

April to the beginning of May. Only when lhe rocks have been sufficienlly

healed hy the sun, around 11-12 o’clock, lhe vipers leave lheir retreat to lie on

the bare rocks until more or less 6-7 o’cloek, the loose folds of skin on their sides

giving them an emaciated appearanee. Approximately at lhe beginning of July,

the malure speeimens adjust lo crepuscular and nocturnal aclivity and are nol

to be seen in daytime at all. The knovvledge of th is fact helps lhe population

of those areas to lake every precaution dnring springtime, not even turning out

cattle to grass; everyone hecomes careless in summer-time, since the return of

the vipers does not occur hefore the end of October. In case of some evident

danger, for instance when suddenly faeed hy ils human enemy, the viper tries

lo dive into lhe nearesl eover produeing charaeteristic jerky hissing sounds in

(juick succession. Having sueeeeded in intercepting its retreat, the snake takes

up a peculiar threatening posture raising lhe for-part of its body almost to an

upright position. making rapid ihrusts wilh a wide open mouth into the direction

of its enemy. Large full-grown males are particularly aggressive in sueh cases.

Fe EDI MG HA BITS - Afler emerging from hihernation the vipers don t feed at

all or only on some insects, explaining lhe remainders of some orlhoperans found

in the stomach contents of vipers, caught and opened hy Chernov in May of

1939. Later on the snakes feed on mouse-like rodenls. Many vipers caught hy

lhe author at the beginning of June disgorged lhe swallowed rodenls of the day

hefore, mostly Microtus arvalis, less frequently M. nivalis. The eonsumed number

of rodenls is very large. Il was checked lhat a single snake during the season

consumes not less lhan one hundred small rodenls co-existing wilh them in the

same region. Less frequently the snakes feed on lizards and young birds which

nesl on the ground. The very young ones feed on insects and small lizards.

Repkoduction The mating of vipers coincides wilh lhe firsl shedding of

skin. in springtime and may be prolonged til the end of May or even until

migration starls. While lhe snakes hask in lhe sunshine the males erawl rest-

lessly around until finding a fernale, gliding around her agitating the lail at

higli speed trying lo hook the female’s tail which hy this time also begins to

stir. Now and than the tnale succeeds in his endeavor hui the fernale frees her

lail immediately forcing lhe male lo start all over again. At least both snakes

move convulsively wilh interloeked tails. this movement spreading gradually wilh

violenee over the whole body. Frequently the fernale breaks loose, being pursued

elosely hy the male. which body twitches violently from time lo lime. After

protracted chase during which the male or leaves or seeks the fernale, both snakes

interlaee the posterior paris of the body. Both, one third of lhe body erected

in “S” forni, start swaying, the male trying now and then to push lhe fernale s

head lo lhe ground hy violenl ihrusts. Afler sueceeding lo force the head down

for several times both drop ahruplly, interlwine lheir hodies rope-like leaving free

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 81-83, 1966

I. S. DAREVSKY

a small pari near the head. Somo lime later they begin coupling their cloacas

lighlly joined together. Copulation oflen occairs about 1-2 hours alter lhe be-

ginning of courtship and lasts 20-30 minutes, the departure of the snakes follow-

ing soou. The young ones are born in general during the first days of Septem-

Depending 011 size

egg membrane vvhile

her, hut hirth may be delayed until lhe end of the month.

lhe females may give birth lo 3-9 youngs whicb break the

still in the womb, dispersing immediately inlo all directions after birth. Females

of 501-500 mm length give birth to 3, oftener 4 or 5 youngs, vvhile those of

570 mm and Ionger bring forth 6 youngs. In one case there were recorded 9

youngs. Data of Groubant and Koudneva (1956) discovered 10-13 eggs in the

oviducts of examined females. An investigation proved however, that besides the

living youngs the female cast off 1-2 unfertilized eggs. The youngs are born

large in size, about 204-214 mm length of which 11-19 mm consist of the tail.

They are of dull color until the first shedding, about a week later, acquiring

the most brilliant color with the sharpest outline of lhe pattern, normal to mature

snakes.

Perfiliev ( 1941 I made a

Venom

raddei venom, applying it to various animais.

]>erisb within few seconds or some minutes. A

motion faculty after 2 minutes and died after

4 hours hut it took 24 hours for a bitten dog

author during 1951-1953 in Armênia, show that

in severe cases. One of those cases was a mature man, bitten

shoulder, death occurring within 12 hours. Two children aged 1

in the leg. There are no more data in literature on the action

venom on humans.

number of experiments with Vipera

His data show that bitten mice

lizard Lacerta agilis lost its Ioco-

40 minutes. Rabbits died after

lo die. llecords collected by the

the bite of those vipers are fatal

on his righl

, were bitten

of rock-viper

RKEERKNCES

1 .

Ghernov, S. A. — Herpetological fauna of Armênia and Nahichevan. Assr.

Zool. sbornik Akad. Nauk Armenian SSR. 7:77-194, 1939.

Perfiliev, P. P. — The action of the poison of Vipera raddei and Vipera ursini

on the animais. Pharmacology and Toxicology, Moscow, 2:53-56, 1941.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 85-92, 1966

R. MERTENS

11. UNTERLAGEN ZUR ÕKOLOGIE, ETHOLOGIE UND EVOLüTlON DER

BAUMSCHLANGEN ARBORICOLOUS SNAKES: THEIR ECOLOGY,

GEOGRAPHICAL DISTRIBUTION AND ECOLOGY

R. MERTENS

Natur-Museum und Forschungs-Institut Senckenberg. Frankfurt, Germany

W as mag eine Schlange veranlasst liaben, ihr ursprüngliches Leben in oder

auf der Erde aufzugeben und sich ins Geast, ein ])aar Meter iiber dem Boden,

zu begeben? Eine erfolgreiche Eroberung dieses Lebensraumes ist für ein bein-

loses Geschõpf wie eine Schlange eigentlich eine erstaunliche Leistung. Das

umso mehr, ais es unter den vielen schleichenfòrmigen Eidechsen mit rückge-

bildeten Gliedmassen keine bezeichnenden Baumtiere gibt, wenn mau von einigen

Bewohnern der Epiphytenballen in tropischen Waldern absieht.

Sicherlich waren die ursprünglichen Schlangen, gleich vielen anderen in Ent-

faltung begriffenen Tiergruppen, von einem Ausbreitungstrieb beherrscht, einem

Trieb, der sie in iminer neue, von ihren Verwandten noch nicht besetzte Le-

bensráume geführt hat: so z.B. in die Gewasser verschiedenster Art — vom

Bach bis zuni Ozean —, in die Sand — und Steinwiisten und in die Hõhen der

Pflanzendecke, einschliesslich der Baunikronen. In einem üppigen Walde sind

nâmlich die Licht- und Warmeverháltnisse in den hüheren Regionen für eine

Entfaltung des Kleintierlebens günstiger ais auf dem Waldboden, und es mag

daher verstandlich sein. dass die Schlangen dorthin dem übrigen Getier gefolgt

sind. In einer gevvissen Hohe über dem Erdboden boten sich ihnen nicht nur

neue Nahrungsquellen dar, sondem auch geeignete Wohnplatze.

Dass eine derartige Llmstellung tatsãchlich vorkommen kann, zeigen uns nicht

wenige Bodenschlangen, wie z.B. E l a p h e , die mehr oder weniger regelmassig

den Erdboden verlassen und sich in luftiger Hohe aufhalten. Ja sogar Blind-

schlangen, wie die philippinischen Typhlops dendrophis und longicauta sind im

Epiphytenhumus hoch über der Erde gefunden worden. wohin sie sicherlich den

Bauntermiten oder Ameisen gefolgt sind. In Usbekistan ist Vipera iebctina an

manchen Stellen zu einem haufigen Buschbewohner geworden. Auch auf der

Cycladeninsel Milos verbirgt sich die Levanteotter, laut mündlicher Mitteilung des

Herrn H. Kratzer (Zürich), sehr oft auf und in Strauchern, zweifellos auf der

Suche nach geeigneter Nahrung, die hauptsachlich aus Viigeln bestehen dürfte.

Dasselbe trifft schliesslich für Bothrops insularis auf Queimada Grande zu, deren

Neigung zum Leben ein paar Meter über dem Erdboden sogar seit langern be-

kannt ist. Das wurde neuerdings sogar für Bilis nasicornis in den Waldern

Ostafrikas festgestellt.

Das Leben auf Strauchern und Báumen hat mm, im Laufe von Jahrmillionen,

<ler Erscheinungsform und den Lebensausserungen vieler Schlangen bekanntlich

einen einheitlichen Stempel aufgedrückt. Manche sind nahmlich untereinander so

SciELO

10 11 12 13 14 15

8(5

UNTERLAGEN ZUR OKOLOGIE, ETHOLOGIE UND EVOLUTION DER

BAUMSCHLANGEN ARBORICOLOUS SNAKES. . .

ãhnlich geworden, dass es zu weilen nicht leiclit ist zu entscheiden, ob diese

Âhnlichkeit auf einer Verwandschafl oder auf einer parallelen Enlwieklung (Kon-

vergens) beruht. Vergleicht man z.B. einen brasilianischen Hundskopfschlinger

(Corullus caninus) mit einem papuanischen Baumpython (Chondropython viridis ).

so muss auch der Schlangenkenner sehr genau hinsehen, urn beide auseinander-

zuhalten: beide sind niihmlicb laubgrün und haben auf der Hückenmitte einen

kreideweissen, leilweise in Flecken übergehenden Langsstreifen; beide stimmen in

der seitliehen zusammengedrückten Kdrperform überein und haben anniihernd die

gleiehe Grõsse. Ausserdem ist beiden eine ahnliche, rotbraune oder gelbe Jugend-

farbung eigen, und beide pflegen sicli in Ruhestellung auf einem Ast üfler in

gleicher Weise zusammenzurollen, wobei der Kopf in der Mitte auf den Kõrper-

schlingen liegl (vgl. Mertens 1960. Taf 62). Und doch sind beide Schlangen

nicht naher miteinandcr vervvandt: sie gehoren zvvar zur gleichen Familie der

Boiden, aber zu zwei verschiedenen. durch eraniologische Merkmale gul unter-

schiedcnen Unterfamilien: Corullus zu den Boinae Chondropython zu

den Pythomnae. Beide Arten. an zwei entgegengesetzten Punkten des Erdballs

lebend. rniíssen daher vollig unabhíingig voneinander ihre Âhnlichkeit erworben

haben.

Ilier liegt also ein sehr eindrucksvolles Beispiel einer Parallelenlwicklung oder

Konvergenz vor. Am auffalligsten wird davon die Erscheinungsform der Schlan¬

gen betroffen also etwa die Farbung, die bekanntlich sehr oft laubgrün oder

rindenfarbig ist. wahrend auffalig gefarbte — etwa schwarzgelbe wie Spilotes

pullatus oder Boiga dendrophila — nur vereinzelt vorkommen. Ferner ist für

eine Baumsehlange bezeichnend eine Verlangèrung des Korpers, der lniufig seit-

Iich zusammengedriickt ist. Seine Ausmasse und sein Gewicht sind trotzdem

gcring: iiber 2 m. lange Baumbewohner sind nicht allzu hãufig, bei den meisten

schwankt die Lange um 1-1.5 m. Irn Gegensatz zur schlanken, zierlichen Kõrper-

form mil langem Schwanz der Baumnattern sind kurze und dicke Gestallen mit

verkürztem Schwanz wie sie unter den Boiden und Baumollern vorkommen, sel-

tener. Bei diesen hat der Schwanz die Funktion eines Greiforgans, wahrend bei

einigen Baumnattern der lange Schwanz zuni Umklammern nicht befáhigt ist.

Der schlankeren Kõrperform entsprechen in vielen Fállen die Verminderung der

Langereihen von Rückenschuppen (bei Chironius bis auf 10-12) und Er-

hõhung der Ventralia und Subcaudalia-Zahlen. Man findei diese Verschiebung

der Schuppenzahlen fast immer wenn man eine Baumsehlange mit ihren nachsten

Verwandten unter den Bodenschlangen (z.B. Bseudohaje mit Naja) verg-

leicht. Audi die schmale Kõrperform triigt zu diesem Erscheinungsbilde bei; sie

ist bei einigen Schlangen vorne stark verlángerl und erheblich zugespitzl oder gar

in einen Fortsatz ausgezogen (z.B. bei Ahaetulla nasula und pulverulenta, bei

Rhynchophis boulengeri und den Canga ha — Arten). Auf Grund derartiger

Merkmale kanu man für manche Schlangen. deren Biotop unbekannt ist, mit

einiger Sieherheit ein Baumleben aunehmen.

Wenn es auch Baumschlangen mit glatten Rückenschuppen nicht allzusellen

gibt. so fallen andere, wie z.B. die tropisch-afrikanischen Nattern Gaslropyxis

srnaragdina und llapsidophrys lineutu. dann die Eierschlangen ( Dasypeltis)

und Baumvipern ( Atheris ) durch kraftige Kiele auf. die gewiss ais Gleit-

schulz wirken. Dieselbe Bedeutung kommt auch den kriiftigeii Seitenkielen an

den Ventral- und Subcaudal- Schildchen zu: letztere sind besonders ausgepragt

bei dem afrikanischen Philothamnus semivariegatus und den orientalisehen Chry-

sopelea — Arten schwaeher bei den gleiehfalls orientalisehen Dendrela-

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

■Simp. Internar.

33(1): 85-92, 1966

R. MERTENS

p h is — Arlen und hei der Gonyiphis margarUatus. Bei manchen der erwiihnten

Nattern zeigt jedes Ventrale und Subcaudale ausser dem Kiel an den Seiten noch

eine klcine, aber deutliche Kerbe.

Nieht wenigen Baumnattern vermogen mil einer geradezu unheimliehen Ge-

schwindigkeit im Geasl dahinzugleiten, eine Fortbewegung die wie aiif dem Erd-

boden nuf einer lateralen Schlangelung des Kõrpers beruht. Um einen Naeh-

barzweig zu erreichen, kann eine sehlanke Baumnatter ihren Vorderkorper gerade

ausstreeken, vvobei zu seiner Versleifung sowohl seine seitliehe Kompression wie

die zuweilen verlangerten, wenig beweglichen Wirbel und die geringe Zabl der

dorsalen Schuppenreihen, deren mittelste haufig verbreitert ist, dienen. Peters

(1960:18) hat auf diese Beziehungen hingewiesen, doch ist letzteres Merkmal

nieht auf die Baumschlangen beschrankt. Es kommt namlich auch bei den am

Erdboden lebenden Schlangen (z.B. Bungarus ) vor, bei denen es alter auch

im Zusammenhange mit einer erheblichen Verminderung der dorsalen Schuppen¬

reihen auf eine Versteifung des Kõrpers ankommt. Manche Baumnattern (Chry-

sopelea, Dendrelaphis) sind imstande, durcb das Vorwiirtsschnellen

einige Meter frei in der Luft zurückzulegen, wobei man etwas übertrieben von

"fliegenden Schlangen ” ge sprochen hat.

Beim Erkennen der Beute muss die Zunge hei Baumberwohnern naturgemáss

eine weit geringere Kolle spielen ais hei den Bodenhewohnern: die Schlangen

haben weniger Gelegenheit, mit ihrer Zunge ihre Beuleliere zu berühren und

sie durch “Bezüngeln” ais solche zu erkennen. An Stelle der Zunge treten hei

ilinen die Augen in Funktion: es ist der Gesichtssinn, mit dessen Hilfe wohl

die meisten Baumnattern (weniger die Baumottern) ihre Beute wahrnehmen. Das

zeigen uns am anschaulichsten die indischen “Baumschnüffler” der Gattung

Ahaetulla, welche das Bezüngeln ihrer Nahrungstiere võllig verlernt haben.

Trotzdem pflegen diese langgestreckten, eindrucksvollen Geschõpfe lehhaft zu ziin-

geln, eine Tiitigkeit, die hei ihnen allenfalls zur Aufnahme von Duftstoffen dient

oder eine ganz andere Bedeutung (vgl. weiter unten) hekommen hat.

Besonders grosse Augen sind also für Baumschlangen überaus zweckmãssig.

Das umso mehr, ais ja in den meisten Lebensraumen dieser Tiere die Lichtver-

haltnisse nieht übermassig günstig sind. Es ist daher verstandlich, dass nieht

nur die Dammerungstiere, sondem auch die Tagtiere unter den Baumnattern sich

oft durch eine erhebliche Vergrõsserung der Augen auszeichnen. Anschauliche

Beispiele für diese Eigenart liefern unter den Tagnattern: Dispholidus typiis,

Rhamnophis aethiopissa, Hapsidophrys lineata und Dendrelaphis jormosus sowie

andere Arten dieser Gattung; unter den Nachtnallern: Dipsadohoa duchesnii,

ímantodes cenchoa, Aplopeltiira boa, Dipsas indica und ihre Verwandten. Auch

hei den Baumnattern und Baumelapiden ist die relative Augengrõsse in der Regei

hedeutender ais hei ihren auf dem Erdboden lebenden Stammformen, wie es sich

z.B. aus einem Vergleich von Pseudohaje mit Naja ergibt. Oft sind die

Augen nieht nur gross, sondem auch erheblich vorgewõlbt, um das Gesichtsfeld

zu erweilern, vor aliem nach vorn und unten.

Die in der Dammerung oder hei võlliger Dunkelheit aktiven Baumschlangen

sind in der Regei durch eine senkrechte, hei Lichtmangel stark erweiterungsfahige

Pupille gekennzeichnet. Bei einigen Tagnattern mit verlangertem Vorderkopf

hat sich bekanntlich eine eigenartige Pupillenform ausgehildet: sie ist nahmlich

weder rund noch senkrecht, vielmehr waagerecht langgestreckt. Eine solche langs-

ovale, zuweilen in der Mitte etwas eingeschnürte Pupille haben die indisch-sun-

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88 UNTERLAGEN ZUR OKOLOGIE, ETHOLOGIE UND EVOLUTION DER

BAUMSCHLANGEN ARBORICOLOUS SNAKES...

daischen A h a e tull a und Dryophios sowie die áthiopische T h elo t o r -

n i s, indem sie sicli bei schlafenden Tieren erheblich zusammenziehl. Die Be-

deutung derartiger Pupille für die am Tage jagenden Naltern ist klar: die nach

vorne ausgezogene Pupille ermoglicht den Tieren das für das Erkennen der Beu-

tetiere wichtige binokulare Sehen.

Von vielen Baumschlangen wird die Beute schnell ergriffen und dann, sowcit

ihre Griisse ein bestimmtes Mass nicht überschreitet, obne vveiteres verschlungen.

Ist aber ein Beutelier zu gross, so wird es entweder erdrosselt oder — bei Vor-

handensein von Giftdrüsen — vergiftet. Im letzteren Falle kann es zwischen den

Kiefern so lange festgehalten werden, bis sein Tod eintritl. Es ist verstandlich,

dass für baumbewohnende Schlangen ein Giftapparat von besonderer Bedeutung

sein muss. So sind unter den Colubriden gerade bezeichnende Baumbewobner

(z.B. Thelotornis, Dispholidus, B o i g a , in Afrika; Ahae-

t u l I a , Dryophiops, B o i g a , Chrysopelea in Asien;

O xybelis, Philodryas, Imantodes in Amerika) hãufig opis-

ihoglyph, auch alie arboricolen Nattern Madagaskars ( Mimophis , Lyco-

dryas. Langa ha ) gehoren ebenfalls dazu. Bezeicbnenderweise gibt es sogar

unter baumbewohnenden Aglyphen Arlen, deren Biss- auch eine für den Menschen

spürbare — Giftwirkung hat: so Leptophis ahaetulla in Columbian und Uromacer

oxyrhynchus auf Hispaniola.

Inbezug auf den Schutz vor dem Feinde zeigen die Baumscblangen eine

Fiille von verschiedenartigen Verhaltensformen. Ein wesentliches Scbutzmittel ist

ihre Mimese. Manche Arlen z.B. der Gattungen Ahaetulla, O xybelis

der Imantodes sind sehr schlank und heben sich von ihrer Umgebung in

der Tat so wenig ah, dass sie übersehen werden sofern die Tiere regungslos ver-

harren. Jch habe Thelotornis kirtlandii ais Lianenschlange bezeichnet, so gross

ist ihre Ãhnlichkeil mit diesen Schlinggewachsen. Bei ihr sind übrigens ebenso

wie hei Langaha alluaudi, und bis zu einem gewissen Grade bei Ahaetulla pul¬

verulenta, die Internodien durch eine entsprechende Bãnderung angedeutet. Die

Wirkung der Mimese wird weiterhin unterstrichen durch die Kopfform, besonders

durch die erwahnlen Schnauzenfortsãtze, die bei der madagassischen Langaha

von hõchster Eigenart sind und nicht nur ais Geschlechtsmerkmale eine Bedeutung

haben, sondem zweifellos auch zur Tarnung dienen. Einige Baumschlangen pfle-

gcn — übrigens ahnlich dem rein aquatilen Er peto n liingere Zeit bewegungslos

zu bleiben, auch mit emporgehobenem Vorderkorper, wie die madagassische Mi¬

mophis mahjalensis, was ebenfalls ais Ausdruck oincr Mimese zu werten isl. Von

der Gattung A t h e r i s, den âthiopischen Baumvipern, wird ebenfalls berichtet,

dass sie ihren Vorderkürper aufrichten und in einen spitzen Winkel zum übrigen

Korper bringen, wodurch ein abgeknickter oder halb abgebrochener Zweig vor-

getauscht wird. Dieser Eindruck wird noch verstiirkt, wenn sie sich — wie

A m blycephalus und Aplopeltura — herunterfallen lassen und sich

dann “tot” stellen. Ein ])aar Baumnattern, z.B. Leptophis ahaetulla, die am

Schwanze festgehalten werden, vermogen überraschend leieht und obne grossen

Blutverlust ihre Schwanzspitze einzubüssen, ohne sie allerdings regenerieren zu

kõnnen.

Im Abwehrverhallen spielen bei den Baumschlangen chemische oder akustische

Mittel offenbar keine allzugrosse Kolle. Von den ersteren sei auf das Erzeugen

von Duftstoffen aus den Postanaldrüsen bei der südamerikanischen Gattung L e p -

t o p h i s und bei der indo-australisehen Gattung Dendrelapis aufmerksam

gemacht; in dieser Beziehung scheinen sich jedoch die einzelnen Arten innerhalb

2 3

5 6

11 12 13 14 15

Mem. Xnst. Butantan

Stmp. Internac.

33(1): 85-92, 1966

R. MERTENS

dieser Gattung recht verschieden zu verhalten, indern die Duftstoffe z.B. hei Den-

drelaphis pictus sehr intensiv, hei tristis dagegen kaum oder überhaupt nicht

wahrnehmbar sind.

Ais Ausdruck einer Erregung íasse ich das eigenartige Hin-und Herpendeln

des frei von den Zvveigen hinunterhangenden Vorderkõrpers auf, das ich hei den

Ahaetulla und 0 xy b e I i s — Arten am háufigsten beobachtet habe. Ãhn-

lich ist es mil dem “Warn-Züngeln” (Mertens 1946:24). Dabei wird die Zimge

für geranine Zeit ausgestreckl, wohei die Zungenspitzen sich langsam nach oben

krümmen (Dendrelaphis punctulalus, Displwlidus typus, Thelotornis kirtlandii)

oder gelegentlich sogar vihrieren (Philothamnus se mi variegai us, Ahaetulla prasi-

na) . Besonders eigenartig nimml sich hei den zuerst genannten Baumnattern die

Fárbung der Zimge ans, da sie einen scharfen Kontrast zur Gesamtfarbung hildet:

sie ist tiefschwarz hei dem hlaulichen Dendrelaphis punctulalus, ziegelrot mil

schwarzen Spitzen hei der rindenfarbigen Thelotornis. In manchen Fállen

streckl eine Baumnatter die Zunge gleich einer Sonde für mehrere Sekunden võllig

hewegungslos heraus, wie ich es bei Oxybelis seneus und Opheodrys aeslivus, beim

Anhlick anderer Febenswesen, auch die Beutetiere, heohachtete.

In der Hanptsache hei Baumschlangen ist ais recht hezeichnende Warnhand-

lung das Aufsperren der Kiefer verbreitet, wohei der Rachen durch grosstmogliche

Auseinanderspreizen der Unlerkieferáste erweitert wird. Dieses Ahwehrverhalten,

dem erst sehr viel spáter oder garnicht, ein Biss folgt, ist mir unter Baumnattern

von Leptophis — Arten, Dendrelaphis pictus, Oxybelis aeneus, Uromacer

oxyrhyncus und Dasypellis scabra loveridgei bekannt und ausserdem von Chloro-

phi irregularis, Chironius juscus, Ahaetulla nasuta und anderen heschrieben wor-

dcn. Irgendwelche Schlüsse daraus auf etwaige verwandtschaftliche Beziehungen

dieser Nattern zu ziehen, dürfte verfehlt sein, da sich die einzelnen Gattungsan-

gehorigen recht verschieden verhalten: wohl ist das warnende Aufsperren der

Kiefer z.B. für Dendrelaphis pictus charakteristisch, aber hei dem verwandten

Dendrelaphis tristis hahe ich es niemals festgestellt.

Eine recht hâufige Abwehrhandlung stelll ferner das Aufhlãhen des Vorder-

korpers dar, wie es z.B. von Spilotes, Pseustes und Dispholidus heschrieben

worden ist. Dabei wird die ofl sehr auffalende und mit der übrigen Fárbung

kontrastierenyde Zwisehenschuppenhaut sichthar: sie ist zum Biespiel schwarz-weiss

hei der lauhgrünen Ahaetulla prasina, hlauviolelt bei dem bronzefarbigen Dendre¬

laphis tristis. Indessen ist dieses Ahwehrverhalten hekanntlich ebensowenig auf

die Baumschlangen beschránkt, wie das Abplatten des Kòrpers oder seines vor-

dersten Ahschnittes (D e n d r o a s p i s) Immerhin verdient vermerkt zu werden,

dass die extremsten Formen des Halsaufbláhens, wohei tatsáchlich der Eindruck

eines mit Luft angefüllten Ballons entsteht, bezeichnenden Baumschlangen, wie der

afrikanischen Lianennatter ( Thelotornis kirtlandii), eigen ist.

Nur der Kenner kann in der Regei die Geschlechter hei den meisten Baum¬

schlangen einigermassen sicher unterscheiden. Wie auch sonst bei den Schlangen,

haben die Mánnchen meist weniger Bauchschildchen ais die Weihchen und einen

an der Wurzel gegeniiber der práanalen Region kaum verschmálerten, relativ lán-

geren Schwanz, was in der hoheren Zahl der Unterschwanzschilder zum Ausdruck

kommt. Bei Ahaetulla prasina und nasuta stellte ich fest, dass die Rüchenschup-

pen in der Analregion hei den Mánnchen kráfligere Kiele haben ais bei den

Weibchen. Weitere Beispiele dafür fiihrt Kopstein (1941) von javanischen, auch

bodenbewohnenden Arten an und hemerkt, dass diese Kiele das gegenseitige Fest-

halten wáhrend

der Paarung erleichtern.

Weitaus am stárksten

was im

Zu-

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UNTERLAGEN ZUR OKOLOGIE, ETHOLOGIE UND EVOLUTION DER

BAUMSCHLANGEN ARBORICOLOUS SNAKES. . .

sammenhange niit dem Baumleben verstiindlich ist - - trelen diese Kiele auf dem

Riicken von mannlichen Leptophis ahaetulla und einigen anderen Arten dieser

Gattung und namentlich von Chironius carinatus hervor, dagegen kõnnen sie liei

den weiblichen Tieren überhaupl fehlen.

Auf eitien sehr merkwürdigen Gesehlechtsunterschied stiess ich früher bei

Dendrelaphis pictus und calligaster (Mertens 1937:173). Es zeigte sich, dass bei

diesen in Südostasien und auf den indoauslralischen Inseln weitverbreiteten Strauch-

nattern die ausgewaehsenen Mannchen deutlich grõssere Augen haben ais die Weib-

chen. Obwohl die mannlichen Nattern dieser Arten die Grõsse der weiblichen

nicht erreiehen, betragt der horizontale Augendurchmesser des javanischen pictus

im Mittelwert 4,25 gegen 3,78 mm der Weibchen. Auch Kopstein (1941:164)

bemerkt, dass die Mannchen von pictus “an ihren wesentlich grõsseren Augen

leicht erkannt werden kõnnen.” Àhnliche Verhiiltnisse fand ich neuerdings bei

der athiopischcn Natter Philolhamus sentivariegatus, von der allerdings wenige

Stücke zur Verfügung standen. Laut mündlicher Mitteilung von Dr. A. R. Hoge

trifft dasselbe für den brasilianischen Chironius bicarinatus zu. In welcher Be-

ziehung dieser Gesehlechtsunterschied zur Lebensweise der Tiere steht, weiss man

nicht. Da der Geruehsinn für Baumschlangen von untergeordneter Bèdeutung sein

dürfte, ist es nicht ausgeschlossen, dass die grõsseren Augen der Mannchen zum

Erkennen der Geschlechter dienen. In anderer Weise erfüllen die gleiche Auf-

gabe verschiedene Formen der Schnauzenfortsátze bei den Mannchen und Weib-

chen von Langaha nasuta. Kampfende Mannchen scheinen unter den Baumsch¬

langen bisher nur bei Angehôrigen der Gattung Dendroaspis beobachtet

worden zu sein (Leloup 1964).

Über die Besonderheiten in der Fortpflanzung von Baumschlangen ist nichts

hekannt. Dass hei Baumnattern die viviparen Formen ( Ahaetulla Arten,

Lycodryas gainiardi) gegenüber den eierlegenden überwiegen, wie man es

aus naheliegenden Gründen erwarten würde, scheint sich nicht zu bestatigen.

So hezeichnende Baumnattergattungen wie Chry sopeie a und Dendrela-

p h i s in Asien, P hilot h a m u s , D i spholidus, Thelotorni s und

Dasypeltis in Afrika, Mimophis und Langaha in Madagaskar, Lep¬

tophis und Philodryas in Amerika sind Eierleger. Hingegen bringen

wohl alie Vertreter baumbewohnender Boiden und Oltern (A t h e r i s in Afrika,

Trimeres u r u s in Asien, Bothrops in Amerika) lebende Junge zur Wclt.

T rotz des Eingangs erwahnten Beispiels C o r allu s — Chondropython

kann es keinem Zweifel unterliegen, dass die Ãhnlichkeit mancher Baumschlangen

durchaus nicht immer auf Konvergenz zu beruhen braucht, sondem in vielen

Fallen ganz einfach auf Blutverwandschaft zurückzuführen ist, indem sich aus

einer baumbewohnender Art unmittelbar eine andere entwickelt hat. So dürften

z.B. die gemeinsamen, mil dem Baumleben zusammenhangende Merkmale aller

Arten der Gattung Ahaetulla sicher nicht dureh eine parallele Fntwicklung

jcdesmal von Neuem entstanden sein, sondem sind gewissermassen ein Ausdruck

der verwandschaftlichen Beziehungen der Arten. Dabei kõnnen die einzelnen

Anpassungsmerkmale selbst bei stark spezialisierten Arten eine recht verschiedene

Entwicklungshõhe erreiehen: Ahaetulla pulverulenta erscheint z.B. dureh die grau-

braune Gesamtfarbung primitiver ais die laubgrünen prasina und nasuta, aber

dureh den langen und feinbeschuppten Schnauzenfortsatz fortgeschrittener ais pra¬

sina und sogar ais nasuta. Ãhnlieh wie die Ahaetulla — Arten dürften die

Glieder innerhalb der Gattungen Leptophis , Uromacer, Oxybelis

oder Langaha zu heurlcilen sein. Auch eine ganze verwandschaftliche Gruppe

(Gattung) mil ihren strauch — oder baumhewohnenden Angehôrigen braucht

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 (1) :85-92. 1966

R. MERTENS

nicht unbedingl von einer bodenbewohnenden l rform abzustammen, sondem kann

ais Vorfahren bereits einen bezeichnenden Strauch oder Baumbewchner aus einer

anderen Gattung gehabl haben: Ahaetulla dürfle z.B. von einer Dryo-

p h i o p s — Form, C h r y s o p eles von einer Dendrelaphis — Forni

und Philothamus von einer Chlorophis — Form den Ausgang genom-

men haben.

Im Gegensatz dazu konnen jedoch nahe verwandte Arten vollig unabhangig

voneinander ahnlieh gevvorden sein Das zeigt uns das Beispiel gewisser Baumot-

tern unter den Crotaliden: Rothrops in Amerika, Trimeresurus in Asien.

Beide Gattungen stehen sich ganz nahe, so nahe, dass man sie verschiedentlieh

zu einer einzigen zusammengefasst hat, wie es zuletzt Parker (1965) gelan hat.

Und doch miissen die kleineren, meist griin gefiirhten Arten trolz ihrer Ahnlich-

keil von den entsprechenden Bodenottern ais ihren Stammformen in beiden Kon-

tinenten sicli selbstandig ausgehildet haben. Bei vielen anderen verwandschaft-

liehen Grnppen liegen indessen die Beziehungen weniger klar. Hat sich z.B. die

hispaniolische Baumnatter-Gattung U r o m ac e r von einer bodenbewohnenden

Stammform ausgehildet oder isl sie ein Abkõmmling einer anderen neotropischen

Baumnatter? Es lasst sich nicht bestreiten, dass sie ais ganzes ani meisten an

das Genus L e p t o p h i s erinnert, leilvveise aber aueh an die Nattern der Gat-

tung Oxybelis. Dass letztere opisthoglyph sind, würde nicht gegen das an-

gedeutete Verwandschaftsverhaltnis

Uromacer oxyrhynchus (ahnlieh

wirksames Gifl besitzt.

sprechen, da ja aueh zumindest der aglyphe

wie L e p I o p h i s ) ein für seine Beutetiere

Eine weitere Frage: bilden die Gattungen Ahaetulla und Thelolornis

engere verwandschaftliche Gruppe oder handelt es sich hier um einen Fali von

Ihre iiussere Ahnlichkeit ist unbestritten und kommt sovvohl

1 in der eigenartigen waagerechten Pupille und bis zu einein

Was jedoch die Pupillen-

Pa rallelentwieklung?

in der Kürperform wie

ge.wissen Grade im Abwehrverhalten zum Ausdruek

form betrifft, so kann sie im Laufe der Schlangen-Evolution mehrmals entstanden

sein: bei Dispholidus typus einer ebenfalls opisthoglyphen Art mit Hundpupille.

kann ais eine individuelle Variation die Pupille nach vorne ausgezogen sein (Filz

Simon 1935:320), ein bemerkenswerter Befund. den ieh bestatigen und aueh bei

Philothamus semivariegatus nachweisen konnte. Ais noch nicht gesichert darf

hingegen die friiher angenommene Verwandtschafl zwischen den athiopischen

Eierschlangen (Dasypei.tinae) und dem indischen Elachistodon westermarmi gel-

ten (Gans & Williams 1954).

Reich an ungelõsten Ratseln isl schliesslich die so überaus bemerkenswerte

Schlangenfauna Madagaskars. Da zur Eidechsenfauna dieser Insel bekanntlich

die sonsl anf die Neue Well und die Fidschi- und Tonga-Inseln beschrankle Fa-

tnilie der Leguane (IGUANIDAE) zahlt und aueh die Sehildkroten dureh eine Art

der siidamerikanischen Gattung Podocnem is vertreten sind, liegl der Gedanke

nahe, dass sich aueh bei den madagassischen Schlangen Beziehungen zur neo¬

tropischen Herpetofauna verhergen. Das ist in der Tat der Fali. und zwar im

Hinhliek auf die Kiesenschlangen (BOIDAE), deren madagassisehe Vertreter

(Acrantophis, Sanzinia) den amerikanischen, ebenfalls überwiegend

baumbewohnenden Gattungen Boa und Coral lus zumindest sehr nahe stehen,

wie man es seit langem weiss. Unter den Baumnattern sind für Madagaskar 3

Gattungen bezeichnend, die ja alie zu den Opisthoglyphen zahlen: Mimophis.

Lycodry as (inel. Stenophis ) und Langa ha. Ihre verwandtschaftli-

ehen Beziehungen sind noch ganz ungeklart. man mochte sie aber ebenfalls unter

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UNTERLAGEN ZUR ÜKOEOGIE, ETHOLOGIE UND EVOLUTION DER

BAUMSCHLANGEN ARBORICOLOUS SNAKES...

6 .

8 .

i Nallern des tropischen Amerika sucheu: vielleichl unter deu Angehorigen

i Leptodeir a für Lycodryas und Oxybelis für Langa ha.

[ jeden Fali ware die Klarung der stammesgeschichtlichen Beziehungen der

ílangen Madagaskar’s für einen Morphologen eine sehr dankbare Aufgabe.

SCHRIETE \

Fitz Simons, V. — Scientific results o£ the Vernay-Lang Kalahari Expedition,

March to Sept., 1930. Ann. Transvaal Mus., Cambridge, lfi:295-397, 1935.

Gans, C. & Williams, E. E. — Present knowledge of the snake Elachistodon

westermanni Reinhardt. Breviora, Cambridge, Mass., 36, 1954.

Kopstein, F. — Uber Sexualdimorphismus bei maiaiischen Schlangen. Tem-

ninckia, 6:109-185, 1941.

Leloup, P. — Observations sur ia reproduction du Dendroaspis jamesoni kaimo-

sae (Loveridge). Buli. Soc. r. Zool., Anvers, 33:13-27, 1964.

Mertens, R. — über áussere Geschlechts-Merkmaie einiger Schlangen. Sencken-

bergiana, Frankfurt a.M., 19:169-174. 1937.

Mertens, R. — Die Warn-und Droh-Reaktionen der Reptilien. Abh. senckenb.

naturf. Ges., Frankfurt a.M., 471, 1946.

Mertens, R. — The World of Amphibians and Regtiles. London, Harrap & Co.,

1960.

Parker, H. W. — Natural History of Snakes. London, British Museum Nat.

Hist., 1965.

Peters, J. A. — The snakes of the subfamily Dipsadinae. Misc. Publ. Mus.

Zool. Univ. Midi., Ann Arbor, 114, 1960.

6 .

8 .

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 93-99, 1966

AIIARON SHULOV

12. BIOLOGY AND ECOLOGY OF VENOMOUS ANIMALS IN ISRAEL

AHARON SHULOV

The Hebrew University of Jerusalem, Israel

The variety of elimatic conditions in lhe land of Israel ranging from 1,000

meter high hills rich in rainfall to dry deserts and lhe Dead Sea valley situated

deep below the sea levei provides differenl hiolops for a very wide spectrum of

animal life. In addition ihis tiny plol of land situated in the meeting point of

Asia, África and Europe serves as a passageway for many migrating animais

and hirds. Nevertheless the zoogeographical analysis of the animal population

of the land of Israel shows quite a high pereentage of endemic species. Some

of them are venomous.

The knowledge ahout the venomous animais of the land of Israel originates

from the passages of the Old Testament in which references were made to venomous

suakes, scorpions and spiders either descrihing the fatal results of their hites or

as a warning.

Over the ages, information ahout the appearanee and the way of life of these

ereatures and the methods of treatment of ])oisonous hites was accumulated in

Talmudic and Jewish Medieval Literature. Hovvever these references were as a

rule commentaries to the passages of the Bihle without adding any new knowledge.

Although some of the dangerous ereatures mentioned in lhe Bihle became

through the world wide use of the Rible. fabulous and mystical heings, never¬

theless in zoologists living in and visiting lhe land of Israel succeed in identifying

at least some of them.

This reporl deals only with terrestrial venomous ereatures particularly wilh

suakes. scorpions and spiders.

I. SNAKES:

Of seven poisonous snakes in Israel only one is of importance; this is the

Palestine viper, Vipera xanthina palestinae. The others, Echis colorala, Pseudo-

cerastes fieldi, Aspis cerastes, Aspis vipera as well as the Black cobra, IValterin-

nesia aegyptia, and Ein Gedi mole viper, Atraetaspis eingaddensis, are comparative-

ly rare and live only in remole and scarcely-populated areas of lhe Negev — the

Southern part of lhe land of Israel.

The Palestine viper which is the Bihlical “Zerfa” is translated in lhe King

James version as Basilisc. Il is lhe most common Israeli poisonous snake, the

distribution of which is connected with the Mediterranean zoolgeographical regions

of Israel. lt seems lhat the highest concentration of this snake is in the Coastal

plain of Sharon where the most intensive agricultural cultivation is taking place

with consequenl abundance of rural rodents on which this viper preys. Its

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10 11 12 13 14 15

BIOLOGY AND ECOLOGY OF VENOMOUS ANIMALS IN ISRAEL

maximal length is 120 cm. This viper is frequenl also around the fish ponds

where il fcerls on fish. Tliis snake lives in hurrows of rodents, and is as a rule

nocturnal. However, in winler il can be mct during lhe warm days when bast-

ing in the sun. Il is sluggish in movements and does nol slrike as a rule when

not provoked. Many cases are rcportcd about lhese snakes being found and held

in lhe hands by ignorant adults and children who wcre not bilten by the snake.

as they treatcd it gently. However this snake is responsible for more than 99%

ol all ophidian liites in Israel. The Palestine viper is oviparous and. the hatch-

ing of the eggs takes slightly more than a rnonth and a lialf at the lemperature

of the Coastal plain. The yonng vipers have already developed a venomous ap-

paratus and are able to kill their prey.

The seeond snake in order of importanee is lhe saw-sealed sand viper, Echis

colorata. It is smaller than the Palestine viper, nol exeeeding 40-50 em. Its

basie color varies from yellovv lo reddish rose with distinet oval markings in

darker frames. It is found in and around the Dead Sea and along the Jordan

and Arava valley, reaching the eastern slopes of the Judean and the (iilboa hills

bordering the Jezreel valley. This snake inhabits stony areas with very scaree

vegelation. being found under stones. As a rule, it does not live in hurrows.

Il is very quick in its reaetions, striking often instantly when approaehed and

is therefore popularly believed more dangerous than the Palestine viper, allhough

the comparison of the lelhal dose of this snake venom with that of the Palestine

viper shows that the venom of the Palestine viper is almost twiee as strong as

that of Echis. Il preys on small rodents, reptiles, oecasionally on young desert

birds. Its diet eonsists also of some inseels. Il is oviparous, the development

of eggs takes at 3T’C. 43 days.

The Pseudocerastes fieldi vipers are yellow ish-grey in colour. On each side

of lhe body there is a row of light hrown rhomhoid hlotehes and the ventral

side of it is pure white. The length of lhe body ranges between 60 lo 90 em

and the weight of a well fed adult speeimen may reaeh 500 gm. All specimens

of this viper have “horns” eonsisting of small seales ahove lhe eyes and are easily

reeognized by the black tip of their lails.

Pseudocerastes vipers live in areas of the sandy soil with stretches

of hard ground and a eertain amount of vegetation. It seerns that they prefer

spaeious hiding places under stones and rodents’ hurrows. Thcse vipers are found

quite often among vegetation during daylight allhough they live on small rodents.

birds and lizards. Il seerns that they feed also on already dead animais and

birds. In Israel, this snake is found in Jordan, Sinai, lhe central pari of the

Neger with the main population in the Nahel Ramon and northern Arabia.

Copulation has heen ohserved in May and June. The number of eggs in a

eluleh ranges between 14 lo 21. As the eggs are laid in more advanced stages

than in Echis colorata and Vi pera xanthina palestina, they develop relatively rapid-

ly hatehing in 30-31 days at 31°C. The duration of development until the adult

stage is nol yet known.

The venom of Pseudocerastes is quite a strong one and among lhe

Israeli venomous snakes, it is seeond oídy to that of lhe black cobra W al¬

ter itine si a. There are no authenlic reports regarding lhe influenee of this

snake’s bi te upon human beings.

The horned sand viper. Aspis cerastes is smaller than Pseudocerastes.

being 50-70 em long and 100 gms in weight. Its colour is yellow with hrown

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Mem. Inst. Butantan

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AHARON SHULOV

and grey markings. The speeimens found in Jsrael are hornless in contrast to

the horned ones found in Norlh Afriea. the Arah península, Iraq. Sinai and

Jordan.

It is found in sandy areas of the Arava valley. This snake hurrows itself

into lhe sand by sidewise movements of its ribs so that only its head and the

nostrils remain above the surfaee. Sometimes the whole hody is hidden in the

sand. It seems that in this position it is amhushing its prey. However its

permanent ahode is usually lhe rodents’ holes in which it exhihits the same

hurrowing habit.

The horned viper feeds on mice and lizards. It is oviparous. The female

lays up lo 20 eggs which liatch after more than two months in summer. The

period of development until maturity lasts year for males and 214 for females.

The venom of the horned viper seems to he quite strong. However, no

authentic records of causalties have yet been reported in this land.

The Aspis vipera is the smallest of the sand vipers, reaching 30 cm in length

and 50 gm in weight. It is the plumpest of all the venomous snakes of Israel.

Its colour is similar to that of the horned viper. being yellowish wilh brown

spots. It is possible to distinguisb between males and females of this species by

the hlack tip of the tail of the latter.

The distribution of the Aspis vipera is similar to that of the horned viper.

In Israel, it has heen found only in the sandy areas bordering Sinai, being

sympatric wilh the horned viper.

This viper seems lo he very well adapted to life in sand. Its ahility lo bur-

row itself in the sand is very well developed. When alarmed, instead of running

away it hurrows deeper into lhe sand. Like the horned viper. it digs itself vertical-

ly into the sand.

No authentic reports on bites in human beings have so far heen published.

The hlack cobra ( Walterinnesia aegyptia) is the only Elapid snake found

in Israel. It is quite vvidely distributed in the south of Israel without reference

of any type of soil or vegetation. It ranges from Egypt to Iran in lhe deserl

and steppe areas. As a rule its length doesnT exceed one meter. Its color is

shiny hlack. This fact causes some trouble when the necessity arises to distinguisb

between the venomous hlack cobra and the harmless hlack coluhrid snake ( Colu-

ber jugularis) in the border areas of their distribution. As a rule the hlack

cobra does not appear farther norlh than a few miles north of the city of Beer-

sheva, whereas Coluber jugularis does not occur to the south of this town. The

other useful difference is in the hody length — that of the hlack coluber is more

I han one meter.

The hlack cobra is a nocturnal animal and is a subterranean dweller, whose

ahility to burrow can easily be detected by the form of its head and the smooth-

ness of its scales. Its sight is very poor and it locates its foods by smell only.

It preys on a variety of creaturcs such as frogs, toads, mice, small birds and

various reptiles. ll also eats dead animais and even those already decomposing.

It likes to drink water and it is found many times in humid places near or within

settlements.

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BIOLOGY AND ECOLOGY OF VENOMOUS ANIMALS IN ISRAEL

There are no authentic reports regarding reproduction in this snake. How-

ever, many papers have been devoled lo lhe properties of its venom.

There are some lethal accidents attributed lo Cohra biles in Sinai and Egypl

Inil there have been no casualties in Israel, although several eases of hites were

reported.

The Ein-Gedi Mole-Viper was discovered only in 1944 in lhe oásis of Ein-

Gedi, situated on the western shore of the Dead Sea. Il belongs to lhe Mole-Vipers

of the genns Atractaspis living in several regions of tropical África. Atrac-

taspis engaddensis has since been reported from other places in the Negev and

in the Sinai península.

Il is a typical burrowing snake with a small head, withoul any marked cons-

triction between the head and the body. Its color ranges from black lo brown,

lhe eyes being very small and lhe sight exlremely poor.

The effect of the venom on human being and animais is neurotoxic. How-

ever, certain hemorrhagic effects exvasations have also been observed in aulopsies

of laboratory animais killed by ihis snake. No exact chemical and toxicological

data regarding ihis venom have yel been published.

The relative toxicity of venom of 5 Israeli snakes is presented in the table I.

The comparison was carried out by injeeting dissolved venom subcutaneously into

white mice 14-16 gr in weighl. A similar investigation carried ont with fresh

pooled venom of lhe same snake gave similar results. However the differences

in the strength of venom do not reflect the degree of potential danger of lhese

snakes as the main factor in tliis respect would be the probabilily of human beings

meeting wilh bearers of the different venorns. In this respect the Palestine Viper

is important being responsible for almost aII'cases of ophidian bites in the

population in Israel.

II. SCORPIONS:

In comparison lo snakes, the scorpions are considered less dangerous. How¬

ever. it appears that in Israel as well as in México and in Algeria the number

of casualties caused by scorpions is at least equal to the number of deaths caused

by snakes. The lack of knowledge ahout the potential danger of the bite of a

scorpion quite often causes delay in Irealment and leads lo lethal consequences.

There are some 12 species and subspeeies of scorpions already described

from Israel. Stings of al least two of which may cause death.

lt is interesting to mention that lhe use of lhe poisonous sting by scorpions

is not necessary connected with lheir feeding. As a rule a scorpion does not

start using its sting when hunting. It does it only when lhe prey is too hig

and il cannol be crushed wilh pedipalps enabling sucking and feeding on it.

Therefore the scorpion stings only when treaded on or otherwise seriously disturbed.

Similarly to snakes there is a wide range of differences in the strength of scor-

pioiTs venorns with regard to their potential danger to human beings, ranging

from almost harmless species to lhose whose sting mighl be fatal. The amount

of venom injected at each sting varies considerably and its influence fluctuates

according to the part of the body stung, to the proximity of the spot of sting

to nerves or blood vessels, and many other conditions. Many other factors in-

íluencing the effect of the sting depend on the scorpion. Among these, secondary

2 3

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Mem. Inst. Butantan

Simp. Internac.

33(1): 93-99, 1966

AIIARON SHULOV

only lo the specific qualities of ils venom is llie fact lhat according to observations

based ou thousands of specimens, the pointed lip of the sting is frequently hroken,

thus enahling its bearer lo sting lhe soft-skinned prey hut rendering such a sting

completely harmless lo human heings.

The most dangerous of the scorpions of Israel is the common yellow scorpion

Leiurus quinquestriatus H. et E. It is quite widely distributed, ranging from

North África through the Arah península and the countries of lhe Levant to

Turkey and Pérsia. In Israel il is the most common species of scorpion. ranging

throughout the country with the exception of lhe Coastal plain vvhere il is replaced

by the hlack scorpion Buthotus judaicus E. Sim. In lhe Medilerranean pari of

lhe Judea and the Gallilee, both species appear lo he sympatric. In the hilly

region appears as a rule the biggest scorpion of lhe Near East Ncho hierochonticus

E. Sim. which reaches 16 cm length. Two species of lhe genus Prionurus cras-

sicauda Ol. and bicolor H. et E., are foimd in isolated groups mostly in the bilis.

In the Neger, lhe Southern part of Israel, there they occur togelher with Leiurus

quinquestriatus, the small hlack desert scorpion Orlhochirus Innesi F. Sim. as well

as with endemic Buthus occitanus. Mardochci var. Israelis Shuiov et Amitai which

is foimd only in few isolated localities.

Although as a result of intensive collection we now have considerahle know-

ledge about the dislribution of scorpions in Israel it would nevertheless be dif-

ficult lo descrihe exactly lhe ecological habitais of each of the species mentioned

above, especially in localities where as many as four species are foimd. It may

he stated in general, lhat Leiurus quinquestriatus inhahits dry stony calcareous

ground, often prefering the Southern and eastern slopes of the hills. The Judean

scorpion is foimd under stones in the Medilerranean region to the west of the

watershed of the Jordan and Mediterranean water systems. Sometimes il is also

foimd under the bark of trees. Orlhochirus Innesi is found under small stones

on light soils. The burrows of some of the scorpions are highly characteristic

and can be easily identified especially those of Scorpio maurus pulmatus Seurat

and Scorpio maurus juscus H. et E. as well as the typical entrance under a stone

to the burrow of Nebo hierochonticus E. Sim.

All the scorpions of this country appear to be nocturnal, their activity being

directed by a biological clock. Only sporadically, a scorpion may be foimd dur-

ing the day apparently disturbed from its ahode. They seem lo he active through

the whole year with the exception perhaps of the coldest days of the winter in

the hill region. During the hottest part of lhe summer they burrow deep into

their retreats or remain under large stones and in the crevices of rocks. The

copulation of three species of scorpions ohserved in our laboratory almost simulta-

neously with the observations made in Germany, South África, Brazil. and Uruguay

show quite an elaborate process of transfer of lhe spermatophore previously formed

within a eomparatively short period within the hody of the male. The maturation

of the eggs before ovipositiou within one hody of the scorpion as described by

Pavlovsky as early as 1923, revealed the possibility both of viviparity and ovo-

viparity in various groups of scorpions. According to our observations the period

of development of local scorpions rangei! between 6-7 years with the exception

of the small 0 r tho c h i r u s where this period may be much shorter. The

period of reproduction and appearance of the young scorpions on the back of

their mothers is July-August. The experiments carried out in our laboratory

showed quite a wide range of toxicity of various local scorpions. The results

are presented in the table.

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BIOLOGY AND ECOLOGY OF VENOMOUS ANIMALS IN ISRAEL

III. SPIDERS:

llnlike definite indications regarding potential danger of snakc and scorpion

biles is no direcl reference of this kind in lho Rible regarding the spiders. Only

one problematic passage about the anger of an unidentifiable creature named

Achshuv may bear somo relation lo spider venom.

It is remarkable that in sucb a liny country as Israel tbree different species

of Latrodectus spiders have beeh found, as well as a few specimens of

Latrodectus mactans possibly recently introduced. The Karakurt palaearctir: species

of Latrodectus tredecimguttutus is found all over the country, reaching in several

localities of the northern Negev and Arad, a density of almost one adull female

spider per square meter. As a ride its retreal is situated under médium size

slones from which a corridor-like shiny threads reveal the presence of a live

spider. It preys mostly on beetles of the family TENEBRIONIDAE. Otber in-

sects as grass-hoppers, crickets and bugs are also found. It is worth mentioning

the small SOLIFTJGA and small and médium size scorpions that are also found in

its snares. In spring lhese spiders are found together vvilh numerous eocoons

eontaining each up to 500 eggs or already hatched spiderlings. The cycle of

development of the female spider depends on the supply of food and extends

from one to l\vo years. The development of a male spider is much shorler and

takes a couple of months only. Despite its abundance, there are only a few

records of lliis spider’s bites and among them only one willi a seientifieally proved

lethal result.

The second Latrodectus spider originally recorded from this country

and later also from lhe Arab península is Latrodectus pallidus. This spider was

found in quite dense population in several localities in the valley of Jesreel south

of lhe city of Beer-Sheva and along the Coastal plain.

It feeds mainly on ants and for this purpose it builds its snare in a peculiar

way, with threads extending as a rule hetween two or tbree shrubs at the height

of 40-60 cm. This thread bridges over the path of the ants, and the spider

catches them by descending on a thread from above, seizing its vietim and lifting

it through lhe bridge into its ahode. The spider’s retreat consists of a peculiar,

very elaborate strueture at the highest point of which is a small thumh like

strueture in which the spider sits awaiting its prey or digesting. The venom of

this Latrodectus species is comparatively weak, although it may cause dcalh

to white mice as well as to field mice. Occasionally insects other than ants can

be found in its snare.

Latrodectus revivensis is anolher species which up lo presenl has been de-

scrihed only from the land of Israel. Its general appearance resemhles that of

lhe Latrodectus Iredecimguttatus and it can he distinguished from it by lhe form

of lhe hairs covering its abdômen and on close observation, by the general hue

of lhe body which ranges from dull black lo heavy brown with exceptionally

occuring light coloured specimens. The markings and lhe colour of the young

spiders are com|)letely different from these of the other Latrodectus species.

The adull male retains its peculiar markings, hut the adidt female loses all ju-

venile designs and hecomes dark as descrihed above. The snares of Latrodectus

revivensis are similar to a certain extent to lhese of Latrodectus pallidus hut the

reatreat is much shorter and broader and lhe whole snare is situated obliquely

in contrast to lhe almost vertical position of lhe snare of Latrodectus pallidus.

Its height is 35-40 cm above the ground. The food of Latrodectus revivensis is

2 3

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Mem. Inst. Butantan

Simp. Internac.

33(1): 93-99, 1966

AÍ1ARON SHULOV

similar lo lhal of lhe Latrodectus tredeciinguttatus and all investigaiions carried

onl willi dry and fresh venom of both species indicate that lhey are similar if

not equal. No study both of pallidus and revivensis venom lias yet been carri¬

ed onl.

Hecently several specimens of Latrodectus mactans bave been found along

the eoastal plain and it is suggested lhal they have been introduced by immi-

granls from lhe American continents.

Several cases of bite witb quite severe symptoms have been reported as a

resull of the bite of the common honse spider Loxosceles rufescens found frequently

in houses, eellars and caves, investigations carried onl on the white mice sbowed

symptoms of neurotoxic envenomation, which seem to be similar to those described

for the bite of some spiders of Loxosceles genns described in South America

but not showing any kind of histopathological effects as described for there.

Sporadic observations carried out on a specimen of Hagna narbonnensis lyco-

sid spider showed a low degree of venom neurotoxic influence but clear histolytic

and vasolytic effects around the location of bite.

Altbough it seems quite amazing that such a small land as Israel, barbours

so many venomous creatures, it must however, be kept in mind that in addition

to its geograpbical position as a meeting point of tbree continents the intensive

zoological research carried out here by scienlists both driven by an interest in

the creatures mentioned in the Bible as well as fostered by secular scientific

interest made the land of Israel one of the most studied countries of the entire

globe.

Bibliography

1. Mendelssohn, H. — On the biology of the venomous snakes of Israel I. Israel

J. of Zoology, 12:143-170, 1963.

2. Mendelssohn, H. — On the biology of the venomous snakes of Israel II. Israel

■I. of Zoology, 14:185-212, 1965.

3. Shulov, A., Weissman, A. & Ginsburg, H. — Observations on the lyophilized

venom of the Egyptian Black Snake Walterinnesia aegyptia. Harefuah, 55:54-

57, 1958.

4. Shulov, A. & Weissmann — Notes on the life habits and potency of the venom

of the three Latrodectus spider species of Israel. Ecology, 40:515-518,

1959.

5. Shulov, A., Flesh, D., Gerichter, Ch., Eshkol, Z. & Shillinger — The anti-scor-

pion serum prepared by use of fresh venom and the assesment of its efficacy

against scorpion stings. Fifth International Meeting for Biological Standard-

ization, Jerusalem, pp. 489-492, 1959.

6 Shulov, A. & Amitai, P. A. — Key to Israeli Scorpions. Tera veavez, 5, 1960.

7. Swarocrp, S. & Grab, B. — Snake bite mortality in the world. Buli. Org. mond.

Santé, 10:35-76, 1954.

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33 ( 1 ): 101 - 103 , 1966

K. KLEMMER

101

13. OBSERVATIONS ON THE BIOLOGY OE SEA SNAKES — HYDRO-

PHIIDAE — WITH REMARKS ON THEIR SYSTEMATICS

K. KLEMMER

Natur-Museum und Forscliungs-Institut Senckenberg, Frankfurt, Germany

Sea snakes are proteroglyphous snakes with vertically flattened tail. I gave

this provisional definition because we have just heard from Dr. Iloze this morning

that lie regards some genera, or at least one genus, of the sea snakes as belong-

ing to the ELAPIDAE. Well. all sea snakes, we are snre, have ancestors among

the old primitive stock within lhe Elapids. It is also snre that they have this

stock in the south-eastern Aegean region. All lhe recent species within lhe sea

snakes, nearly all of the 50 species known, can be found in the sea, only in lhe

Indo-Pacific Ocean, not in the Atlantic. As you know some of the species often

penetrate the mouth of the rivers, living in the brackish water, but only two have

become inhabitants of fresh water: one species in the Tahal-sea on the Philip-

pines and another one on the Renon Island on the Salomons. We know that

the sea snakes are poisonous snakes and have a very effective venom. The oídy

important ohservations we have had, however, in the last few years, were made

by Saint Girons in New Caledónia, who is with ns this evening. In the last

seven years we had in Frankfurt the opportunity to import some specimens of

living sea snakes, mainly of the genus Laticauda laticauda laticaudata, Laticauda

colubrina, Laticauda semifasciata and Lapemis hardwicki. The main prohlem in

keeping sea snakes in captivity is to find how to feed them. When the first sea

snake reached us, we offered all smaller fish species available from lhe Cerman

sea, but none of these fishes arose the slightest interest on behalf of the sea

snake. B l e n i s , D o b i s , viviparous B l e n i s , for example, lived together

in the aquarium with the sea snake for weeks without being swallowed or even

bitten. This changed rapidly when we were able to offer eels, the common

European eel, Anguilla anguilla. The first eel is put into the tank for the sea

snake and immediatcly the snake begins searching movements and when the snake

meets lhe eel, lhe fish is immediately bitten, but suddenly released. After the

sting of the sea snake, it is quitened down. It was never possihle to provoke an¬

other hite of the sea snake on the eel or on any other fish within the next ten

to fifteen minutes. The eel becomes motionless within 3-10 minutes, depending

on lhe weight of the eel and the place where the snake has bitten and then after

a quarter of an hour or even later the snake begins seeking again for the eel

and swallows it, head first usually. Sea snakes are highly specialized in their

diet, we know this from new investigations on the stomach contents of the freshly

captured sea snakes in the Indo-Pacific on the Javan coast, espeeially. But usually

we cannot find any stomach content in sea snakes, although the specimens have

heen killed immediately after capturing. From about 450 sea snakes Bergmann

has studied in Java, he only could find in three of more than 400 specimens,

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102

OBERVATIONS ON THE BIOLOGY OF SEA SNAKES -

HYDROPHIWAB — WITH REMARKS ON THEIR SYSTEM ATICS

rests of their food. Very recent sludies made by Worries, showed us that sea

snakes seem to be more specialized than we could imagine before. He found

tliat sea snakes seem lo be more specialized than we could imagine before. He

found that sea snakes of the genus Emydocephalus feed exclusively on fish

eggs. very small eggs. This species also has a very small head and a slender

neck and then a relatively heavy hody. We do not know what lliis species vvill

do with its venom apparatus, as fangs are hardly used for prolection against

larger enemies. Usually sea snakes are very docile. can he handled without much

danger, and il is only reported from some specimens of sea snakes from the

Malayan coast that sometimes will bile when handled.

Sea snakes find lheir prey chemiotectedly, only they do not react on fasl

movements witliin lhe range of their heads, but they react quickly if Chemical

substances, let’s say. lhe smell of the eel, in lhe Laticauda snakes, is brought

into the aquarium and yon can easily provoke lasting search movements of sea

snakes by adding some water from the tank where the eels have been kept. We

had the luck in finding out that Laticauda specimens and also La pernis

can be kept on a diet of eels exclusively. In their natural habitat, the European

eel does not occur, but there are olher species of genus Anguilla and probably

other fish with similar smell. And thus we could keep Laticauda specimens

for more than 5 years in captivity in relatively primitive aquariae, with only

artificial sea water. which had lo be changed very rapidly, and a plate of cork

or wood above the sea water to give the snake the possibility to come out, and

a brick bole within lhe water to give some proteclion to the snake. We could

do also some observations on egg laying in Laticauda and on the skin shed-

ding. Laticaud a is an egg laying genus and in the one female, we kept

for five and a half years, every year 3 eggs were laid, sometimes only 2 eggs,

sometimes 4. The eggs are laid within two days, up to a week, and it was very

curious lo observe that when the snake was laying eggs, it always shed its skin.

This was regularly combined after having laid the first one, sometimes the second

egg; the skin shed and lhe sea snake was found very often outside the water,

but il can also be found there when the skin is shed without laying eggs. The

L a t i c a u d a egg is relatively large, very elongate. The eggs could not hatch.

because we had at this time only a single female of Laticauda laticauda lalicau-

data. The shedding of the skin takes place 4 to 6 times a year. This is com-

pared with terrestrial snakes of similar size, which also show similar frequency

in skin shedding. This is in strong contrast with observations done on the very

advanced sea snake, Pelamys platurus, the only real open sea inhabitant species

and the only one which has crossed the Pacific and is now inhabiting the western

coast of the Américas. In Pelamys the skin sheds very often, more than 15

limes a year, that means less than 4 weeks. Observations done by Shore at

the San Diego Zoo showed this, and we can explain it also as an adaptation for

living in lhe high sea. The skin of the sea snake is often infested by barnacles

or other sessile marine invertebrates, and by shedding the skin regularly, the

snake gets rid of barnacles and other such animais. All observations are done

in adidt specimens.

The sea snake family is usually divided into two subfamilies, the more

primitive one with the genus Laticauda, often mentioned, and with Ai-

pysurus and Emydocephalus. These are egg laying forms as far as

we know. The supporting musculature is not reduced or hardly at all; they

often come to the shore or are found on land since they have to go on land to

deposit their eggs. All other genera and species, 37-40, this depends on the

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Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 101 - 103 , 1966

103

taxonomic decisions, belong to the more advanced subfamily: lhe Hydrophiinae,

which are oviviparous. The more frequent shedding of the skin in the Hydko-

phiinae, data from the biology of the sea snakes, support ihis usual systematic

arrangement which has heeu drawn from morphological data. especially from the

morphology of the skull. I, for the moment at least. will not go so far as Dr.

Roze has proposed this morning, to give the Laticaudlxae the status of an own

family or to incorporate them into the terrestrial ELAPIDAE. Within the ELA-

PIDAE we have also an aquatic genus, Boulengerina, in Central África,

but in fresh water, living Elapids we can not see any adaptations towards all

sea snakes living in the sea.

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33 ( 1 ): 105 - 114 , 1966

H. SAINT GIRONS

105

14. LE CYCLE SEXUEL DES SERPENTS VENIMEUX

H. SAINT GIRONS

Laboratoire d’Ecologie, Museum National d’Histoire Naturelle, Paris, France

] NTRODUCTION

11 existe, éparses <)ans une littérature abondante, de nombreuses données oc-

casionnelles sur les dates de ponte ou de gestation et de ])arturition des Serpenls

femelles, mais bien peu d’études donnent des renseignements complets sur le cycle

sexuel d’une espèce particulière; mêmes les observations de dates daccouplement

sont généralement rares. La situation est encore plus mauvaise en ce qui concer¬

ne les mâles, puisque 1’étude de la spermatogenèse et des caractères sexuels se-

condaires internes exige la mise en ceuvre de techniques histologiques sur des

pièces convenablement fixées au préalable.

D autre part, on ne peut réellement différencier, du poiut de vue de la re-

production, les COLUBRIDAE des ELAPIDAE, Nous serons donc amené à faire

état d’observations concernant le cycle sexuel de Couleuvres.

Le cycle sexuel dans les regions temperees, a latence hivernale

DUE AU FROID

VlPERINAE :

Chez Vipera beras (Volsoe, 1944; Sinith. 1951) et ehez Vipera aspis (Rolli-

nat, 1934), dans les conditions normales les femelles commencent à s’accoupler

peu après la fin de 1’hivernage, le plus souvent durant la deuxième quinzaine de

mars et 1’activité sexuelle se poursuit durant le mois d’avril. La plus grande

partie de la vitellogenèse s’effectue au printemps et 1’ovulation a lieu à la fin de

mai ou au tout début de juin. La gestation est estivale et les dates de partu-

rition s’étagent, selou 1’espèce et la localité, entre le début d'aoúl et la mi-septem-

bre. Chez Vipera aspis, il existe une deuxième période d’accouplement à la fin

de septembre, à laquelle toutes les femelles ne prennent pas part.

Dans les régions plus ou moins proches de la limite nord de 1’aire de dis-

tribution de 1’espèce, ou en montagne, un pourcentage croissant de femelles pre¬

sente un cycle sexuel biennal, voir même tri- ou quadriennal (Vipera berus :

Vainio, 1932; Volsoe, 1944; Saint Girons et Kramer, 1963. Vipera . aspis: Saint

Girons, 1957; Duguy, 1963). Les figures 1 et 2 donnent une idée de ce type

de reproduetion. 11 convient de remarquer que la date de 1’ovulation n’est pas

modifiée. Au contraire, dans ces zones oü les Vipères souffrent d’un déficit

thermique, la parturition a lieu nettement plus tard. Dans quelques cas excep-

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106

LE CYCLE SEXUEL DES SERPENTS VENIMEUX

lionnels, la geslation peut se prolonger jusqu’au printemps suivant. La vitello¬

genèse a toujours lieu. pour la plus grande pari. au printemps de 1’année de

reproduction.

Le cycle sexuel des mâles est toujours annuel. La spermatogenèse correspond

au Lype dit '‘mixte”, c’esl à dire que la spermatocytogenèso a lieu en élé et la

spermiogenèse au début du printemps, juste avant 1’accouplement; l’hiver corres¬

pond à une période d arrêl du développement de la lignée séminale, la majorité

des cellules étant au stade de jeunes spermatides. Toutefois, ehez Vipera aspis,

Ia spermatogenèse tend à être continue, avec deux périodes de vive activité, la

plus longue de juillet à octobre, la plus courte en mars el avril. Dans les deux

cas, il existe un stade de semi-repos en juin. Chez Vipera beras, les caracteres

sexuels secondaires el notamment le segment sexuel du rein présentent un maxi-

mum de développement au moment de 1’accouplement vernal, mais ils ne sont

atrophiés qu’on juillet. Chez Vipera aspis, le stade. de grand développement est

atteint dès le mois de septemhre, lors de 1’accouplement automnal.

Crotai.inae:

Aux Etats Unis, hon nombre de Crolales femelles semblent avoir un cycle

annuel, analogue à celui qui a été décrit chez les Vipères européennes (voir

Klauber, 1956, pour la bibliographie). Toutefois, les observations d’accoup!e-

ment sont relativement rares et, surtout, difficilemenl interprétables car elles s'é-

tendent sur la plus grande partie de la vie active, à 1’exception du mois de juin.

II existe cependanl deux périodes préférenlielles, ITine en mars-avril, 1’autre en

aoüt-septembre. Corame la gestation dure en général jusquYn septemhre, il est

probable que heaucoup d’accouplements d’automne sont le fait de femelles avant

un cycle biennal.

En effet, chez plusieurs espèces ( Crotalus viridis viridis, Rahn, 1942; C. v.

luiosus, Glissmeyer, 1951; Crotalus atrox, Tinkle. 1962), 1’étude de populalions

captureés dans les abris dhivernage montrc que les femelles peuvent se classer

en deux catégories, l’une composée de post-parturienles à petits ovules, 1’autre

(Lanimaux pourvus de gros ovules el qui ne se sont pas reproduits 1’année pré-

cédenlc. Chez C. v. viridis, ce dernier groupe a des spermatozoides dans le tube

vaginal et scmble s’être accouplé Fété précédent. Chez C. v. oreganus el C. v.

luiosus, Fitch, 1949 et Glissmeyer, 1951 estimenl que la copulation a lieu au

printemps seulement. II cn est de rnême chez Agkistrodon contorlrix (Fitch.

1960); de plus, 1’élude de spécimens caplurés durant la saison active prouve

que, chez cctte espèce com me chez les Vipères européennes, la majeure partie

de la vitellogenèse a lieu au printemps qui précède 1’ovulation, hien que la fin

de 1’hivernage soit tardive. Au contraire, chez Crotalus viridis el chez C. atrox.

la vitellogenèse se situe l’été oii les animaux ne se reproduisent pas (fig. 2).

Chez les Vipères, comrne chez les Crotales, 1’étude des corps gras montrc

que, dans les populations ayant des cycles hiennaux, les femelles n ont pas le

temps de reconstituer leurs réserves au cours d’une seule année, sans doute en

raison dTine température trop hasse qui ralentit la vitesse de la digestion. Cerles,

Faetivité des enzymes digestives à une température donnée est un caractere spé-

cifique. mais à la limite septentrionale ou altitudinale de la ire de répartition,

un poinl critique est toujours atteint. La baisse de fécondité qui résulle d’une

reproduction aussi espacée explique clairemenl l’un des mécanismes de la limita-

lion de 1’habital d une espèce.

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Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 105 - 114 , 1966

lí. SAINT GIRONS

107

II ídexiste aucune donnée precise sur révolution des caracteres sexuels secon-

daires et de la lignée séminale chez les Crotales. Toutefois, Fitch (1960) signale

la présence, durant toule 1’année, de spermatozoides dans les canaux déférents de

Agkistrodon contortrix. II en est de même chez Vi pera aspis, ce qui est un ar-

gumenl en faveur d’un cycle spermatogenétique voisin.

A u t r e s S e r p e n t s :

Aucun ELAPIDAE ne vit dans les régions tempérées de I hémisphère horéale.

Chez plusieurs COLUBRIDAE d’Europe et des Etats Unis (voir, entre autres, Rol-

linat, 1934; Blanchard et Blanchard, 1940-41; Carpenter. 1952; Peter-Rousseaux,

1953; Fox, 1952 et 1954), le cycle des femelles et révolution des caracteres

sexuels secondaires des males sont analogues à ceux qui onl été décrits chez les

Vipères. Mais la spermatogenèse est toujours estivale, de type post-nuptial, les

spermatozoides étant stockés durant 1’hivernage dans les canaux déférents. On

connait également quelques exemples de cycles biennaux chez des espèces vivipares,

notamrnent des T h a m n o p h i s .

Le problème des ELAPIDAE australiens sera évoqué plus loin.

Le CYCI.E SEXUEI. DANS LES RÉGIONS SUBTROPICALES ARIDES. A DOUBLE LATENCE

ET RARES PLUIES DE SA1SON FROIDE

Le cycle sexuel n"est connu de façon relativement complête que chez une

seule espèce saharienne, ovipare, Cerastes cerastes (Saint Girons, 1962) et il pré-

sente une nelte originalité. En effet, il n’existe qu’une seule période daceouple-

rnent, à la fin du printemps (mi-mai à mi-juin) ; la vitellogenèse est vernale,

1’ovulation a lieu fin juillet, la ])onte début aoül, les éclosions en septembre. Les

earactères sexuels secondaires des mâles ne sont développés qu’au printemps,

d’avril à juillet et la spermatogenèse est de type pré-nuptial; la spermatocyto-

genòse commence en hiver et s’accélère de février à avril; la spermiogenèse se

déroule de mars à juillet. L’été et 1’automne correspondent à une période de

repos sexuel complet pour les deux sexes, phénomène inconnu chez les autres

Serpents.

II semhle bien que le cycle sexuel de Crotalus cerastes, hôte des déserts du

Sud Ouest des Etats Unis dont Fécologie se rapproche beaucoup de celle de Ce¬

rastes cerastes, ne diffère guère de celui des Crotales des régions tempérées.

Klauher, 1956, cite des accouplements en avril-mai et en septembre-octobre. En

captivité, la parturition a lieu à la fin de septembre et en octobre. Le seul point

qui pourrait rapprocher ces deux Serpents désertiques est représenté par les dates

relativement tardives de 1’accouplement vernal de Crotalus cerastes. Mais la pré¬

sence d’une activité sexuelle automnale montre que le cycle des deux espèces est

néanmoins très différent.

La plupart des ELAPIDAE australiens ont une vaste répartition incluant, à

la fois, des zones tempérées chaudes, des régions subtropicales arides et des ré¬

gions subtropicales plus ou moins humides. D après les renseignements disperses

que 1’on possède (voir Worrell, 1963), il semhle que le cycle sexuel des femelles

soit caracterisé par un accouplement et une vitellogenèse vernales et par une ges-

tation ou une incubation estivales. Quelques prélèvements faits en hiver (obser-

vations personnelles inédites), suggèrent que la spermatogenèse peut être estivale

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108

LE CYCLE SEXUEL DES SERPENTS VENIMEUX

et post-nupliale, eomme chez les Couleuvres d'Europe et des Elats Unis ( Deniso -

nia signata, à Sydney; Denisonia suta , à Aliee Spring), ou au contraire hiver-

nale et pré-nuptiale, c’est à dire du même type que chez Corastes corastes, mais

un peu plus precoce ( Pseudcchis mist ralis, à Alice Spring; Acanthophis antarticus,

dans le Nord Queensland).

Le CYCLE SEXUEL DANS LES RECIONS SUBTROPICALES HUMIOES, A FA1BI.E LATENCE

iChiVER ET PLUIES DE SA1SON CHAUDE

Dans ces régions (siluées dans I"hémisphère nord sur le flane oriental des

continents, alors que les régions suhtropicales arides soul localisées aux flanes

occidentaux). les Eézards présentent des eycles sexuels particuliers, avec une lon-

gue période de reproduetion au printèmps et eu élé et une spermatogenèse pré-

nuptiale. Aucun Serpent n a été étudié de façon complête, mais des observations

dispersées faites en Eloride semblent montrer que la reproduetion des Crolales y

diffère j)eu de eelle des espèces des zones tempérées voisines. Ce serail notam-

ment le cas de Crotalus adamanleus ( Klauber, 1956).

Becions tropicai.es et equatoriales

Selon loute vraisemblance, le cycle sexuel des Serpents diffère selou qu il

existe, ou non, une saison sèche accentuée et selon que les pluies lombent en

saison froide ou en saison chaude. En Indes, chez Natrix piscutor (aux environs

de Bénares, par 25"12" Nord), la spermatocytogenèse déhute en juillet, la sper-

miogenèse dès le mois de septemhre et une spermatogenèse continue se poursuit

jusqu’en janvier (Srivastava et Thapliyal, 1965). Le segment sexuel du rein ne

suhit pas des variations significatives, mais il y a tout lieu de penser que la re¬

produetion est annuelle, avec un accouplement hivernal ou vernal et des naissances

en été, saison des pluies. A Java, beaucoup plus près de 1’équateur et ou une

saison sèche peu marquée dure de juillet à septemhre, les observations de Kop-

stein (1958) et de Bergmann (1960), montrent que les espèces vivipares, eomme

Agkistrodon rhodostoma et Triineresiirus gramineus, gardent un cycle annuel, avec

une vitellogenèse vernale de septemhre à novemhre et une gestation estivale, de

décembre à mars. Au contraire, les espèces ovipares (des ELAPIDAE et la plu-

part des COLUBRIDAE ), pondent plusieurs fois par an, le nombre des pontes

variant de 3 à 10 selon la taille de 1’animal. Bien qu'il n’y ail pas de rythme

régidier, il existe une diminution très caractéristique du nombre des pontes du-

ranl la saison sèche.

Chez la Vipère africaine, Causas rhombeatus, dans le sud de la Nigéria.

Woodward (1933) a déerit un cycle sexuel à peu près mensuel, lié cTailleurs au

cycle alimentaire et au cycle des rnues. Les femelles muent, puis pondent 4 à

5 jours plus tard. Elles s’alimentent ensuite avidement pendant 10 à 12 jours,

cessent de se nourrir pendant 11 à 18 jours et muent de nouveau. Ces femelles

captives étant isolées des mâles, on ignore à quelle date de ce cycle très curieux

se place 1’accouplement. 11 est assez probable que la spermatogenèse des mâles

est continue dans les régions équaloriales sans saison sèche — bien que la longuc

conservation des spermatozoídes, soit dans les canaux déférents des mâles, soit

dans les voies génitales des femelles, rende ce phénomène non indispensable.

Nous avons eu 1’occasion d examiner quelques testicules de Serpents provenant de

forêts tropicales humides (Côte d’lvoire et Madagascar) et tous les éléments de

la lignée sérninale élaienl toujours ahondamment représentés.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 105 - 114 , 1966

H. SAINT GIRONS

109

Le cycle sexuel cies Hydrophinae presente un certain nombre de particulari-

tés (Bergmann, 1954 à 1962). Ces animaux passent toute leur vie dans la mer

dont la température ne varie que de 18 à 25° environ; de plus, la haute chaleur

spécifique de 1’eau limite étroitement les possibilites de thermorégulation par

insolation; enfin, la distribution de la plupart des espèces s’étend de part et

d’autre de 1'équateur. Ces conditions ont pour consé(|uence une viviparité obli-

gatoire, une gestation de longue durée (puisque le développement des embryons

esl directement proportionnel à la température) et une indépendance relative par

rapport aux saisons. II semble toutefois que chaque population ait un cycle an¬

imei plus ou moins régulier. Dans une région proche de Java, Enhydra schistosa

(Bergmann, 1955) présente une vitellogenèse davril à juin, une ovulation en

juillet et une parturition en novembre. Aux environs de Ceylan (Wall, 1921),

la même espèce effectue sa vitellogenèse d’octobre à décembre, 1’ovulation a lieu

en juin ou juillet, la parturition entre février et mai. Avec cbassez nombreu-

ses exceptions, à Java, chez Thalassophis anomalus (Bergmann, 1954), la vi¬

tellogenèse se situe (1’avril à juin, 1’ovulation en juin-juillet et la parturition en

novembre. Chez Hydrophis jasciatus, dans la même région, Bergmann (1962)

signale l’accouplement en juin, lovulation en juillet, la parturition en décembre.

II ressort de ces données que les Hydrophinae présentent un cycle reproductif

annuel, avec dMmportantes variations dans le lenips en fonction de 1’espèce et de

1’habitat des différentes populations. Le cycle spermatogénétique des mâles est

inconnu.

Chez les Laticaudinae ( Laticauda laticaudata et L. colubrina) qui chassent

dans 1’eau rnais passent une bonne parlie de leur temps à terre et y pondent,

1'hiver correspond à une période de repos sexuel des femelles et d’involution des

earactères sexuels secondaires des mâles (Saint Girons, 1964). La vitellogénèse

est vernale, Ia ponte a vraisemblablement lieu entre décembre et février, 1’éclosion

2 mois plus tard. La spermatogenèse est soit continue, soit, plus probablement,

hivernale et de type prénuptial, bien que d assez longue durée.

Fecondation retardee et conservation des spermatozoides

Le fait que, chez de très nombreuses espèces, il existe un délai de un à

deux mois entre la fin de la période daccouplement et l ovulation — donc la

fécondation — montre déjà que les spermatozoides peuvent survivre quelque temps

dans les voies génitales femelles. D autre part, de multiples observations ont mon-

Iré que des femelles, séparées des mâles aussilôt après raceouplement crautomne,

jreuvent se reproduire normalement 1’année suivante. Enfin, les femelles isolées

de Causus rhombeatus, étudiées par Woodward. ont pu donner jusqu’à 10 pontes

fécondes successives, bien cpie le pourcentage d’oeufs embryonnés décroisse régu-

lièrement. Les spermatozoides sonl stockés dans la lumière de la partie caudale

de 1’oviducte, ou tube vaginal (Rollinat, 1934; Rahn, 1942) et, peu avant 1’ovu-

lation, ils gagnenl les glandes spéciales situées dans la trompe (Fox, 1956).

Selon les espèces, ces spermatozoides disparaissent après la première ponte, ou

survivent encore pendant un laps de temps variable. Leur présence dans les voies

génitales femelles nlndique donc pas un accouplement récent, ce qui rend plus

difficile encore 1’étude des eycles sexuels.

Chez les mâles, les spermatozoides peuvent être stockés dans les canaux défé-

ronts pendant plusieurs mois. C’est ce qui se passe régulièrement chez les es-

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110

LE CYCLE SEXUEL DES SERPENTS VENÍMEUX

pèces dont la spermatogenèse esl posl-nuptiale. Mais on ne connait auciln cas

ou des spermatozoídes normaux puissent être trouvés en nombre important dans

les canaux déférents plus de 9 mois après 1’arrêt de la spermiogenèse.

Lês FACTEURS EXTRINSEQUES DU CYCLE SEXUEL

La température

Dans les régions tempérées, Fétude comparative des différents cycles sexuels

met en évidence Finfluence primordiale de la température. Chez chaque espèce,

celle-ci est directement responsable de la durée de la gestation. Elle règle aussi

la vitesse de la digestion, done la possibilité de reconstituer les réserves néces-

saires à la vitellogenèse. Sans aucun doute, c’est à un (Jéfieit lhermique qu’est

due la présence de cycles reproductifs s’étendant sur plusieurs années, dans les

régions les plus froides de 1’aire de répartition de chaque espèce. Au contraire,

la spermatogenèse, le développement des caractères sexuels secondaires dans les

deux sexes et l’accouplement ne dépendent guère de la température — ou tout

au moins celle-ci ne joue un rôle qu’à ses degrés extrèmes, alors que toule vie

active est devenue impossible au Serpent.

Dans les régions subtropicales arides, tout au moins au Sahara, Fé té cor-

respond à une période de repos sexuel complet et, a priori, Finfluence des tem-

pératures élevées ne peut être écartée. On sait d ailleurs que les hautes tempé-

ratures inhibent la spermatogenèse bien avant d’avoir un effet léthal sur les

Reptiles. Chez Cerastes cerastes, le développement de la lignée séminale et des

caractères sexuels secondaires commence dès le mois de décembre, c’est à dire

au moment le plus froid de Fannée et duranl la courte latence hivernale.

Dans les régions subtropicales humides, Fhiver reste en général une période

de repos sexuel, sauf sans doute en ce qui concerne la spermatogenèse. Dans

les régions Iropieales et équatoriales, I influence de la température sur le cycle

reproducteur semble faihle, sauf dans le cas des Serpents marins.

Autres jacteurs externes

La lumière ne joue vraisemblablement aucun rôle en tant que facteur quan-

titatif et les différentes phases du cycle sexuel peuvent se dérouler à iFimporte

quelle saison lorsque la température le permet. Mais la fréquence des cycles

annuels suggère Finfluence de la périodicité lumineuse en tant que facteur de

"‘remise au jour”.

L’humidité n'agit sans doute pas directement sur le cycle sexuel des Serpents

dont on connait les capacités de survie dans les zones arides. Mais, tout parti-

culièrement dans les régions tropicales, elle joue un rôle important dans Falimen-

tation. Malgré le peu de données dont nous disposons, la rareté des éelosions

ou des parturitions en saison sèche est un fail bien établi. De plus, la séche-

resse est souvent associée à des tempéralures élevées.

LES FACTEURS INTRINSEQUES DU CYCLE SEXUEL

II n’est pas possible de résumer ici, inêrne brièvement, les données acquises

en ce qui concerne la physiologie de la reproduction chez les Reptiles. Dans

Fensemhle, les corrélalions endocrines sont du même type que celles des Mamrni-

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 105 - 114 , 1966

H. SAINT GIRONS

fères, sanf dans le cas de la gestation; celle-ci s'assimile à une simple rétention

des oeufs dans les oviductes et seul le mécanisme de la parturition pose de véri-

laliles problèmes. Chez les rares espèces ou elle été étudiée (voir Saint Girons

et Duguy, 1962, pour la hibliographie), 1’évolution des glandes endocrines au

coiirs du eycle annuel suggère 1'existence d '1111 rythme endogène spécifique. Dans

les régions tempérées, la stimulation histologique du tissu adrénal et de diverses

catégories cellulaires de 1’adénohypophyse débute avant la fin de 1’hivernage,

donc à 1’obscurité constante et à une température proche du minimum annuel.

La fréquence d’une activité sexuelle automnale chez les Serpents exclut 1’hypo-

thèse d’un rôle déclencheur de la luniière, tel que celui qui a été mis en évidence

chez beaucoup dOiseaux et de Mammifères (voir Benoit, 1953, pour la biblio-

graphie). II convient également dinsister sur la date très constante, dune année

à 1 autre, de lovulatiorí, alors que les dates des premières sorties et de la partu¬

rition sont sujeites à dlmportantes variations. Lors des cycles tri- ou quadrien-

naux, on constate même que les ovules, s'ils n’ont pas eu le lemps d’arriver à

maturité au moment voulu, s’atrésient en masse à 1’époque normale de 1’ovulation.

La périodicité annuelle de la plupart des cycles sexuels, sensible même

lorsque la reproduction ne s’effectue que tous les 2 ou 3 ans, indique clairemenl

que des facteurs cosmiques interviennent au moins pour assurer une '"remise au

jour” qui interdit les déealages progressifs. Mais on ignore la nature de ce sti-

mulus chez les Reptiles et la date à laquelle il agit. Chez Vipera aspis, un cer-

tain nombre de faits indiquent 1’importance du rnois d’octobre, plutôt que des

premières sorties. II est probable que des travaux ultérieures démontreront 1’exis-

tence, chez les Reptiles comine chez d’autres Vertébrés, de périodes réfractaires

des glandes endocrines et de leurs récepteurs, ainsi que de périodes neutres pen-

dant lesquelles les facteurs externes (principalement la température et Ia lumière)

peuvenl accélérer ou ralentir Tévolution. C est ce qui se passe, notamment, du-

rant la deuxième partie de 1’hivernage. Mais, dans les condilions naturelles, les

grandes lignes du cycle sexuel (spermatogenèse, périodes d accouplement, date

de 1 ovulation ) sont sans doule déterminées par des facteurs endogènes, innés et

spéci fiques.

SlJMMARY

The sexual cycle of lhe COLUBROIDEA is only known in some species of

lhe temperate regions. In lhese zones, the vittelogenesis is generally vernal, the

ovulation takes place at lhe end of the spring and there is a second period of

sexual activity in autumn. The secundary sexual characters of the males are

developed lhe whole year, except for a short involution in July. There are two

kinds of spermatogenesis, one estival for the COLUBRIDAE and probably among

certain EL.APIDAE, and another mixted one among the Viperenae. The evolution

of lhe spermatogenesis is unknown for the Crotalinae,

As lo the Snake of Sahara, the sexual cycle is characterized by a late re¬

production period, at the end of spring, and by a complete sexual resling period

in suramer and autumn.

In lhe tropical regions, the sexual cycle of the viviparous snakes is generally

yearly, but lhe differences between specimens and populations can be quite large.

The oviparous species have generally several yearly laying periods, at irregular

intervals, but rarely during lhe dry season. In lhe humid equatorial forests,

Causas rhombeatus presents a very interesting monthly cycle, where reproduction,

molting, and feeding are joined.

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112

EE CYCLE SEXUEL DES SERPENTS VENIMEUX

F M A M J J A SOND JFMA-AJ J ASON D

Fig. 1

Fig. 2

Fig. 3

F/í/. 1 — Cycle sexuel des femelles chez divers Serpents venimeux.

A à E: espèces des régions tempérées. F et G: espèces des régions subtropi-

eales arides.

A: Vipera aspis. Cycle annuel. C’est le type le pius répandu ehez les Ser-

pents vivipares, venimeux ou non, dans les régions tempérées.

B: Vipera aspis. Cycle biennal, à la limit Nord de 'laire de répartition de

1’espèce.

Vipera berus. Cycle biennal, en montagne.

Crotalus viriitis viridis. Cycle biennal, d’après Rahn (1942).

Agkistrodon contortrix. Cycle biennal, d’après Fitch (1960).

Crotalus cerastes. Cycle annuel, en région subtropicale aride à hiver froid.

Cerastes cerastes. Cycle annuel en région subtropicale très aride (Sahara)

et hiver doux.

Durée de 1’hivernage.

Durée de la gestation.

Date de 1’éclosion chez les espèces ovipares.

Période d’accouplement principale.

Période d’aecouplement secondaire.

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10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):105-114, 1966

II. SAINT GIRONS

íií

Fig. 2 — Croissance des plus gros follicules ovariens au eours d’un eycle sexuel bien-

nal, chez une Vipère et un Crotale.

A: Vipera aspis. Vitellogenèse vernale.

B: Crotalus viridis. Vitellogenèse estivale.

En abseisses: temps en mois.

En ordonnées: longueur du plus gros follieule, en millimètres.

Fig. 3 — Evolution des caraetères sexuels secondaires (iei le segment sexuel du rein)

au eours du cyele annuel.

A: Vipera aspis (région tempérée).

B: Cevastes cevastes (région subtropicale très aride).

En abseisses: temps en mois.

En ordonnées: diamètre du segment sexuel, en p.

Fig. // — Différents types de cycles spermatogénétiques chez les Serpents. Type post-

nuptial (Natrix natrioc), mixte (Vipera beras) et pré-nuptial (Cevastes cevastes).

La flèehe blanche correspond à la spermatocytognenèse, la flèche noire à la

spermiogenèse, les triangles à la période d’accouplement.

En abseisses: temps en mois.

En ordonnées: poids du testicule et diamètre des tubes séminifères.

Bibliographie

1. Benoit, J. & Assenmacher, I. — Action des facteurs externes et plus particuliè-

rement du facteur lumineux sur 1'activité sexuelle des Oiseaux. Publication

des Annales d’Endocrinologie, Paris, Masson et Cie. Editeur, 1953.

2. Bergmann, R. A. M. — “Thalassophis anomalus Schmidt”. Universidad Na¬

cional de Córdoba, Rep. Argentina, 16 p., 1954.

3. Bergmann, R. A. M. — L’anatomie des Enhydra schistosa D. Arch. néerland.

Zool., 11:127-142, 1955.

4. Bergmann, R. A. M. — The anatomy of some VIPERIDAE (I and II). Acta

Morphol. Neerl.-Scand., 4:196-230, 1960.

5. Bergmann, R. A. M. — The anatomy of Hydrophis fasciatus atriceps. Biol.

jaarb. Dodonaea, 30:389-416, 1962.

6. Blanchard, F. N. & Blanchard, F. C. — Factors determining time of birth

in the garter snake, Thamnophis sirtalis sirtalis. Pap. Mich. Acad. Sei., 26:

161-176, 1940-41.

7. Uarpenter, C. C. — Comparative ecology of the common garter snake ( Tham¬

nophis s. sirtalis ), the ribbon snake (.Thamnophis s. sauritus ) and Butler’s

garter snake ( Thamnophis butleri ) in mixed populations. Ecol. Monogr., 22:

235-258, 1952.

8. Duguy, R. — Biologie de ia latence hivernale chez Vipera aspis. Vie et Milieu,

14:311-444, 1963.

9. Fitch, H. S. — Study of Snake population in central Califórnia. Amer. Midi.

Nat., 41:513-579, 1949.

10. Fitch, H. S. — Autecoiogie of the copperhead. Univ. Kans. Publ. Mus. Nat.

Hist., 13:85-288, 1960.

11. Fox, W. — Seasonal variations in the male reproduetive system of pacific

coast garter snake. J. Morphol., 90:481-553, 1952.

12. Fox, W. — Genetic and environmental variation in the timing of the re¬

produetive cycle of male garter snake. J. Morphol., 95:415-450, 1954.

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LE CYCLE SEXUEL DES SERPENTS VENIMEUX

13.

Fox, W. — Seminal receptacles of snakes. Anat. Rec., 124:519-533, 1956.

14.

Glissmeyer, H. R. — Egg production in the great basin rattlesnake. Herpe-

tologica, 7:24-27, 1951.

15.

Klauber, L. M. — The Rattlesnakes. Berkeley, Univ. of Calif. Press, 1956,

vol. I.

16.

Kopstein, F. — Ein Beitrag zur Eierkunde und zur Fortpflanzung der Ma-

iaiischen Reptilien. Buli. Ra ffies. Mus. Singapore, 14:81-167, 1938.

17.

Peter-Rousseaux, A. — Recherches sur la croissance et le cycle d'activité tes-

ticulaire de Natrix natrix helvetica (Lacépède). La Terre et la Vie, 100:175-

223, 1953.

18.

Ralin, H. — Sperm viability in the uterus of the greater snake, T h a m n o -

phi s. Copeia, pp. 109-115, 1940.

19.

Rahn, H. — The reproductive cycle of the prairie rattler. Copeia, pp. 233-

240, 1942.

20.

Rollinat, R. — La vie des Reytües de la France centrale. Paris, Delagrave,

1934.

21.

Saint-Girons, H. — Le cycle sexuel chez Vipera aspis (L.) dans 1’Ouest de

la France. Buli. Biol., 153:284-350, 1957.

22.

Saint-Girons, H. — Le cycle reproducteur de la Vipère à Cornes, Cerastes

cerastes CL.), dans la nature et en captivité. Buli. Soc. Zool. France, 87:41-

51, 1957.

23.

Saint-Girons, H. — Notes sur 1’écologie et la structure des populations des

Laticaudinae (Serpents, HYDROPHIIDAE), en Nouvelle Calédonie. La Terre

et la Vie, 111:185-214, 1964.

24.

Saint-Girons, H. & Duguy, R. — Données histophysiologiques sur le cycle an-

nuel de 1’hypophyse chez Vipera aspis (L.). Zeit. Zellforsch., 56:819-853, 1962.

25.

Saint-Girons, H. & Kramer, E. — Le cycle sexuel chez Vipera berus en mon-

tagne. Rev. Suisse Zool., 70:191-221, 1963.

26.

Saint-Girons, H. & Saint-Girons, M. C. — Cycle d'activité et thermorégulation

chez les Reptiles (Lézards et Serpents). Vie et Milieu, 7:133-226, 1956.

27.

Smith, M. — The British Amphibians and Reptiles. London, Collins, 1951.

28.

Srivastava, P. C. & Thapliyal, J. P. — The male sexual cycle of chequered

water snake, Natrix piscator. Copeia: 410-415, 1965.

29.

Tinkle, D. W. — Reproductive potential and cycles in female Crotalus atrox

from northwestern Texas. Copeia: 306-313, 1962.

30.

Vainio, I. — Zur Verbreitung und Biologie der Kreuzotter, in Finnland. Ann.

Soc. Zool. Bot. Fenn., 12:1-19, 1932.

31.

Volsoe, H. — Structure and seasonal variation of the male reproductive organs

of Vipera berus. Spolia Mus. Zool. Hauniensis, 5:1-157, 1944.

32.

Wall, F. — The snakes of Ceylon. Colombo, Cottle, 1921.

Woodward, S. J. — A few notes on the persistance of active spermatozoa

in the African night-adder, Causus rhombeatus. Proc. Zool. Soc. London: 189-

190, 1933.

34.

Worrel, E. — ■ Reptiles of Australia. Sydney, Angus & Robertson, 1963.

3 4 5 6 SciELO 10 -Q 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

83(1):115-120, 1966

T. W. BETZ

115

15. THE OVARIAN CYCLE OF NATRIX RHOMBIFERA — AN APPARENTLY

GENERALIZED CYCLE OF SNAKES OF TEMPERATE LATITUDES

T. W. BETZ *

Department of Biology, Carleton University, Ottawa, Canada

Complete descriptions of ovarian morpliology in repliles are available for

only a few species. However, acounts of gross ovarian morphology have been

reported for V i p e r a (Loyez, 1906), Bothrops, Crotalus, X e -

nodon (Fraenkel and Martins, 1938; Fraenkel et al., 1940), Crotalus

(Tinkle, 1962), Opheodrys (Tinkle, 1960), Thamnophis (Bragdon.

1952; Cieslak. 1945; Tinkle, 1957), Thyphlops (Guibé, 1948), Enhydri-

na (Kasturirangan, 1951) and Natrix (Loyez, 1906); Bragdon, 1952; Tinkle,

1959; Betz, 1963a). Aecounts have also been reported for A n g u i s , L a -

certa, Platydactylus, Terrapene, T e s tu d o , C r o c o d y ■

l u s (Loyez, 1906), Lacert a, Lygoso ma, M a b u y a , T ili q u a

(Weekes, 1934, 1935), Lacert a (Hett, 1924), Hoplodactylus (Boid,

1940), Zoo toca (Panigel, 1956), Xantusia (Miller, 1958), Cnemido-

phorus (Carpenter, 1960) and Holbrookia (Johnson, 1960). Others ap-

pear in lhe reviews of Kehl and Combescot (1955) and of Matthews (1955).

Generally the criteria for grouping the animais in these kinds of studies

depend upon lhe size of the ovarian folheies; the size and presence or absence

of corpora lulea and embryos; and the condition of the uterus.

The ovaries of mature Natrix rhombijera (Betz, 1963a) are elongate, thin-

walled. saccular structures with an irregular, lymph-filled central cavity. They

of a loose, semitransparent stroma in which the oval, creamy-white, re-

avascular ovocytes are seen in contradistinction to the yellow, vascular

folheies and corpora lutea. Each ovary is suspended hy a mesovarium

pleuroperitoneal cavity belween the dorsal mesentery and the mesotuba-

oviduct. They generally extend from lhe levei of the oviducal

limits of the kidneys. The right ovary is typically

placed than the left. The length of lhe ovary is

length of lhe animal (Fig. 1) and longer ovaries

than shorter ones. The positive correlation of the

body length has been reported for Natrix sipedon

some lizards (Carpenter, 1960). The smaller ovocytes

lateral aspect of the ovary.

The follieles oecur in four size

consist

latively

atrelic

i n I lie

rium of the

infundihulum lo the posterior

heavier and more anteriorly

posilively correlated with the

generally have more follieles

length of the ovary with

(Tinkle, 1959) and for

of Natrix are usually restricted lo lhe

mature follieles are pale yellow in color.

one group (I) of folheies 0.1 mm long; another group (I

a third (111) 10-20 mm long; and a fourth (IV) 20-46 mm

The largei

rroups:

I) 5-10 mm long;

long. The number

Supporteci by a Travei Grant from the National Research Council of Canada.

SciELO

10 11 12 13 14 15

116

THE OVARIAN CYCLE OF NA TRIX RHOMBIFERA — AN APPARENTLY

GENERALIZED CYCLE OF SNAKES OF TEMPERATE LATITUDES

of follicles decreases as they become mature. In Missouri ovulation occurs be-

tween 15 May and 15 June as specimens caught at ihis time, are immediately

preovular. Postpartum females are caught between 15 August and 15 September.

Consequently, geslation is approximately three months long. Deviations of several

weeks from lhe ahove dates are probably not imcommon, The size-frequeney

distribution of follicles at various times of the reproductive season indicates that

the |>roduclion of mature ova probably requires 2.5 years, i.e., the neonatal late

summer and early fali months, followed by two full years and the spring of the

third year (Fig. 2). The ovary at the end of lhe first year (following the neo¬

natal fali) would contain follicles of groups I and II. At the end of the secomf

year the ovary would contain follicles of groups I. II. and III; group II follicles

of the first year have become the group 111 follicles of lhe second year; group I

follicles of lhe first year have become the group II follicles of the second year;

and a new crop of group I follicles lias emerged from lhe germinal epithelium.

Immediately prior lo ovulation. in lhe spring of the third year, group III fol¬

licles of lhe second year grow rapidly and become group IV follicles. During

geslation the ovary, except for corpora lutea, appears identical lo the two-year-old

ovary in follicular sizes. Apparenlly follicular growlh occurs gradually over a

tvvo-year period but most of the growth occurs rapidly in the spring of the third

year. The ovarian cycle is apparently annual (Fig. 2). In other forms lhe time

required for the production of mature ova may be two years as in Xantusia

(Miller, 1948, 1958); three years as in Thamnophis (Bragdon, 1952; Cie-

slak. 1945) and Crotalus (Rahn, 1942; Tinkle, 1962). Although annual

ovarian cycles are commonly reported, Weekes (1934) reported that Amphi-

boi u r u s may have two ovarian cycles a year. Also nonseasonal cycles occur

in some Javanese snakes I Kopstein, 1938), house geckos (Church, 1962 ), and

in Lygosoma (Baker, 1929, jide Miller, 1958). Yolk deposition may occur

most of the year, as in L acerta (Regamey, 1935), or gra-

long growth period with a final immediately preovulatory in-

deposition as in Phrynosoma (Blount, 1929), Ho pio-

cmidaclylus (Dutta, 1944), X a n I u s i n

T h a m n o p h i s (Bragdon, 1952).

gradually during

dually during a

creased rate of

d a c t y l u s I Boyd, 1940), //

(Miller, 1958), X a I r ix , and

Atretic follicles are hyperemic and more flaccid than developing follicles.

The yolk is more fluid and paler than in developing follicles. The follicle wall

is thin, fragilc and easily broken. Follicles of all sizes in various stages of

astresia are

lhe follicles

pale, more

is replaced

commonly present. As astresia proceeds (as seen in selected stages)

become progressively smaller, more hyperemic, and the yolk more

fluid, and diminished in amount. Eventually the corpus atreticum

by stromal tissue leaving an indefinite, variously persislent scar.

Atresia probably accounts for the decrease in lhe number of follicles as they

mature. Also any follicles of group IV that are not ovulated undergo atresia.

Il is not uncommon lo find ovaries during early pregnaney with large group IV

atretic follicles. Follicular atresia is cominou in most reptiles and has hecn re-

ported by Mingazini (1893), Dubisson (1905), Loyez (1906), Boyd (1940).

Bragdon (1946. 1952), Bretschneider and Duyvene de Wit (1947), Miller (1918.

1958), and Altland (1951).

Occasionally irregularly shaped. creamy-white, viscous masses of ectopic yolk

material occur bolh in lhe coelom and in lhe ovarian stroma. These masses are

of the same consistency irrespective of their size. Apparently the ectopic yolk

masses in lhe coelom or ovarian stroma are from bursl atretic follicles. The

shape and size would depend on lhe space available among the viscera or follicles

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):115-120, 1966

T. W. BETZ

117

and the amount of yolk in the follicle at the time of Intrsting. The position of

lhe masses wouhl depend on lhe site of rupture of the follicle wall. Judging

from lhe amount of yolk usually presenl in these masses, hursting atresia is more

common in large follicles, probably because of lhe greater thinness and fragilily

of their walls. These ecto])ic yolk masses are apparently quickly reabsorbed since

they do not occur in the preovulatory females hut only in pregnant and early

postpartum females. Hursting atresia has also been reported in T h a m n o p h i s

(Bragdon, 1952). One factor which is a source of error in studies of reproductive

cycles which are based oídy on gross morphology is that follicles which have just

begun lo undergo atresia could easily he miscounted as growing follicles. Hovv-

ever, the two types of follicles are easily distinguished from each other on a

histological basis (Betz, 1965b).

The corpus luteurn of the first month of gestation is pale yellow. The sur-

face is indented by a puckered umbilicus which may contain a transient ecto|)ie

hlood mass. The gland is approximately 10-12 mm long. The shape is essentially

oval hut is variable and depends on the space available between the adjacent

developing or atretic follicles. The gland is placed well within the ovarian

slroma and does not appreciably project from the surface of the ovary. The

gland is more vascular than lhe developing follicles hut less so than well-advanced

atretic follicles. There is a 1:1 correspondence between the number of corpora

lulea and lhe number of emhryos or yolk masses in the uterus. It is common

to find a disparity between the number of corpora lutea in the ovary of one

side and lhe number of conceptuses in the uterus of the same side which is

probably due to the extrauterine migration of ova to the contralateral uterus as

reported by Legler (1958).

During the second month of gestation the corpus luteurn is darker yellow,

smaller (5-7 mm) and the umbilicus and eclopic hlood mass are not presenl.

In the Iast month of gestation the corpus luteurn is deep yellow and less than

5 mm long. The corpora lutea usually degenerate rapidly; in the two- or three-

week postpartum animal lliey are srnall II mm), orange patches in the ovarian

stroma. Usually by the following spring all traces of lhe glands have disap-

peared as evidenced by a general lack of these structures in the preovulatory

females; however, occasionally the scars persist until the next spring. Corpora

lutea have been described for oviparous, ovoviviparous, and viviparous species of

reptiles (Lucien, 1903; Hett, 1924; Weekes, 1934, 1935; Fraenkel and Martins,

1938; Kasturirangan, 1951; Bertin, 1952; Amoroso, 1955; Miller, 1958). The

length of time that the corpora lutea are maintained can he positively correlated

with lhe egg-retaining habits of a species (Miller, 1958). In oviparous and ovo¬

viviparous species, a corpus luteurn develops which begins to regress before ovi-

position occurs (Weekes, 1934; Rahn, 1938; Harrison, 1918). In most viviparous

fornis the length of gestation is two to three months. Typieally the corpora lutea

begin to regress during the lasl third of gestation (Weekes, 1934; Cieslak, 1915;

Miller, 1948; Bragdon, 1952), hut in some species regression is not apparent

until after parturition (Rahn, 1938, 1939. 1942).

SciELO

10 11 12 13 14 15

AVERAGE OVARIAN LENGTH (mm)

200

ISO

100

•V •

• • • •

700 SOO 900 IOOO 1100 1200

BODY LENGTH (mm)

1300 1400 ISOO

Fig. 1

Scatter diagram ot the correlation betvveen average ovarlan length with bociy

length of Natrix rhombifera.

YE AR 2

YEAR 3

fali

winter

spring

summer

fali

winter

spring

summer

fali

winter

spring

summer

fali

Fig. 2 — A diagram of the hypothetical sequence of follicular maturation in Natrix

rhombifera. Roman numerais refer to follicle size groups.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 115 - 120 , 1966

T. W. BETZ

119

Literature

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of the box turtle. J. Morph., 89:599-621, 1951.

2. Amoroso, E. C. — De Ia signification du placenta dans 1’évolution de la ges-

tation chez les animaux vivipares. Ann. Endo., 16:435-47, 1955.

3. Bertin, L. — Oviparité, ovoviviparité, viviparité. Buli. Soc. Zool. France, 77:

84-8, 1952.

4. Bete, T. W. — The gross ovarian morphology of the diamond-backed water

snake, Natrix rhombifera, during the reproductive cycle. Copeia, 4:692-97,

1963a.

5. Betz, T. XV. — The ovarian histology of the diamond-backed water snake,

Natrix rhombifera during the reproductive cycle. J. Moryh., 133:245-60, 1963b.

6. Blount, R. F. — Seasonal cycles of the interstitial cells in the tests of the

horned toad ( Phrynosoma solare). Seasonal variation in the number and

morphology of the interstitial cells and the volume of the interstitial tissue.

J. Moryh., 48:317-44, 1929.

7. Boyd, M. M. M. —• The structure of the ovary and corpus luteum in Hoplo-

dactylus maculatus. Quart. J. Micro. Sei.. 82:337-76, 1940.

8. Bragdon, D. E.

542, 1946.

Follicular atresia in ovoviviparous snakes. Anat. Rec., 90:

9. Bragdon, D. E. — Corpus luteum formation and follicular atresia in the

common garter snake, Thamnophis sirtalis. J. Morph., 91:413-46, 1952.

10. Bretschneider, L. H. & Duyvene de Witt, J. J. — Sexual endocrinology of

non-mammalian vertebrates. Monographs on the Progress of Research in

Holland during the War, 11:77-145, 1947.

11. Carpenter, C. C. — Reproduction in Oklahoma Sceloporus and Cnemi-

dophorus. Herpetologica, 16:175-82, 1960.

12. Church, G. — The reproductive cycles of the Javanese house geckos, Cosym-

botus platyurus, Hemidactylus frenatus and Peropus mutilatus. Copeia, 2:262-

9, 1962.

13. Cieslak, E. S. — Relations between the reproductive cycle and the pituitary

gland of the snake Thamnophis radix. Physiol. Zool., 18:299-329, 1945.

14. Dubisson, M. — - Degenerescence des ovules chez les reptiles. Compt. Rend.

Soc. Biol., 59:473-4, 1905.

15. Dutta, S. K. — Studies on the sexual cycle in the lizard Hemidactylus flavi-

viridis. Allahabad Univ. Stud. Zool. Sect., pp. 57-153, 1944.

16. Fraenkel, L. & Martins, T. — Sur le corps jaune des serpents vivipares. Compt.

Rend. Soc. Biol., 127:466-8, 1938.

17. Fraenkel, L. & Mello, R. F. — Studies on the pregnancy of viviparous snakes.

Endocrinology, 27:836-7, 1940.

18. Guibé, J. — Contribution à 1’étude de 1’appareil genital des typhlopides (ophi-

ciiens). Buli. Soc. Zool. France, 73:224-8, 1948.

19. Harrison, R. J. — The development and fate of the corpus luteum in the

vertebrates series. Biol. Rev. Cambridge Phil. Soc., 23:296-331, 1948.

20. Hett, J. — Corpus luteum der Zauneidechse Lacerta agilis. Z. mikr. Anat.

Forsch., 1:41-8, 1924.

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THE OVARIAN CYCLE OF NATRIX RHOMB1FERA — AN APPARENTLY

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Johnson, C. — Reproductive cycle in females of the greater earless lizard

Holbrookia texana. Copeia, 4:297-300, 1960.

Kasturirangan, L. R. — Placentation in the sea snake Enhydrina schistosa.

Proc. Indian Acad. Sei., Sec. B., 34:1-32, 1951.

Kehlj R., Combescot, C. — Reproduction in the reptiles. Mem. Soc. Endo.,

4:57-74, 1955.

Kopstein, F. — Ein Beitrag zur Eierkunde und zur Fortpflanzung der Ma-

laiischen Reptilien. Buli. Raffles Museum, 14:81-167, 1938.

Legler, J. M. — Extra-uterine migration of ovain turtles. Herpetologica, 14:

49-52, 1958.

Loyez, M. — Recherches sur le développement ovarien des oeufs méroblasti-

ques a vitellus nutritif abondant. Arch. Anat. Micro., 8:69-398, 1906.

Lucien, M. — Note préliminaire sur les premières phases de la formation des

corps jaunes chez eertains reptiles. Compt. Rend. Soc. Biol., 55:1116-7, 1903.

Matthews, L. H. — The evolution of viviparity in vertebrates. Mem. Soc.

Endo., 4:129-48, 1955.

Miller, M. R. — The seasonal histological changes occurring in the ovary,

corpus luteum and testis of the viviparous lizard Xantusia vigilis. Proc. Calif.

Acad. Sei. Zool., Sec. 47, pp. 197-224, 1948.

Miller, M. R. — The endocrine basis for reproductive adaptations in reptiles.

In: Comparative Endocrinology, N.Y., J. Wiley & Sons Inc., 1958, pp. 499-516.

Mingazini, P. — Corpi lutei veri e falsi dei Rettili. Ric. Lab. Anat. Norm.

Univ. Roma, 3:105, 1893.

Panigel, M. — Contribution à l'étude de 1’ovoviviparité chez les reptiles: ges-

tation et parturition chez le lézard vivipare Zootoca vivipara. Ann. Sei. Nat.

Zool., ser. 11, 18:569-668, 1956.

Rahn, H. — The corpus luteum of reptiles. Anat. Rec., 72:55, 1938.

Rahn, H. — Structure and function of placenta and corpus luteum in vivi¬

parous snakes. Proc. Soc. Exp. Biol. Med., 40:381-2, 1939.

Rahn, H. — The reproductive cycle of the prairie rattler. Copeia, 4:231-40.

1942.

Rcgamey, J. — Les caracteres sexuels du lézard < La certa agilis). Rev. Suisse

Zool., 42:84-168, 1935.

Tinkle, D. W. — Ecology, maturation and reproduction of Thamnophis sauri-

tus proximus. Ecology, 38:69-77, 1957.

Tinkle, D. W. — Observations of reptiles and amphibians in a Louisiana

swamp. Am. Midi. Natl., 62:189-205, 1959.

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Copeia, 1:29-34, 1960.

Tinkle, D. W. — Reproductive potential and cycles in females Crotalus atrox

from northwestern Texas. Copeia, 2:306-13, 1962.

Weekes, II. C. — The corpus luteum in certain oviparous and viviparous rep¬

tiles. Proc. Linn. Soc. N. S. W., 59:380-91, 1934.

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Mem. Tnst. Butantan

Simp. Internac.

33 ( 1 ): 121 - 133 , 1966

H. SCHILDKNECHT

121

16. VERTEBRATE HORMONES AS DEFENCE SUBSTANCES IN

DYTISCIDES

H. SCHILDKNECHT

Organisch-Chemisches Institui der Universitat, Heidelberg, Germany

Introduction

As we have shown (1), many arthropods defend lhemselves against their

enemies with special glandular substances. The existing research lias concerned

ilself with secretions which serve lo repulse small invertebrates, vertebrates and

microorganisms. In continuation of this work we have examined for the first

time, in the case of Dytiscus marginalis, a group of protective substances which

react specifically against vertebrates. This glandular protective system is found

in the breast section of lhe beetle (Fig. 1). Figure 2 shows one of the two

secretion reservoirs, each about 3.5 mm long and 1.4 mm wide, on which lie a

thick layer of glandular cells.

Blunck (2), (3) describes lhe formation of the milk-white secretion in the

glandular cells and its transmission to the reservoir. Merely by holding or by

a lighl pressure to lhe head, the beetle secretes the glandular fluid through

muscular pressure.

It was of particular advantage, for the successful identification of the loxic

factor in the secretion, that this crystallised out without further assistance after

several weeks from lhe secretion, absorbed in a glass capillary.

Identification of the defence sudstance as an steroid

In agreement with the already published analytical data of the loxic com-

ponent of the defence secretion we also found, at the onset of our recontinued

work, in lhe UV-spectrum of the secretion taken in optically pure ethanol, an

absorption maximum at 240-241 m/x typical of a conjugated chromophore.

According to Woodward, mono-alkylated enones absorb on an average at

224 m/x. By the calculation of similar chromophores one adds to this value 11 m/x

for an additional alkyl group or a cyclic residue in the /J-position, and a further

5 m/x for the exo-cyclic position of the C = C-double bond. Thereby, e.g. a

maximum of 210 m/x is obtained for testosterone which is alrnost identical with

lhe maximum of the hormone as found by us. This agreement between testo-

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10 11 12 13 14 15

122 VERTEBRATE HORMONES AS DEFENCE SUBSTANCES IN DYTISCIDES

sterone and the hormone from Dytiscus is especially good wlien one compares

lhe values of the absorption bands of hotli infra-red spectra (Tahle 1).

TABLE 1 — COMPARISON OF THE IR-ABSORPTION

BANDS OF TESTOSTERONE AND THE HORMONE

Type of

vibration

Position of the

bands with testo¬

sterone in cm-'

Position of the

bands with the

hormone in cm- 1

" O-H

3520

3476

1693

1668

" c=c

1612

1613

1272

" c-c=o

1230

1233

" C = 0

1063

1074

1053

1061

7C-H

867

870

Alone, a superficial consideration of Tahle 1 indicates that the Dytiscus

secretion constituent could !>e a steroid. Also the position of the y c — H vihrational

hand at 870 cm -1 and the v c = c vihrational band at 1613 cm -1 as well

as the v c = o vihrational hand at 1668 cm -1 indicates a A 4 -3-keto-steroid. More-

over, as with testosterone, an OH-group could be detected by an IR-spectrum

taken in carhon disulphide (Fig. 5).

The intensity of lhese bands is very low and from this one could infer that

the hydroxyl group is hydrogen bonded to a carbonyl oxygen atom. lt is pos-

sihle of course to rule out honding with the carbonyl group in position 3. There

is, however, a further discernable band at 1693 cm -J in the IR-spectrum of

the Dytiscus hormone which does not appear in the IR-spectrum of testo¬

sterone and must be assigned to a saturated aliphatic ketone (Fig. 6 & 7).

In the UV-absorption spectrum, however, this carbonyl chromophore was not

detectable. It shoúld, on the other hand be detectable with the help of circular

diehroism (CD), when the hypothesis that it is a steroidal ketone is correct i.e.

a ketone with several optically active centres. In fact, the CD-spectrum taken

showed two extrema at 283 m/x with lhe AF lllnx = + 3.076 and at 321 m/x with

the Ae milx = —1.16 (Fig. 8).

According to Velluz and Legrand (4), the A 4 -3-keto-steroids possess a mini-

mum at 334 m/x with lhe Ar lmix = — 1.35 dr 0.12 (ind dioxan) which conforms

with our interpretation of the minimum at 321 m/x (in ethanol) found by us.

This finding confirms again that the Dytiscus hormone may be a A 4 -3-keto-

steroid. For the second maximum at 283 m/x (in ethanol), two possibilities are

indicated in the literalure (4) :

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H. SCIIILDKNECHT

123

a) 20-keto-steroids: 295 m/x (clioxan)

h) 17-keto-sleroids: 303 m/x (dioxan)

An assignation was made more diffieult in lhal lhe two relevanl Ae raax values

at 3.459 ± 0.12 and 3.290 ± 0.10 are also not so very different. We must

al lliis point, however, introduce a correction for the use of differenl solvents.

Probably our compound should have its maximum at (283 + 13 =) 296 m/x in

dioxan. Since the eorrected value of 296 m/x lies nearer to 295 than to 303,

we concluded that the hormone is a 20-keto-steroid.

On considering that the ketone group at C 2 o can be bridged and that its

accompanying absorption band in the IR-spectrum occnrs, not as usual, at

1710 em -1 but al 1693 cm -1 , one may suppose that the OH-group is bonded

to lhe C 21 atom.

On lhe basis of lhe partial structure just proposed, we can thus assume that

the Dytiscus hormone is A 4 -pregnen-3,20-dion-21-01 (11-desoxycorticosterone,

cortexon).

We eonfirmed this postulation by a comparison of the CD of cortexone

(Fig. 9) and the secretion constituent.

Cortexone shows the two extrema at 282 and 322 m/x vvith lhe correspond-

ing Ae max values of 3.275 and —1.16. Small differences in the Ae max values

are known to occur — according to the literature (5), the influence of temperature

frequently plays a big role.

In 1959, Heller published a paper on the lR-spectra of steroids(6). His

information on hydroxyprogesterone can be referred to for the further identifiea-

tion of our natural product. He was concerned witli lhe wave numbers of lhe

bands to be found between 1000 and 1150 cm -1 . The curves obtained for the

spectrum of the secretion are shown in Fig. 10.

A simple comparison of these spectroscopic data with those which were found

by us for the secretion constituent reveals that the latter is most probably identical

with cortexone iTable 2).

TABLE 2 — COMPARISON OF THE IR-

SPECTRAL BANDS IN THE REGION OF

THE

-C-O

-VIBRATION BETWEEN

1000 AND 1150 cm-'

Bands found for

Cortexone (cm- 1 )

Bands found for

the Hormone

(cm- 1 )

1005 (w)

1009

1039 (w)

1040

1060 (st)

1061

1072 (st)

1074

1093 (w)

1093

1117 (w)

1117

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124 VERTEBRATE HORMONES AS DEFENCE SUBSTANCES IN DYTISCIDES

Since cortexone is commercially availalile, il vvas ]>ossiI>k- lo obtain an IR-

speetrum of a synthctic sample from Fluka 1 >1 <I. for further information.

The spectrum of this sample of cortexone (Fig. 11) agreed so completely

with that of our compound. ohtained hy thin-layer chromatography, that an

identity was therehy suggesled.

The UV-spectrum of cortexone (Fig. 13) agreed likewise with that of lhe

secretion constituent (Fig. 14) isolated hy ns.

We ohtained a further agreement hetween cortexone and our natural product

hy the comparison of the physieal data of lhe corresponding 2,4-dinitrophenyl-

hydrazones (2,4-DNP). The 2,4-DNP of cortexone was prepared following the

method of Reich and Samuéis (7). In the literalure, different melling points

are reeorded for this product — Wettstein et al. (8) give a decomposition tem¬

peratura of 278 to 284°C and Reich and Samuéis (7) record that lhe 2,4-DNP

of cortexone mells at 251"C to 254°C. The 2,4-DNP of cortexone prepared

hy ourselves decomposed hetween 267° and 269 U C. The UV-spectra had, how-

ever. the same appearance.

For the preparalion of the 2,4-DNP of the heetle secretion, the secretion

was added lo a solution of 2,4-DNP in 2N hydrochloric acid, left lo stand for

several hours and lhe crystals formed were then filtered off. By lhe ihin-layer

chromatographic separation we ohtained two zones which were scratched apart.

eluted and crystallised from aqueous ethanol.

Reich and Samuéis (7) record the UV — maxima and minima of the cor-

texone — 2,4-DNP as follows: maxima at 257 and 383 mp and a minimum at

313 m/u We found for the secretion — 2,4-DNP (decomp. 269"), maxima at

254 and 378 mp and a minimum at 310 m/u This is a satisfactory agreement.

Finally, there was still lhe agreement hetween lhe mass spectra of the syn-

thetic cortexone and the material contained in the heetle secretion lo he tested.

In the mass spectrum of the heetle suhstance (Fig. 16), an additional mass peak

at 316 was ohserved which could not he assigned. This may be caused hy lhe

contamination which also influeneed the melting point. This was found at 135”C

and not, as with cortexone, at 141°C.

The mass peak of 330 showed us lhe molecular weight of cortexone. Further,

only the peak at 299 can be elearly assigned the moleeule having lost a CfPOH

group with mass 31. In then losses a carhonyl or an ethyl gronp of mass 28

resulting in a fragment of mass 271. An oxy-

gen alom is removed as water leaving a residue

of mass 253.

The mass spectrum of cortexone (Fig. 17)

shows the same fragmentation pattern.

All these descrihed results permit the con-

clusion that the non-volatile, ethanol-soluble

compound from lhe water beetle’s proteetive

secretion is A 4 -pregnen-3,20-dion-21-01.

ch 2 dh

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II. SCHILDKNECIIT

125

ExPERIMENTS O N THE PHYSIOLOGICAL EFFECT OF

BIOLOGICAL IMPORTANCE

THE SECRETION \\T> 1TS

As previously mentioned in lhe introduction, lhe physiological effect of lhe

beetle-secretion ou various animal was irivestigated hy Blunck(3). He íound

lhat il was a strong narcotic and poisou effective in very small amounts, especially

on cold-hlooded vertebrates. On invertebrates, it produced only liltle or no ef-

íecl. To begin wilh, we repeated some of Bhmck’s important experiments. In

lhese, goldfish ( Carassius auratus), OLIGOCHAETA ( Enchytreen ) and Dytiscides

('Colymbeles fuscas and Dytiscus marginalis ) were placed in an aqueous solution

of the secretion. In agreement wilh Blunck’s residis, lhe goldfish were stupified

whilst the worms and the heetles showed no reaction at all. The water beetle

Colymbetes even ale meat, which had been treated with the poisonous

substance, without harrn.

Later, as it was found that the constituent of the beetle-secretion was eor-

texone, it was necessary to examine whether this substance had the same effect

as the secretion. For this purpose we used cortexone from Flnka Ftd. The

aqueous solution of cortexone is of 0.001% strenght and contains 10.39 mg of

lhe substance per litre. From this, dilutions of 1:10 and 1:100 were prepared.

Into these three Solutions were placed the test fish (goldfish and young tench

[Finca vulgaris ]). The fish reacted to the concentrated solution exaclly as lo

lhe natural secretion. Afler a shorl phase of exeitement, the fish tired rapidly.

After about eighl minutes they could be turned onto lheir backs with a glass-rod

and after twenty minutes they displayed severe equilibrium disturbances and lay

immobile on lheir sides. Finally, they were completely motionless except for

weak mouthing motions. When the stupified fish were placed in fresh water

they recovered completely over a period of ten to twelve minutes and later could

be used again in other experiments. Fish in the 1:10 dilution permitted them-

selves lo be touched and turned over with a glass-rod without swimming away -

Cortexone, in this instance, had no further action. In lhe case of the 1 :100

dilution, the test fish showed no noticeable reaction.

Cortexone is a hormone of the córtex of mammalia. It possesses a mineral-

corticoidal effect, i.e., it regulates the sodium-potassium content of the cells. On

administration of an overdose, sodium is retained and potassium secreted, and

so the ionic equilibrium is disturbed. This dislurbance in the salt concentration

ctrresponds lo a dislurbance in the sensitivity of the nerve and muscle cells by

which. the physiological effect of lhe beetle-secretion and cortexone may be ex-

plained. This agreement of the biological effect of the secretion and cortexone

again confirms the results of the Chemical analysis.

In order lo determine the biological importance of lhe secretion in nalure.

we carried oul a series of further experiments. To this purpose we used synlhctic

cortexone as this is easier to administer and can be, more easily obtained than

the natural beetle-secretion.

There are two possibilities for the natural use of the poisonous secretion by

lhe beetle. In the first instance, the predatory Dytiscus could stupify its

prcy with lhe cortexone eontaining secretion in order to overcome il more easily;

one must then speak of an attacking-secretion. If. however, the beetle only de-

livers up the secretion when it is under attack in order to protect ilself from an

enemy, then it may be called a defence-secretion.

Initially we investigated the possibility of the Dytiscus secretion being

used as an attacking weapon.

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10 11 12 13 14 15

126

VERTEBRATE IIORMONES AS DEFENCE SUBSTANCES IN DYTISCIDES

Experiment a: Water heetles were placed together with young tench

and goldfish in small containers (100 ml) where the íish had no room lo escape

from the heetles. Soon after they were put together there was a fierce fight

which ended in the death of the fish. In tliis experiment it was. however, im-

possihle lo observe the appearance of the white secretion. A following investigation

of the water in order to detect cortexone by chemical means was also without

success. In order to lest whether the heetles had full reservoirs after all, they

were so provoked after the experiment that they all gave up plentiful amoimts

of secretion.

Experiment h: In an experiment lasting several month, goldfish and

water beelles were kept together in an aquarium (20 litre). Althongh the

Container was over-occupied by the water heetles and the heetles had not been

fed for a long lime, and already had begiin to eat each other, lhe goldfish were

not killed.

From these experiments il is concluded that lhe beetle-secretion is not used

as an attacking weapon. It is therefore very probable that the beetle employs

the secretion for protection from vertebrates. If a fish or an amphibian tries

lo eat a beetle, then the beetle immediately gives out a large quantity of secretion.

This penetrates the gills of the fish and also, when the beetle has been swallowed,

into the stomach-intestinal tracl. The described experiments show that cortexone

penetrates through the hody surfaces and is effective.

In order to prove the effect, especially on lhe gills of the fish, we smeared

300 /xg of cortexone on lhe gills of a large trout (Salmo gairdneri) of 750 g

weight. Afler some lime il was almost unable to move and could only with

difficulty retain its balance. Only after more than six hours did it recover. It

is interesting that this illness was immediately utilized by a small trout. Whilst

before, this smaller fish had been continuously bitten by the larger one, now

the order was reversed and the small fish attaeked the larger continuously and

pressed it into the very corner of the aquarium. Such examples could also be

of importance in nature if a predatory fish ale a beetle.

Finally, we examined the effect of cortexone on stomach-intestinal tract of

the fish. To this end, goldfish were fed with bread-crumbs in which 100 pg of

cortexone was embedded. The same symptoms appeared as when cortexone pe¬

netrates through the hody surfaces (skin and gills) of the organism. The effecl

takes place only a little later but on lhe other hand remains for a much longer

time. Translated to the natural slale, this means that a vertebrate that has eaten

a water beetle temporarily suffers severe discomfort and probably will not capture

further heetles in the future.

To sum up, it may be sliown through lhe afore mentioned experiments lhat

the prolhoracic glandular secretion of the water beetle is a defence secretion that

serves as protection againsl vertebrates. The physiological efficacy is caused by

the contained cortexone.

Experimental Seclion

All IR-spectra quoted in the following work were taken on the Perkin-Elmer

Speclralphotometer 221 and all UV-absorption speclra on lhe Beckman 1)15 Spectro-

photometer.

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H. SCHILDKNECHT

127

The concentration determination of cortexone in the beetle-secretion was

carried out using a speclralphotometer PMQ II from Cari Zeiss Lts. Also, using

tliis apparatus, lhe log e value of corlexone was determined as 4.24.

A dichrograph from Roussel-Jouan Ltd., Paris was used for the circular

dichroism measurements.

The mass spectra were taken with a inass spectrometer of the type CH 4

from the Atlas MAT Company, Bremen.

All melting poiuls were determined ou the Bock-monoscope.

The water beetle (Dy ti seus) were compelled to give up the secretion by

ligbt pressure with lhe finger against the head. The secretion was taken up in

a capillary and squirted inlo pure ethanol. Alter a few days the insoluble com-

ponents collected on lhe bottom and could be centrifuged off using a microfuge.

The insoluble components were dried in air and without preliminary treatment

were examined spectroscopically in potassium bromide. The hormone was separal-

ed from the alcoholic solulion and purified by the use of preparative thin-Iayer

ehromatography using plates coated with Kieselgel “G” and chloroform as the

solvent. The plate was developed from a distance of 10 cm. Under a UV lamp

of wave-length 366 mp, the compound is observable by fluorescence. Since the

Bf-value is relatively low, the plate was redeveloped using chloroform. After

this, the compound had moved 4 cm. The zone was scraped off and eluted with

chloroform in a microcolumn. The compound so obtained was used without

further preliminary treatment for spectral investigations.

Preparation oj the 2,4-dinitrophenylhydrazones of the beetle-secretion

and cortexone

The secretion of the Dytiscus was added to a solution of 2,4-DNP in 2N

hydrochloric acid and allowed lo stand for several days. The crystals formed

were filtered off. dissolved in chloroform and the two 2,4-DNP derivatives pre-

paratively separated on Kieselgel “G” thin-layer ])lates. As developing agent,

chloroform was used and the plate was run for an hour. Although, after this

time, the separation was noticeable we were unable to separate the zones by

scraping them apart as they lay too closely together, and therefore the chromato-

graphy was carried out twice again each time for an hour. lt was then easy

to separate the two 2,4-DNP derivatives by scraping apart and eluting them

separately on micro-columns. The produets crystallised from aqueous ethanol with

melting-points: 269°C.

The 2,4-DNP of cortexone was prepared following the method of Reich and

Samuéis (7). 10.7 mg of Cortexone and 15 mg of 2,4-DNP were dissolved in

1.8 ml of ethanol and 3 drops of concentrated hydrochloric acid were added.

After 2.5 hours the crystals formed were filtered off, washed with ethanol and

crystallised from chloroform/ethanol. Melting-point: 267-269°C.

Experiments to test the effects of the beetle secretion and cortexone

Experiments with the natural secretion were carried out on goldfish ( Caras -

sius auratus) and invertebrates. The secretion from seven beetles was dissolved

in 100 ml of water and the goldfish was placed in the solution. After 30

SciELO

10 11 12 13 14 15

128 VERTEBRATE IIORMONES AS DEFENCE SUBSTANCES IN DYTISCIDF.S

minutes lhe excitation phase commenced. 75 minutes after lhe start of lhe ex-

perimenl lhe reactions of the fish had heen slowed down considerably, and after

a further 90 minutes the fish was unable lo react any more.

OLK.OCHAETA and Dytiscus exhihited no reaction. The effecl of cor-

texone on fish was tested using young tench (Finca vulgaris).

Experiment a: A tench of 1.18 g weight was placed into a saturated

sohition of eortexone in water. As a blank-test, another fish was placed into tap-

water. Afler 5 minutes the movements of the test-fisli became unnatural. After

a total of 9 minutes it swam on its back and had grown pale. 13 minutes afler

the start of the experimenl it was unable to swim to lhe surface and Iay on its

side. After 18 minutes, it was unable to move and mouthed only seldom. After

24 minutes it Iay, as if dead, on the botlom of the Container. The control-fish

was completely normal.

Experiment b: Young tench were tested in various concentrations of

eortexone in water. A saturated sohition of eortexone was made, and from this,

dilutions of 1:10 and 1:100 were prepared.

250 ml of each of the three Solutions of differing concentration were poured

into separate containers and at the same time a young tench was placed into

them. In Container 1, with the concentrated eortexone sohition, the fish swam

immediately here and lhere in an agitated manner. After 5 minutes it reeled.

It was possible after a period of 8 minutes to turn it onto its back with a glass-

rod. After 11 minutes it Iay down but now and then swam quite normally to

the surface. 9 minutes laler it had completely lost its sense of balance. The

gills bled 28 minutes after the start of the experiment and after 60 minutes the

fish Iay unmoving on lhe bottom — it seldom made mouthing movements.

In Container 2 with the dilution 1:10 the first reaction from lhe tench carne

after 11 minutes. It swam agilatedly, allowed itself to be louched with a glass-

rod, and Iay on its side. The influence of the eortexone did not proceed any

further.

The fish in Container 3 with the dilution 1:100 displayed no noticeable re¬

action throughoul lhe duration of lhe experiment.

Experiment c: A young tench was placed in a saturated solution

of eortexone and after some lime was llien placed into fresh water. 7 minutes

after it was placed in the solution, the fish becamc disquitened. Afler a total

of 16 minutes, it Iay on its side and after a further 2 minutes it Iay unmoving

on the bottom. After 20 minutes it was mouthing only weakly and was then

replaced in fresh water. Within 11 minutes the fish had recovered and then

behaved again as normal. The experiment was repeated with the recovered-fish.

In lhe eortexone sohition it again displayed stupefaction symploms and again

recovered in fresh tap-water. This action may ostensibly be repeated as often

as desired.

Experiment d: A young tench was kept for a longer lime in a

saturated eortexone solution.

After 18 minutes the fish tilted onto its side and afler 26 minutes did not

move any more. Soon it swam spontaneously (after 2 hours), but always Iay

down again. This lying-down occurred less and less frequently and the next day

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 121 - 133 , 1966

H. SCHILDKNECHT

129

lhe fish swam quite normally in its Container. 26 hours after the commence-

ment of lhe experiment a second fish was placed with the test-fish. This fish

showed no reaction. After 10 minutes, holh fish swam frequently lo lhe surface

probahly due to an oxygen-shortage caused l*y the presence of two fish in the

Container.

Experiments to test the secretion as an attacking weapon

Experiment a: Eleven water beetles were placed singly into 100 ml

of water together with goldfish and young tench. After a fierce battle the

creatures were separated. The total amount of liquid (1100 ml) was reduced

in a rotary evaporator and extraeted with chloroform. The residue was treated

with a solution of 2,4-DNP in 2N hydrochloric acid but a thin-layer chromato-

gram showed no evidence of the presence of cortexone-2,4-DNP.

Experiment b: Three goldfishes and up to 30 water beetles were kepl

in an aquarium, 39 by 25 by 22 cm with a capacity of ca. 20 litres. The fishes

were fed continuously with dry forage and no beetle managed to kill a fish. If

lhe fishes were not fed any more they soon hecame weak and unable lo excape

the beetles and were killed.

Experiments to test the secretion as a dejence weapon

Experiment a: The effect of cortexone on the gills.

A trout ( Salmo gairdneri) of 750 g weight was orally given 300 pg of cor¬

texone in 10 ml water. The duration of the operation was one minute. After

9 minutes the fish began to pale and already after 13 minutes it succumbed in

a battle with a smaller trout. After 85 minutes the trout stood inert on its head

and it was possible to pull it out of the water by its tail.

Experiment b: The action of cortexone ihrough the stomach-intestinal

tract.

Different goldfishes were given cortexone embedded in bread by pushing it

into their mouths with a glass-rod. A control fish was given only bread in lhe

same amount.

In the case of the test-fishes they became weaker after 25 minutes. This

weakening was so intense after 45 minutes lhat lhe fish was unable to turn back

any more when one lurned it over with a glass-rod. The fish was certainly not

helpless but was constantly very slow to react. After 11 hours all the goldfishes

behaved normally again.

The water beetles carne from all parts of the Bundes-republik. They were

kept in aquaria and were fed on meal-worms. As long as one changes the water

frequently enough and give the beetles ample nourishment, also in form of meai

and fish, they can be kept alive for a longer time. As previously stated, the

beetles were “milked” by a light pressure on the head. They regenerated the

secretion in about three weeks.

SciELO

10 11 12 13

VERTEBRATE HORMONES AS DEFENCE SUBSTANCES IN DYTISCIDES

Offense glands

Aõ

<T>

Defense glands

Fig. 1

Fig. 1 — • Site oí the protective glands of Dytiscus.

Fig. 2 — Secretion reservoirs with the overlying glandular cells

R = reservoir

Dr = glandular cells

Ao = secretary opening

3500 3000 2500 2000 1500 1000 Icm-1]

Fig. 3

300 i Im>u)

Fig. 3 — IR-Spectrum of the crystailine crude secretion in KBr.

Fig. 4 — - UV-Spectrum of the whole secretion in ethanol when taken immediately and

after >4 one years exposure to air.

Xmax 240-241 m/í

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H. SCHILDKNECHT

131

6 I>i]

6 M

Fig. 6

1100 [cm-1] non

Fig. 7

Fig. 5 — IR-Absorption of the D ytisc u s hormone in the region of the v 0 _ H vibra-

tion (upper curve in CS 2 ; lower curve pure CS 2 ).

Fig. 6 — IR-Absorption o£ testosterone in KBr.

Fig. 7 — IR-Absorption of the hormone in KBr.

v y \.

282

IO' /v

/ \

300 1 [nyi]

321

Fig. 8

350 X lm/i)

_J _ lOií)

Fig. 9

1100 1000 [cm-1 ]

Fig. 10

Fig. 8 — The circular dichroism of the Dytiscus hormone.

Fig. 9 — The circular dichroism of cortexone.

Fig. 10 — IR-Absorption of the hormone in the region 1000 to 1150 cm- 1 taken in KBr.

2 3

6 SciELO 10 11 12 13 14

132 VERTEBRATE IIORMONES AS DEFENCE SUBSTANCES IN DYTISCIDES

10 [/il

2000

1500 1000 [cm-')

Fig. 12

2.0

250 300 3 (m/i)

Fig. 13

Fig. 11 — IR-Spectrum ot cortexone from Fluka Ltd., in KBr.

Fig. 12 — IR-Spectrum ot the compounct obtained by thin-layer chromatography using

chloroform also taken in KBr.

Fig. 13 — UV-Speetrum of cortexone taken in ethanol Xm«x 240 m/i, log c = 4.24.

' 3,0

, o

250 300 1 Imul

Fig. 14

Fig. 15

Fig. 14 — UV-Speetrum of the compound obtained by thin-layer chromatography taken

in ethanol Xm»* 240-241 m/i.

Fig. 15 — UV-Spectrum of the 2.4-DNP of cortexone in ethanol \ ml i 227, 256 and 376 m/i.

Fig. 16 — Mass spectrum of the compound obtained by thin-layer chromatography.

Fig. 17 — Mass spectrum of cortexone.

2 3 4

6 SciELO 10 11 12 13 14 15

Mem. Inst. Butantan

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33(1): 135-150, 1966

GIORGIO SCHREIBER et al.

135

17. PESQUISAS DE CITOLOGIA QUANTITATIVA. XIX DNA E VOLUME

NUCLEAR NOS TECIDOS SOMÁTICOS DOS VERTEBRADOS

GIORGIO SCHREIBER et al.

Instituto de Biologia Geral, Faculdade de Filosofia da U.F.M.G.,

Belo Horizonte, Brasil

Introdução

O presente trabalho visa o estudo das relações quantitativas entre o conteúdo

em DNA e o volume nuclear. O trabalho pioneiro de Walter Jacobj veio intro¬

duzir na citologia o conceito de “quantum”. As velhas pesquisas sôbre a assim

chamada “relação núcleo plasmática”, tateavam sem uma diretriz teórica esclare¬

cedora de um mecanismo determinante desta relação. A cariometria de Jacobj

teve o valor de esclarecer que o crescimento da matéria viva se dá por valores

descontínuos e com a regra de duplicação. As pesquisas de Jacobj, retomadas à

luz da cilogenélica, que vinha se desenvolvendo então, levaram imediatamente a

esclarecer que o “quantum” de reduplicação da matéria viva é o genoma. As

variações do volume nuclear deveriam, portanto, ser paralelas às do DNA. Su-

cessivamente, porém, apareceram vários casos nos quais a relação entre genoma

e volume nuclear não se mantém e núcleos com o mesmo DNA podem ter volu¬

mes diferentes. O primeiro caso de discrepância entre o teor em DNA e o vo¬

lume nuclear foi descoberto por Schrader e Leuchlenherger (1956) na série es-

permatogenética de um hemíptero ( Arvelius ), onde os vários túbulos testi-

culares apresentam todos o mesmo valor de DNA nas fases correspondentes da

meiose, porém, o volume nuclear é, em alguns túbulos, respectivamente, 2 ou 4

vêzes maior que os volumes dos correspondentes estádios dos túbulos normais.

O teor de proteínas segue de perto as variações do volume nuclear independente

do teor em DNA.

Variações da relação entre DNA e o volume nuclear, em condições fisioló¬

gicas normais ou experimentais, foram observadas por Bern, Alfert e colaborado¬

res no estudo da ativação de determinados órgãos por hormônios específicos.

Alfert e Bern (1951) verificaram que uma variação do volume nuclear (com

módulo 2 ou às vêzes 1.5) se dá na tireoide ativada pelo hormônio hipofisário

e nas células da parede uterina ativada pelos hormônios estrogênicos, sem que

haja variação do conteúdo de DNA. Foram, prevalentemente, êstes fatos que le¬

varam Bloeh a considerar a existência dos dois tipos de interfase dos quais fala-

Êste trabalho é apresentado em colaboração com Norma M. B. Melucci, Silvia E.

Gerken, Yeda X. SanCAna, Luís Alexandre Fallieri e Flávia de O. Amorim. O trabalho

é dedicado à memória da co-autora Norma M. B. Melucci, falecida em 1963. As pesqui-

sas foram realizadas com o auxilio do Conselho Nacional de Pesquisas, CAPES e Fun¬

dação Rockefeller.

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136 PESQUISAS DE CITOLOGIA QUANTITATIVA. XIX — DNA E VOLUME

NUCLEAR NOS TECIDOS SOMÁTICOS DOS VERTEBRADOS

remos adiante. Outro caso de desvio das relações entre DNA o volume nuclear

é aquele descrito por Fautrez (1957-8) e chamado de "décalage”, no qual o

núcleo da classe diplóide do pâncreas nos roedores tem um volume igual à me¬

tade do correspondente volume do núcleo diplóide do fígado.

Na Escola de Lison (Valeri — 1962), foram evidenciados outros casos de

independência do volume nuclear em relação ao conteúdo em DNA, nos tecidos

patológicos. No estudo dos tumores do colo do útero, Valeri verificou que. os

núcleos podem ter volume maior ou menor em relação ao teor em DNA. e, pela

primeira vez, foi empregado o têrmo “megetismo” para indicar o excesso (hiper-

megetismo) ou deficiência (hipomegetismo) do volume nuclear em relação ao te¬

cido normal.

No estudo da ploidia somática das glândulas dos Moluscos, Schreiber e cola¬

boradores (1964-1965) verificaram mais um caso de exceção à constância das

relações entre DNA e volume nuclear. Os tecidos glandulares, especialmente o

hepatopâncreas, e a glândula salivar, nos Gaslerópodos, apresentam intensos fe¬

nômenos de endopoliploidismo (ou politenia), verificáveis por classes superiores

(múltiplas de 2) no conteúdo em DNA (Schreiber e colaboradores — 1964-1965).

Estas glândulas, portanto, representam um mosaico de células de valores múltiplos

de ploidia. Schreiber e colaboradores demonstraram um fato nôvo neste tipo de

fenômenos: a relação entre DNA e volume, na série dos meiocitos, é. respectiva¬

mente, 1:1, 2:2 e 4:4, ao passo que, nas células glandulares poliplóides dêstes

tecidos, a relação DNA/volume é respectivamente 1/2, 2/4, 4/8, 8/16, etc. Isto

quer dizer que as células dos tecidos especializados têm um conteúdo em proteí¬

nas não histônicas duas ou quatro vêzes maior do que as células germinais.

0 estudo de todos êstes casos, nos quais a relação entre conteúdo em DNA

e volume nuclear não fica constante, foi considerado por nós (Schreiber e colabo¬

radores — 1965) de forma sintética, introduzindo o conceito de “relação plóido-

megética”. Estas considerações nos permitiram reconsiderar, sob nôvo aspecto, o

trabalho dos antigos autores de cariometria.

A base bioquímica destas variações foi elaborada por Bloch (1958), que

chegou à conclusão de que o núcleo pode seguir duas formas de crescimento in-

lerfásico. A primeira é a “autossintética”, na qual duplicam lodos os compo¬

nentes bioquímicos e morfológicos do núcleo. Assim, duplicam o DNA, as pro¬

teínas histônicas que o acompanham em quantidade sempre constante, e as pro¬

teínas não histônicas, que constituem parte dos cromosomas e o suco nuclear.

Êste tipo de interfase se verifica antes da divisão mitótica ou da endomitose. ()

segundo tipo de interfase, “heterossintética”, verifica-se quando o DNA não du¬

plica, mas trabalha na função de produzir o UNA e sucessivamente as proteínas

eitoplasmáticas específicas da função celular. Neste tipo de interfase, porém, du¬

plicam as proteínas não histônicas e o volume nuclear segue êste ritmo de au¬

mento. As proteínas não histônicas, que constituem o suco nuclear, determinam

pela sua constituição físico-química o volume nuclear, que adquire geralmente

valores múltiplos de duplicação (Schreiber — 1964). O volume do núcleo re¬

presenta, portanto, uma “constante” celular que é característica de cada tipo de

células. As variações do volume nuclear refletem fundamentalmente a função do

núcleo, e, no presente trabalho, em falta de uso de métodos específicos para de¬

terminar as proteínas não histônicas, consideramos o volume nuclear como ex¬

pressão morfológica embora grosseira do conteúdo destas proteínas. Deixamos

aqui de considerar o problema das variações do conteúdo em RNA. que é um

componente mais “móvel" e provavelmente em estado de equilíbrio durante o

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Slmp. Internac.

33(1):135-150, 1966

GIORGIO SCHREIBER et al.

137

ciclo fisiológico (“steady slate”j e ligado às variações quantitativas do nucléolo.

Do ponto de vista morfoquantitativo, a participação do DNA, histona e RNA,

para a constituição do volume do núcleo, deve ser relativamente menos conside¬

rável do que a das proteínas não histônicas. Talvez os fenômenos de estado fí¬

sico-químico (embebição, etc.) destas proteínas sejam os responsáveis pela deter¬

minação de um volume específico e constante nas diferentes fases da vida celular.

O esquema apresentado por Alfert (1958, Fig. 3) esclarece a participação dos

vários componentes na determinação do volume nuclear e as suas variações nas

duas interfases, respectivamente, reprodutiva e funcional da célula. Pouco ou

nada se conhece acerca da origem destas proteínas, se endonuclear on citoplas-

mática; o fato é que elas se encontram em quantidade e estado físico-químico

constante após uma divisão e duplicam antes da divisão sucessiva (interfase au-

tossintética), on durante o ciclo funcional da interfase heterossintética. A quan¬

tidade destas proteínas não histônicas está de certa forma ligada ao genoma, mas

cias podem variar independentemente dêle e a sua variação se dá, conforme a

feliz expressão de Pollister, sob forma de “multiple sets of proteins”. Neste fato

está a base de todo o sistema de pesquisa cariométrica e do fenômeno fundamen¬

tal que foi verificado por Jacobj sob a forma de “Verdoppelungsgesetz”) do núcleo.

A função das proteínas não histônicas (proteínas acídicas, fosfoproteínas, etc.)

parece ter uma importância fundamental, pois a estas proteínas parece estar con¬

ferida a função de desligar a união do DNA com as histonas. Esta ligação que

parece ser a base de repressão da atividade do gen seria rompida pela nova li¬

gação com as proteínas acídicas que agiriam como "depressors” do loeus gê-

nieo, permitindo a sua atividade específica. Por esta razão nos parece interessan¬

te estudar quantitativamente a relação entre estas proteínas (provisoriamente ex¬

pressas pelo volume nuclear) e o conteúdo em DNA nos diferentes tecidos espe¬

cializados na série dos Vertebrados.

Uma comparação das variações de DNA e do volume nuclear entre vários

grupos de tecidos e em vários grupos de animais nos levou a fazer uma classifi¬

cação destas variações conforme a concepção de Bloch. O esquema aqui apresen¬

tado compara a relação entre DNA e volume nos tecidos de alguns Artrópodos,

nos quais as variações entre tecidos se dão por polilenia ou poliploidia e, portan¬

to, as variações do DNA são paralelas às do volume nuclear (autossíntese), com

a dos Vertebrados e Moluscos, nos quais o DNA é sempre diplóide na classe

básica dos núcleos, mas o volume pode ter valores múltiplos nos diferentes tecidos

(heterossíntese). A êste fenômeno se sobrepõe, em certos tecidos, como por exem¬

plo o fígado, o poliploidismo somático que forma classes múltiplas nucleares do

valor de DNA e do volume. O que varia entre os tecidos é, portanto, o valor

da relação plóido-megética que será constante nas differentes classes de endopoli-

ploidismo de cada tecido. Pode-se dizer que nos Artrópodos se dá um mosaico

de ploidias entre os tecidos com relação plóido-megética 1:1, ao passo que nos

Vertebrados se dá um mosaico de núcleos com relação plóido-megética diferente,

em cada qual pode-se dar também endopoliploidismo (Fig. 1).

Podemos concluir, portanto, que do ponto de vista das relações entre DNA

e volume nuclear (megetismo), os tecidos podem apresentar séries de volumes

nucleares que classificamos como se segue:

I. A série “eumegética ”, na qual o DNA e o volume variam paralelamente,

devido ao mecanismo de interfase “autossintética”. Esta série se verifica nos

meiócilos (ide = 1. gônios e eitos segundos = 2 e eitos primeiros = 4), nos le¬

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138 PESQUISAS DE CITOLOGIA QUANTITATIVA. XIX — DNA E VOLUME

NUCLEAR NOS TECIDOS SOMÁTICOS DOS VERTEBRADOS

eidos que apresentam poliploidia somática (polissomatismo), como em algumas

glândulas nos Vertebrados e nos Moluscos, ou entre tecidos com ploidia caracte¬

rística como uos Artrópodos.

II. A série “Idpnrmegélica’ somática , como se verifica na comparação de

núcleos com igual grau de ploidia de órgãos diversos nos Vertebrados e Moluscos.

Nestes, cada tecido tem um volume característico da classe diplóide e os volumes

básicos dos vários tecidos formam uma série de duplicação (série “polimegética”.

■‘Verdoppellungs-gesetz” de Jacobj — 1925). Às vêzes. porém, podem ter valo¬

res múltiplos de 1.5 (“sesquifase” de Schreiber — 1960).

III. A série “liipomegética.”, representada pelos linfócitos e núcleos de eri-

tróeitos (Jacobj — 1925) que, embora tenham teor diplóide de DNA, podem

apresentar volumes 1/2, 1/4 ou até 1/8 do volume básico do gônio.

Materiais e técnicas

O material foi sempre fixado, parte em Bouin e corado com hematoxilina-eosina

para cariometria, e parte em formol neutro a 10% e corado com Feulgen sem con¬

traste para citofotometria. Os cortes foram feitos com espessuras muito variáveis

conforme o material (de 2 micra para os peixes até 20 micra para o Sipho-

ii o p s ). Foram tomados como base os seguintes tecidos: testículo, fígado, pân¬

creas e baço. Nos Peixes foram feitas medições orientadoras no tecido nervoso e,

quando possível, foi medida a série espermatogenética completa (gónios, eitos I,

eitos II e espermátides), mas às vêzes foi difícil encontrar todos os estádios. Con¬

tudo foi fácil encontrar eitos I e ides, com os quais foi feita a regressão DNA/vo-

lume. No baço foram medidos os linfócitos, e em Rato foram medidos os timó-

citos e linfócitos grandes. Do pâncreas, nesta pesquisa, foi considerado somente

o tecido exócrino. Nos anamniotas foram medidos os eritrócitos nos vasos capila¬

res, escolhendo os núcleos bem orientados para a medida. As medidas cariométri-

cas foram feitas seja com o micrômetro filar do citofotômetro, seja com desenho

à câmara.

As medidas de DNA foram feitas com citofotômetro, montado com Microscópio

Ortholux Leitz, com objetiva 124 Nachet, imersão e ocular 10, montado num su¬

porte Aristophot Leitz. Na cabeça binocular, uma das oculares é usada para a

escolha do campo e a outra, substituída por um micrômetro filar de Zeiss com

ocular K16, para a medição dos dois diâmetros, máximo e mínimo, do núcleo, e a

centração do núcleo a ser fotometrado. No Aristophot foi adaptada a caixa do

fototubo (RCA 931 A) com um disco de 4 diafragmas de superfície variável. No

foco do visor lateral foi introduzida uma chapa diapositiva com as imagens con¬

cêntricas dos diafragmas para o "plug”. O fole de Aristophot foi ajustado para

obter uma imagem do núcleo a ser medido ampliado a 1.500 diâmetros. Utiliza-se

uma área do “plug” com cêrca de 20% da área do núcleo. Foi feita uma medida

de absorção de cada núcleo; para cada medição foi determinada a luz padrão em

zona fora do núcleo e ajustado o valor do galvanómetro a 100 com pequenos mo¬

vimentos do diafragma do condensador do microscópio. Com êste método cada lei¬

tura de absorção é dada em percentagem da intensidade da luz padrão usada. A

iluminação foi dada por um monocromador Bausch & Lomb, a retículo, com lente

frontal supletiva usando uma luz monocromática de 540 angstrons ou com filtro

Wratten n* 58. Foi usado um micro-amperímetro "Norma”, modelo 251, de sensi¬

bilidade 2,10 <. Com as medidas de absorção de cada núcleo, e o diâmetro médio

dois dois diâmetros cruzados, foi calculada a quantidade de DNA para cada núcleo,

de acordo com a fórmula DNA — D- (2 — log In), sendo D- a superfície óptica

do núcleo, In a medida de absorção e 2 o log 100. Dos dados de DNA assim obti¬

dos foram calculados a média, desvio-padrão e êrro-padrão da média. Na elabo¬

ração estatística dos dados foi indicado sempre o valor da média e de ± S para o

DNA. Nas medidas cariométricas geralmente foi indicada a moda, pois é a me¬

dida mais adequada nos casos de histogramas bimodais ou com modas encobertas.

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 135-150, 1966

GIORGIO SCUREIBER et ai.

139

Quando as medidas eram excessivamente dispersas, como em certos casos de volu¬

mes medidos ao micrômetro durante a citofotometria, foi indicado o valor mediano

como o mais apto a dar uma indicação utiliâzável.

Nos diagramas de cascavel e parelheira aqui publicados utilizamos um nôvo tipo

de gráfico, ou seja, as “áreas plóido-megéticas". Estas áreas (Figs. 4 e 8) resultam

de quadriláteros, cujos lados correspondem ao valor total de dispersão dos dados

respectivamente de DNA e volume. Embora se esteja ainda iniciando o estudo

dêste tipo de representação, os diagramas exprimem com grande clareza a situação

dos núcleos dos diferentes tecidos em relação ao DNA e ao volume, e a sua com¬

paração entre os diferentes tecidos nos parece de grande utilidade.

Descrição dos resultados

Nas pesquisas precedentes desla série foram estudados vários tecidos de Ver¬

tebrados, determinando o volume nuclear e o DNA. Êstes trabalhos foram exe¬

cutados no decorrer de vários anos, cada um com banhos de Feulgen diferentes.

Os resultados não podem ser confrontados quantitativamente entre as espécies, mas

podem-se verificar as relações quantitativas entre estas duas variantes celulares

somente entre os tecidos de cada espécie.

Os materiais estudados nas pesquisas anteriores foram os seguintes: Peixes

(Tilapia melanopleura) , por Yeda X. Sanl Ana (1965). Foram estudados o fí¬

gado, pâncreas, rim. eritrócitos e neurônios. O estudo foi feilo não somente em

adultos, mas também em fases de larvas recém-eclodidas e alevinos. Os dados

desta pesquisa estão ainda em elaboração. Somente foi publicado o estudo da

variação do tamanho do núcleo e seu conteúdo em DNA durante o desenvolvi¬

mento pós-embrionário. Anfíbios: (Siphonops annulaius ), por N. Melucci

(1962), no qual foram estudados o fígado, pâncreas, rim e esperma togônios.

Répteis : (Tupinambas sp.) , Melucci (não publicado), no qual foi estudado

o fígado, rim e pâncreas. Mamíferos : (Cricetus auratus e Mus rattus). Em

Cricelus, Gerken (1962) estudou o fígado, pâncreas, rim e glândulas salivares.

Em Rattus, Yeda X. SanFAna estudou o fígado, pâncreas e os elementos do

timus. relacionando estas medidas aos gônios (1961).

Ofídios : 1. Crotalus durissus terrificus (Laurenti) — Apresentamos na

Fig. 2 os diagramas de regressão DNA/volume nuclear da série espermatogené-

tica e na Fig. 3 os histogramas, respectivamente, do DNA e do volume nuclear

com a tabela dos valores numéricos relativos. A Fig. 4 representa a regressão

entre DNA e volume da linha germinal (linha contínua) e dos tecidos somáticos.

Representamos de uma forma sintética os vários tecidos por meio da área ocupa¬

da pelos dados de cada tecido. Os retângulos abrangem a totalidade dos pontos

do diagrama de regressão de cada tecido indicando assim a área de variabilidade.

Chamamos êsle diagrama de "áreas plóido-megéticas” por dar uma expressão geo¬

métrica da dispersão dos dados nos tecidos e indicar com grande evidência a

situação de hiper e hipo megetismo em cada tecido. Destes diagramas aparece

bem claro que os tecidos somáticos têm um teor em DNA geralmente na faixa

diplóide. As variações dentro desta faixa provavelmente são determinadas seja

pela variação interfásica de uma certa percentagem de células em movimento mi-

tótico ou endomitótico, seja por variações devidas ao estado fisiológico, como foi

amplamente indicado na série de trabalhos da Escola de Fautrez ( Roeis- De Schrij-

ver — 1961, Roeis — 1954, Anteunis & Liu — 1960, Venvoerd-Verhoef & Ver-

woerd — 1962).

SciELO

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140 PESQUISAS DE CITOLOGIA QUANTITATIVA. XIX — DNA E VOLUME

NUCLEAR NOS TECIDOS SOMÁTICOS DOS VERTERRADOS

Sc observarmos, agora, as variações cio volume nuclear, resulta com grande

evidência que os vários tecidos têm uma variação específica, dentro de determi¬

nados limites, e diferente em cada tecido. Êste é o fenômeno chamado de “dé-

ealage” por Faulrez na comparação dos volumes nucleares das classes diplóides do

fígado e do pâncreas.

O pâncreas tem um volume mais ou menos duplo do espermatogônio e o fí¬

gado quatro vêzes maior. Às vêzes êstes volumes encontram-se na relação de

1 :1,5. Mandamos os trabalhos anteriores desta série para o estudo dêste pro¬

blema, que chamamos de "sesquifase” (Schreiber — 1960).

Nos Képteis, em geral, não se dá, como nos Mamíferos, a existência de classes

múltiplas de endopoliploidia no fígado e às vêzes no pâncreas; por isto. a varia¬

bilidade dos núcleos é estritamente limitada à faixa diplóide.

2. Philodryas schottii (Schlegel) — A í ig. 5 representa a regressão DNA/

volume na série meiocítica e as Figs. 6 e 7 representam os histogramas de DNA

e do volume nos seguintes tecidos: fígado, pâncreas, supra-renal, tireoide, linfóci-

tos e monócitos. A Fig. 8 representa a regressão entre DNA e volume nos tecidos

somáticos com a representação da “área plóido-megética”, como no caso precedente

do Crotalus. A regressão entre DNA e volume dos valores nos meiócilos é

perfeitamente regular. O DNA, como na cascavel, está na faixa diplóide para

todos os tecidos, com exceção dos monócitos, nos quais tanto o DNA como o vo¬

lume estão distribuídos na faixa 4c-6c, indicando um valor tetraplóide dos seus

núcleos. Os volumes dos tecidos, como na cascavel, se encontram decididamente

deslocados para uma situação de hipermegetismo, com exceção dos linfócitos, que

se encontram com um valor 2c e 2, portanto exatamente como os espermatogônios.

Os demais tecidos têm volumes nucleares altamente superiores ao volume da cé¬

lula diplóide típica. Os dados numéricos dêstes volumes estão indicados na ta-

Muitas vêzes, devido â grande dispersão dos dados, os valores do volume não

refletem o valor teórico da série de duplicação. As exceções a esta série, como

já dissemos, se devem fundamentalmente a estados de “movimento” da população

de núcleos, seja em atividade mitótica, seja em atividade fisiológica.

Na Philodryas, os valores da tireóide são um pouco discordantes do

valor teórico, provavelmente por serem os núcleos dos folículos tireoidianos de¬

formados, dando um êrro na valulação do volume.

Análise geral dos resultados

Pelos dados das pesquisas precedentes e da nova série sôbre Ofídios, podemos

verificar alguns fatos gerais que resumimos como segue:

a) o estudo comparativo do teor em DNA e do volume nuclear nos tecidos

somáticos e na espermatogênese dos Vertebrados revelou o falo já constatado em

trabalhos precedentes nos Moluscos que a relação plóido-megética é diferente na

linha germinal masculina e nos tecidos adultos somáticos. A regressão entre DNA

(' volume na série dos meiócitos espermatogenéticos é uma regressão muito regu¬

lar e quando as coordenadas são escolhidas com as mesmas unidades de medida,

“b” = 1, isto é, as variações de DNA são iguais às do volume. Chamamos an¬

teriormente a êste tipo de regressão de “série eumegética”, conforme a nomencla¬

tura de Valeri e Lison adotada com ligeiras modificações;

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 135-150, 1966

GIORGIO SCHREIBER et al.

141

l>) os vários tecidos somáticos aqui examinados apresentam todos a mesma

forma de variação da relação plóido-megética, embora em grau quantitativo dife¬

rente. Pràticamente todos estão com um teor em Dl\A dentro do que chamamos

de “faixa diplóide isto é, com uma variabilidade de 2c-4c devida, em parle, aos

fenômenos multiplicativos de uma certa percentagem de células e, em parte, aos

desvios do teor em DNA que se verificam nos diferentes estados funcionais. Os

volumes dos núcleos de cada tecido, pelo contrário, têm um campo de variação

muito mais amplo e característico para cada um. Nas pesquisas precedentes, nos

Mamíferos em geral, os valores modais dos histogramas de volume têm uma re¬

lação de duplicação entre si. Nos Ofídios esta regra, às vêzes, é mascarada por

fatores ainda não esclarecidos, mas que, por exemplo, para o fígado poderiam

ser relacionados a condições fisiológicas excepcionais (jejum prolongado). Não

podemos aqui examinar êstes desvios mais detalhadamente; limitamo-nos a cons¬

tatar que os diferentes tecidos somáticos têm volumes que desviam, às vêzes, for-

temente do valor que deveriam ter em base à ploidia. Isto significa que os nú¬

cleos se encontram naquela situação que Bloeh define de interfase heterossintética.

O volume nuclear do pâncreas é geralmente o dobro do volume da série eu-

megétiea (espermatogônio) ; o do rim, nos Hépteis, maior e não excluímos que

possa ser afetado por medidas feitas no assim chamado segmento sexual caracte¬

rístico do rim dos Répteis.

() fígado se encontra mais ou menos com um volume quatro vêzes maior

que o do correspondente estádio eumegético.

Os linfócitos e os eritrócitos, embora com ligeiros desvios no conteúdo em

DNA, talvez devido a dificuldades técnicas da medição, têm nos Ofídios volume

correspondente aos dos gônios e, portanto, têm uma relação eumegética. Nos tra¬

balhos precedentes de Schreiber e SanfAna, nos timócitos do rato encontramos

um valor do volume dos timócitos de 1/2 o valor da célula diplóide típica, con¬

firmando quanto tinha encontrado Jacobj nas suas pesquisas cariométricas (“Un-

terklassen”). Nos iKonócitos de Ofídios, pelo contrário, se verifica um caso de

endopoliploidismo, estando o DNA na faixa de 4c e os volumes correspondentes

exatamente na linha de regressão eumegética.

c) O estudo da relação “plóido-megética” do tecido hepático durante o de¬

senvolvimento embrionário e pós-embrionário nos Peixes e Anfíbios revelou um

fenômeno nôvo. Pesquisas cariométricas precedentes, de Bruno Schreiber e An-

geletti, nos Peixes, e de Giorgio Schreiber e Maria Romano Schreiber, nos Au-

fíhios, mostraram uma redução progressiva do volume nuclear por etapas de di-

midiamenlo (chamado por G. Schreiber de “elaxis”). Nos Peixes (SanPAna)

foi estudado, também, o teor em DNA e verificou-se estar a diminuição somente

a cargo do volume, ficando constante o valor diplóide do DNA. Os núcleos em¬

brionários e larvários, neste caso em situação polimegética, alcançam, por etapas

de dimidiamento, o valor básico do núcleo no adulto. Êste fenômeno foi por nós

comparado com o progressivo dimidiamento do volume nuclear dos blastômeros

durante a segmentação, sendo, provavelmente, a diminuição progressiva em certos

tecidos pós-embrionários, um prolongamento do tipo de divisão característico dos

blastômeros.

O megetismo dos blastômeros, porém, poderia ser devido a um tipo de pro¬

teínas diferente daquele que aparece na diferenciação histógena pós-embrionária.

Devemos lembrar que o núcleo dos blastômeros em segmentação não possui ainda

SciELO

10 11 12 13 14 15

442 PESQUISAS DE CITOLOGIA QUANTITATIVA. XIX — DNA E VOLUME

NUCLEAR NOS TECIDOS SOMÁTICOS DOS VERTEBRADOS

genes ativados, salvo casos especiais, e, como demonstraram Bloeh e Hew (1960),

as liistonas destes núcleos até à gastndação pertencem a uma categoria diferente

('‘cleavage histones”).

O caso do pâncreas, órgão altamente produtor de proteínas citoplasmáticas,

e que geralmcnte apresenta hipermegetismo menor do que o fígado, deve-se talvez

relacionar com um valor quantitativo do nucléolo, excepcionalmente superior aos

demais tecidos (Schreiber e colaboradores). O caso do bipomegetismo dos linfó-

eitos e eritrócitos deve ser idteriormente estudado, estando a origem destas célu¬

las ligada a processos característicos de maturação histógena permanente e não

somente embrionária ou pós-embrionária.

O estudo de todos êstes fenômenos deve ser estendido aos estádios histogené-

ticos embrionários de cada órgão para se determinar o momento em que, para

cada tecido, se estabelece uma dose múltipla de proteínas acídicas do suco nu¬

clear (“multiple sets of proteins” de Pollister) e subseqüentemenle um volume

nuclear específico.

Conforme foi dito antes, a base bioquímica de todos os fenômenos aqui re¬

latados (hiper e bipomegetismo) deve-se encontrar no conceito de interfase bele-

rossintética de Blocb (1958), durante a qual, a célula está empenhada em trabalho

de síntese de materiais não genômicos que constituem fundamentalmente o suco

nuclear (RNA e proteínas não histônicas ou acídicas), cuja variação é geralmcnte

proporcional à variação do volume nuclear. Êste tipo de proteínas acídicas são

recentemente consideradas como os fatores que deslocam a histona do DNA

(depressores), permitindo a função hcterossintética dos genes e. portanto, a pro¬

dução de proteínas específicas da função de cada tecido. Markcrt recentemente

(XVI Int. Cong. of Zoology, Washington) escreve textualmente: “If histones are

indeed inhibitors of gene function, then lhe removal of this inhibitors through

complexing with non histonie proteins (or RN A) may be lhe basic event in gene

activation”.

Opinião análoga é expressa por Moore (1966), em base às pesquisas de

Langan, que mostram ter a cromatina ativa o dôbro de fosfoproteínas por uni¬

dade de DNA do que a cromatina repressa. Frenster (1966) também acha que

as fosfoproteínas (não histônicas), que se encontram na cromatina expandida

ativa em quantidade maior do que na cromatina condensada, têm a função de

deslocar as ligações do DNA com as histonas que o reprimem.

Parece-nos, portanto, decididamente concordante o fato de que alguns tecidos

somáticos (pelo menos nos Vertebrados e Moluscos) têm volume nuclear maior e,

portanto, mais proteínas não histônicas do que a linha germinal. O fígado, de

modo especial, se distingue nesse sentido; devemos lembrar que êste órgão está

empenhado em um metabolismo extremamente mais complexo que as demais glân¬

dulas e, portanto, seu genoma deve ter um número maior de genes ativados em

interfase hcterossintética.

A êste propósito, uma observação na literatura sôbre a distribuição das en¬

zimas nos diferentes órgãos (tabela à página 452 e seguintes de West e Todd)

nos indica grosseiramente a quantidade de enzimas diferentes nos vários tecidos

dos Vertebrados. Resumimos aqui muito superficialmente esta situação, indican¬

do que existem 13 enzimas específicas do fígado, 8 do pâncreas e 3 do rim.

Êstes valores estão na relação em que se encontram os volumes nucleares destes

orgaos.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan GIORGIO SCHREIBER et al. 143

Simp. Internac.

33(1) :135-150, 19(i(i

Tudo isto parece se dar nos Vertebrados. Nos Artrópodos, pelo contrário,

como já mencionamos antes, o diferenciamento histógeno é acompanhado por um

grau específico de poliploidia ou politenia, e as variações de volume dos núcleos

entre os diferentes tecidos é paralela à variação do teor em DNA. Não quere¬

mos, portanto, generalizar demais o critério acima exposto da significação bio¬

química do hipermegetismo no diferenciamento histógeno. Devemos, talvez, lem¬

brar que os núcleos poliplóides dos Artrópodos têm uma grande parte de DNA

sob forma de heterocromatina; é possível pensar que os genomas múltiplos não

estejam lodos funcionantes contemporâneamente, permitindo à totalidade das pro¬

teínas não histônicas correspondentes manifestar a sua ação ativadora somente

sôbre os genomas funcionantes.

Podíamos traduzir a descrição destes fenômenos em termos da genética mo¬

lecular, considerando a “relação plóido-megética” como a expressão morfoquan-

litativa dos fatores de repressão e ativação dos genes durante a diferenciação dos

tecidos somáticos.

StJMMARY

In the precedent papers of this series, we have studied the DNA content per

per nucleus in the nuclear volumes in different tissues of Vertebrates and Molluscs.

The present paper deals with the same problems in Ophidia.

The theoretical basis of these researches consists in facts studied by Bloch in

which appears the existence of two types of interphasic growth of the nucleus

(autosynthetic and heterosynthetic). In the first one, the three fundamental cons-

titutions of the nucleus, i.e., DNA, histonic and non histonic proteins, are re-

duplicated. The nuclear volume follows, always, the quantitative variations of lhe

non histonic proteins. In the second type of interphase, there is a reduplication

of the non histonic proteins, only, and not of the DNA and histones. In this case,

the volume of the nucleus increases without the increase of the DNA content.

The comparative study of the DNA and of the nuclear volume reveals, thus, the

variations of the ratio “genome non histonic proteins”. These proteins have been

recently considered as the factors that free the genic DNA from its bound with

the histone and, thus, acting as “de-repressors”, allow the DNA to synthetize the

RNA, specific for the genic action.

The present research considers the ratio DNA/Volume in comparison with the

spermatogenetic stages (in which the DNA and nuclear volume are always in fixed

ratio), and with the somatic tissues, in which the nuclear volume can vary in-

dependelly from the DNA content (heterosynthesis).

Thus, in C r o t alu s and Philodry a s, we present here the regression line

between the DNA and the nuclear volume in the spermatogenetic stages, and in

some tissues (liver, pancreas, kidney, etc.). It is well evident, by these diagrams,

that the somatic tissues have, all of them, characteristic nuclear volume greater

than that of the spermatogonia (diploid typical cell). We call this DNA/Volume

ratio, as the “ploido-megetic ratio”.

We can correlate this excess of volume of the nucleus with the content in

non histonic proteins, in each somatic tissue, that can explain the “de-repressing”

action upon the specific gene loci.

Agrad eci meti los

Queremos, aqui, agradecer ao Dr. H. E. Belluomini, do Instituto Butantan,

pelo fornecimento do material ofiológico.

Agradecemos, também, ao nosso colaborador Prof. Luis Alexandre Fallieri, pela

revisão definitiva do texto.

SciELO

10 11 12 13

[vertebrados]

dosses de DNA

dasses de VOLUME

ARTROPODOS

Hipoderma

Intestino

Fibra muscular

Glândulas

Aaa

AAA

2n 4n 8n

1 1 1

I 2 4

dasses de DNA

I 2 4

classes de VOLUME

Fig. 1 — Curvas de freqüência do DNA e do volume nuclear

em diferentes tecidos de Vertebrados e de Artrópodos.

VOLUME

Fig. 2 — Diagrama de regressão entre DNA e volume nuclear na série espermato-

genética de Crotalns durissus terrificus (Laurenti).

SciELO

10 11 12 13 14 15

]46 PESQUISAS DE CITOLOGIA QUANTITATIVA. XIX — DNA E VOLUME

NUCLEAR NOS TECIDOS SOMÁTICOS DOS VERTEBRADOS

Fígodo

1)4 S < 7 9 9 10 II 12

i i t D II 'i IS 13 20 22 24 29 20 70 92 MK 1940

r .. h

1 3 4 5 t 7 • 9 10 II 12

024 I I iO 2 a

2 J 45 9 79 9I0III2

24 6 I 10 « 4 4 4 2022 24 29 M»l,

> J 4 5 « 7 I 9 10 II II

.Cl.

DNA VOLUME

Fig. 3 — Histogramas do DNA e do volume nuclear nos

diferentes tecidos de Crotalus durissus terrificus (Laurenti).

2 3 4

6 SciELO 10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 135-150, 1966

GIORGIO SCHREIBER et al.

147

Regressõo meiocitos

Fig. 4 — Diagrama de regressão entre DNA e volume nos tecidos somáticos de

Crotalus durissus terrificus (Laurenti). Cada retângulo representa a área de dis¬

tribuição de todos os valores de um tecido (“área ploido-megética”). A linha de

regressão representa a série dos meiocitos.

Fig. 5 — Diagrama de regressão entre DNA e volume nuclear

da série espermatogenética em Philodryas schottii (Sehlegel).

2 3

6 SciELO 10 11 12 13 14 15

PESQUISAS DE CITOLOGIA QUANTITATIVA. XIX — DNA E VOLUME

NUCLEAR NOS TECIDOS SOMÁTICOS DOS VERTEBRADOS

ONA ™ LU " £

Fig. 6 — Histogramas cio DNA e do volume nuclear na série

espermatogenética de Philodryas schottii (Schlegel).

ONA VOLUMt

Fig. 7 — Histogramas do DNA e do volume nuclear nos

tecidos somáticos de Philodryas schottii (Schlegel).

SciELO

10 11 12 13

dna

Mem. Inst. Butantan

Simp. Internac.

3»(1):135-150, 1966

GIORGIO SCIIREIBER et al.

149

Fig. 8 — Diagrama de regressão entre DNA e volume nuclear nos tecidos somá¬

ticos de Philodryas schottii (Schlegel). Os retângulos representam a área de dis¬

tribuição de todos os dados para cada tecido. A linha de regressão representa

a série espermatogenética.

Bibliografia

1. Atfert, M. — Variations in cytochemical properties of cell nuclei. Exytl. Cell.

Res., (Suppl. 61:227-235, 1958.

2. Alfert, M. & Bern, H. A. — Hormonal influence on nuclear synthesis. I, Es-

trogen and uterine gland nuclei. Proc. Nath. Ac. Sei., 37:202-205, 1951.

3. Anteunis, A. & Liu, S. L. ■—• Variations de la teneur en DNA des noyaux du

tissu interstitiel du testicle du rat blanc en fonction de 1’activité cellulaire.

Arch. de Biologie, 71:228-233, 1960.

4. Bloch, D. P. — Changes in desoxyribonucleic complex during the cell cycle.

In: Frontiers in Cytology, New Haven Univ. Press, Ed. L. Palay, 1958, pp.

113-166.

5. Bloch, D. P. & Hew. H. Y. C. — Changes in nuclear histones during fertiliza-

tion and early embryonic development in the pulmonate snail Helix aspersa.

J. Biophysical and Biochemical Cytology, 8:69-81, 1960.

6. Fautrez, J., Pisi, E. & Cavalli, G. — Teneur en acide desoxyribonucleique et

volume du noyau dans le pancreas exoerine du rat. Annales de la Société

Royale Zoologique de Belgique, 88:315-320, 1957/8.

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10 11 12 13 14 15

8 .

10 .

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27.

PESQUISAS DE CITOLOGIA QUANTITATIVA. XIX — DNA E VOLUME

NUCLEAR NOS TECIDOS SOMÁTICOS DOS VERTEBRADOS

Frenster, J. H. -— Ultra structural continuity between active and repressed

chromatin. Nature, 205:1341-1342, 1965.

Gerken, S. E. — Ploidia somática em Cricetus auratus (Citometria e DNA).

Ciência e Cultura, 14(4) :214-215, 1962.

Jacubj, W. — Uber das rhytmische Wachstum der Zellen dureh Verdoppelung

ihres Volumens. Roux’ Arch., 106:124, 1925.

Markert, C. L. — In: Ideas in Modern Biology. New York, The Nat. Hist.

Press, 1965.

Melucci, N. M. B. — Ploidia somática em Siphonops (Anf. Ap.) Citometria

e DNA). Ciência e Cultura, 14(4):214, 1962.

Moore, R. D. — Gene activation. Science, 152:548-549, 1966.

Roeis, H. — Cell activity and desoxyribonucleic acid content of the nuclei of

the thyroid gland of the white rat. Nature, 174:514-516, 1954.

Roels-de Schrijver, M. P. — Influence de 1'activité ceilulaire sur le teneur

en ADN et le volume des noyaux dans la medulle surrénale du cobaye. Arch.

de Biol., 72:174-184, 1961.

SanVAna, Y. X. & Schreiber, G. — Relação entre volume nuclear e DNA nas

classes submúltiplas cariométricas de Jacobj. Ciência e Cultura, 16(2) :179-

181, 1964.

Schrader, F. & Leuchtenberger, D. — A cytochemical analysis of functional

interrelations of various cell structures in Arvelius albopunctatus (De Geer).

Exptl. Cell. Res., 1:421-453, 1956.

Schreiber, B. & Angeletti, S. — Rhythmic increase and decrease of nuclear

volume of the hepatic cells of the Carp ( Cyprinus carpio var. spec.). Anat.

Record, 76:431-439, 1940.

Schreiber, G. — Ricerche di citologia quantitativa. Zeit. f. mikroskopisch-

nnatomische Forschung, 66(3) :301-328 (com bibliografia completa).

Schreiber, G. — The duplication series of some nuclear components. Anais

Acad. Bros. CL, 36(1) :73-84, 1964.

Schreiber, G. & Schreiber, M. R. — Diminuição rítmica do volume nuclear

do fígado e do pâncreas nos girinos de anuros. Boi. Fac. FU. Ci. Letr. Univ.

de São Paulo, 22.° — Zool. 5.°:234-264, 1941.

Schreiber, G. & Schreiber, M. R. — Researches on quantitative cytology. XVII.

The somatic ploidy in gland tissues of Gastropods. Rivista di Biologia, 57:265-

310, 1964.

Schreiber, G., Schreiber, M. R. & Carnevalli, N. E. D. — Researches on

quantitative cytology. XVIII. Somatic ploidy in the tissues of the “multifide

gland” of Vaginüla taunaysii Ferrussac. Riv. di Istochimica Nornmle e Pato¬

lógica (1965 — no prelo).

Schreiber, G., Melucci, N. M. B., Kaiserman-Abramof, I. R. & Memória, J.

M. P. — Researches on quantitative cytology. XIII. Quantitative variations

of the nucleolus in secretory cells. Symposium on Cell. Secretion — Belo

Horizonte, 1955- pp. 100-120.

Schreiber, G. & SanVAna, Y. X. — Volume nucleare e DNA negli epatociti

di Tilapia melanopleura (Duméril) durante lo sviluppo postembrionale. Acc.

Naz. Lincei, série 8, 39:348-351, 1965.

Valeri, V. — Estudo da relação volume nuclear/ácido desoxyribonucleico em

células normais e patológicas. Tese Fac. Med. Ribeirão Prêto, U.S.P., 1962.

Verwoerd-Verhoef, H. L. & Verwoerd, C. D. A. — Start and progress of in¬

crease of desoxyribonucleic acid content after stimulation of cell activity.

Nature, 193:1208, 1962.

West, E. S. & Todd, W. R. — Text book of Biochemistry, 2nd. ed., New York,

1956.

8 .

10 .

11 .

12 .

13.

14.

15.

16.

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18.

19.

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21 .

22 .

23.

24.

25.

26.

27.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 151 - 152 , 1966

WILLY BEÇAK, MARIA LUIZA BEÇAK and

HELENEIDE NAZARETH

151

18. EVOLUTION AND SEX CHROMOSOMES IN SERPENTES

WILLY BEÇAK, MARIA LUIZA BEÇAK and HELENEIDE NAZARETH

Secção de Genética, Instituto Butantan, São Paulo, Brasil

A basic karyotype consisting of eight pairs of macrochromosomes and ten

pairs of microchromosomes appeared to be possessed by the majority of snakcs.

Most of the species were studied by utilization of short term culture techniques

(Beçak et al., 1968, 1961). The diploid number of 88 chromosomes was found

in the family BOIDAE, species Boa constrictor amarali, Boa constrictor constrictor.

Eunectes murinus and Epicrates cenchria crassas; in the family COLUBRIDAE,

species Spilotes pullatus anomalepis, Spüotes pullatus maculatus, Drymarchon co¬

rais corais, Dryadophis bijossatus bijossatus, Chironius bicarinatus, Chironius qua-

dricarinatus, Philodryas olfersii olfersii and Philodryas aestivus, and in the familv

CROTALIDAE, species Lachesis mula, Bothrops jararaca, Bothrops alrox, Bothrops

alternatus, Bothrops jararacussu, Bothrops pradoi, Bothrops insularis and Crotalus

durissus terrijicus. Deviations from th is were encountered in lhe species Corallus

caninas (2n = 44) of the family BOIDAE, Micrurus lemniscatus (2n = 42) of

lhe family ELAPIDAE, and in the species Clelia occipitolulea (2n = 50). Oxy-

rhopus petola (2n = 46), Phrynonax sp. (2n = 88, Thamnodynastes strigatus

(2n = 82), Tomodon dorsatus (2n = 82), A enodoa merremii (2n = 80), Eri-

throlampus aesculapii venustissimus (2n = 28), Liophis milliaris (2n = 28).

Tropidodryas serra (2n = 28), Hydrodinastes bicinctus |2n = 24) and Lejosophis

gigas (2n = 24) of the family COLUBRIDAE (Beçak et al., 1962; Beçak. 1965).

The analysis of the diploid number and of lhe karyotypes of these snakes of the

family COLUBRIDAE, indicates that, the more evoluted lhe species, according

to syslematic criteria, the smaller lhe diploid number. In those species the re-

ductions in number are mainly due to reduction in the number of microchromo¬

somes (Beçak et al., 1965.

Uniformity of lhe suborder SERPENTES with regard lo the lolal genetic

content was estahlished not only for tliose species possessing lhe basic karyotype

hut also for lhose with deviating numhers (Beçak et al., 1964; Alkin et al., 1965).

In the ophidians the fourth largest pair of the basic karyotype is generallv

the sex pair regardless of family. In lhe primitive BOIDAE lhe Z and W are

still homomorphic to each other. Among the COLUBRIDAE initial steps lowards

the development of the heteromorphism hetween the male determining Z chromo-

some and the female determining W chromosome could he seen. The fourth pair

is still represented hy homomor|)hic chromosomes, in Pseustes sulphureus. Both

memhers of lhe fourth largest pair in the female were still the same in ahsolute

size, hut a pericenlric inversion appeared to have oceurred in the W chromosome

This work was supported by grants from FAPESP and FPIB.

SciELO

10 11 12 13 14 15

152

EVOLUTION AND SEX CHROMOSOMES IN SERPENTES

which is a subterminal element in lhe species Spilotes puüatus, Drymarchon co¬

rais, Dryadophis bifossatus, Chironius bicarinatus, Chironius quadricarinatus, Phi-

lodryas olfersii, Philodryas aestivus and Tropidodryas serra. Another approach

toward heleromorphism was taken in Clelia occipitolutea in which lhe W is twice

as large as lhe Z. In Tomodon dorsatus (2n = 32), Thamnodynastes strigatus

(2n = 32). Xenodoa merremii (2n = 30) and Liophis milliaris (2n = 28) of

the family COLUBRIDAE and all species of lhe family CROTALIDAE the highly

advanced poisonous snakes of lhe New World the W has become a distinctly

smaller element comparable in degree of specialization to the minute W of birds

( Beçak et <d., 1964).

Literature

1. Atkin, N. B., Mattinson, G., Beçak, W. & Olmo, S. — The comparative DNA

content of 19 species of placenta] mammals, reptiles and birds. Chromosoma

(Berl .), 17:1-10, 1965.

2. Beçak, W., Beçak, M. L. & Nazareth, H. R. S. — Karyotypic studies of two

species of South American snakes (Boa constrictor amarali and Bothrojis ja¬

raraca). Cytogenetics, 1:305-313, 1962.

3. Beçak, W., Beçak, M. L. & Nazareth, H. R. S. — Chromosomes of snakes in

short term cultures of blood leucocytes. American Naturalist, 97:253-256, 1963.

4. Beçak, W., Beçak, M. L., Nazareth, H. R. S. & Ohno, S. — Close karyological

kinship between the reptilian subordem SERPENTES and the class AVES.

Chromosoma (Berl.), 15:606-617, 1964.

5. Beçak, XV., Beçak, M. L., Nazareth, H. R. S. & Peccinini, D. — Chromosomes

of cold blood animais from whole blood short-term cultures. Microtechnique.

Mamm. Chrom. Newsl., 14:55-56, 1964.

6. Beçak, W. ■— Constituição cromossõmica e mecanismo do sexo em ofídios sul-

americanos. I. Aspectos cariotípicos. Mem. Inst. Butantan, 32:37-78, 1965.

7. Beçak, W., Nazareth, H. R. S. & Beçak, M. L. — Variação da constituição cro-

mossômica em colubrídeos (COLUBRIDAE, SERPENTES). Ciência e Cultura,

17:148, 1965.

1, | SciELO

Mem. Inst. Butantan

Simp. Internac.

33(1): 153-154, 1966

MANUEL O. DIAZ and FRANCISCO A. SAEZ

153

19. KARYOTYPES OF SOUTH-AMERICAN ARANE1DA

MANUEL O. DIAZ and FRANCISCO A. SAEZ

Departamento de Citogenética, Instituto de Investigación de Ciências Biológicas,

Montevideo, Uruguay

The karyotype constitution of eleven species of AIÍANEIDA belonging to

eight families vvas studied. The data ahout chromosome number, and sex de-

termination system obtained from this study are summarized in Table E

TABLE I

Species

2 n

Sex determ. system

Fam. DYSDERIDAE

Dy der a magna Keys

x-o

Fam. SEGESTRIDAE

Ariadna mollis

X-O

Segestria ruficeps

xx-o

Fam. SICARIIDAE

Scytodes maculata

xx-o

Loxosceles rufipes

xx-o

Fam. AMAUROBIIDAE

Amaurobius simoni

xx-o

Fam. SPARASSIDAE

Polybetes pitagorica

xx-o

Fam. LYCOSIDAE

Lycosa erythrognata

xx-o

Lycosa nordenskõlii

x-o

Fam. THERIIDIDAE

Theridium tepidariorum

xx-o

Fam. ARGIOPIDAE

Metepeira lathyrina

xx-o

In the karyotypes of Scytodes maculata and Loxosceles rufipcs all the auto-

somes are metacentric and the Xs acrocentric. The location of lhe centromere

in Dysdera magna, Ariadna mollis and Segestria ruficeps cannot be clearly de-

termined because of the absence of centromeric constrictions or angulalions during

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10 11 12 13 14 15

154

KARYOTYPES OF SOUTII-AMERICAN ARANEIDA

gonial divisions. Meiotic bivalents are very contracted and their configuration

suggest a metacentric nature. Neverlheless, this assumption does not expiain tlie

behaviour of lhe chromosomes in gonial divisions where lhere is no polarization

of lhe eentromeric regions during anaphases. For explaining lhese peculiarities

it is necessary to assume they have a dijjuse centramere of the type observed

in Ti l/y us bahiensis by Piza. (1)

In lhe other species studied all lhe chromosomes are acrocentric.

Mulliple X sex determinalion system was observed in eight of lhe studied

species and X-0 Systems only in three of them. The Xs are acrocenlric in all

species studied except in those belonging lo DYSDERIDAE and SEGESTRIDAE

where centromere position is uncertain. The Xs in Scytodes maculata show a

strong heleropycnosis from diplotene until second anaphase and are paired by

lheir proximal ends during all ihis period. In gonial divisions the Xs of 5 c y-

t o d c s are free and isopyctonic.

The higher chromosome numbers were observed in Amaurobius simoni and

Polybetes pitagorica with n = 21 and 22 respectively, but in account of chromo¬

some arms number Loxosceles rujipes is close lo the former with n = 20. Ly-

cosa nordenskôlii show a sharp reduction in the chromosome number relative to

the modal number of the family n = 12 (Suzuki (2)) and have an aberrant

X-o system for the family. The lower chromosome number was found in Dys-

d e r a and Ariadna wilh an n = 5. Suzuki (2) has described a karyotype

with n — 4 for Ariadna lateralis, a related species. Evidently in lhe families

DYSDERIDAE and SEGESTRIDAE there is a trend loward extreme chromosome

reduction. This may well he the effect of a diffuse centromere system.

The chromosomes of Loxosceles rufipes have been studied previously by Be-

çak and Beçak (3), and our results confirm their findings. The chromosomes

of Theridium tepidariorum have also been studied by other authors: Haekman (4),

Monlgomery (5) and Suzuki (2), and also in this case we do no more than con-

firming their descriplions.

References

1. Piza, S. de Toledo — J. Hered., 32:423, 1941.

2. Suzuki, S. — J. Hiroshima Univ., B.l, 15:24, 1954.

3. Beçak, W. & Beçak, M. L. — Rev. Bras. Biol., 20:425, 1960.

4. Haekman, W. — Acta Zool. Fenn., 54:1, 1948.

5. Montgomery, T. H. — Zool. Jahrb. Anat. u. Ontg., 25:237, 1907.

í, | SciELO

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20. EVOLITION OF VEHTEBRATE GENOMES

SUSUMU OHNO

Department of Biology, City of Hope Medicai Center, Duarte. U.S.A.

The Darwinian eoncept of evolulion lias revealed lhat speciation lias been

dependent upon lhe process of natural selection. Natural selection, in turn, can

lie effective only if lhere is hereditary variahility among individuais comprising

the population. The identical genetie eonstitution offers uo chance for natural

selection to operate; thus, evolulion is <| 11 i te obviously the consequence of genetie

changes lhat have accumulated within the genome. The genome can he defined

as a set of genes contained within the haploid set of chromosomes of an organism.

In the case of speciation from an immediate ancestor, genetie changes are no

doubt due mainly lo allelic mutations at the already existing gene loci. What

used to he a rare mutanl allele of the ohl species would become the wild-type

alicie in a new species. For instanee, a major component of adult hemoglohin

of man and cattle is /? 2 . Alpha- and beta-chains are different polypetides

produced by two independent gene loci. The alpha-chain in man, however, lias

an aniino acid sequence different from lhat of the cattle alpha-chain. Apparently,

a common ancestor to diverse species of placental mammals already had the gene

loci for two component polypeplides of adult hemoglohin. A series of mutations

at each of these two gene loci finally gave rise to lhe genes for alpha- and lieta-

cliains of today’s human. Another series of mutations at the same gene loci,

on the other hand. produced the gene for alpha- and beta-chains of the cattle.

When the scope is

V E R T E fl R A T A as

cannot possihly accounl

past 300 million years.

vertehrates are incapable

hroadened lo consider the evolulion of the sub-phylum

a whole, allelic mutations of already existing genes

for all the genetie changes that occurred during the

There apparently were creations of new gene loci. fn-

of producing anlibodies as such. The gene loci for

light- and heavy-chains whicli comprise y-globulin molecules were obviously created

f/c novo at the heginning of the vertehrate evolulion.

created

tion of

The crealion of new genes i

evolution within the phylum.

faclor

anew out of the hlue

lhe ohl which already

w' emerges as the most important single

In the biological System, however, nothing is

sky. The new material is produced by modifica-

existed.

IMPORTANCE OF GENE DÜPLICATION IN VEHTEBRATE EVOLUTION _ The eX-

tensive study carried out by Margoliash (1963) on molecular struetures of Cyto-

chrome C revealed the extremely conservative nature of the gene. Cytochrome C

lhe

revei

heme-containing

protein which engages in lhe intracellular transportation

This vvork was supported in part by a grant (CA-05138) from the U.S. Public Health

Service.

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EVOL.UTION OF VERTEBRATE GENOMES

oí oxygen. As such, it must have come to the existence soou after the cells

made lhe firsl appearance on lliis eareh as the unit of life. Yet, il was found

that Cytochrome C of diverse organisms, from yeasts lo mau. nol only have

nearly lhe same molecular weight hui also maintain similar amino acid sequeuces,

in eaeh instanee ahout 101 amino acid residues making up a polypeplide ehain.

The elear implieation is that the particular function assigned to the gene produet

imposes a severe limitation on that gene’s freedom to mutate. lf a change in

the hase sequence occurred at lhe wrong pari of the DNA moleeule, a new gene

produet would be unable to function as Cytochrome C. Such a mutation would

quickly be eliminated.

Natural seleclion eonserved only lhose mutations which were not deteriorative

lo the gene product’s assigned function. The extremely conservative nature of

the already existing genes indicate lo us that the redundancy of genetic material

was the prerequisite for the ereation of new genes. By duplication, if the old

gene had been represented twice within lhe genome. one of the duplicates was

now free to mutate lo an independenl direetion and acquire a new function.

Jn man and probably most other mammals, there are five independent gene

loei for component polypeptides of hemoglohin. They are for «-, c-, y-, /?-, and

8-ehains.

It is lhe view of Ingram (196H) that the aneestry of all the five genes for

five differenl eomponents of hemoglohin can be traced haek to a single ancestral

gene. Firsl, there was a duplication of this gene and by subsequent mutations

to independent direetions, one became a gene for myoglobin while the other be-

eame a gene for «-ehain of hemoglohin. The ancient vertebrate when firsl

emerged may have heen ahle lo produce only one type of a hemoglohin moleeule

which should be a 4 . The genes for four other chains of hemoglohin are thought

lo have heen derived from the multiplicates of the gene for «-ehain.

Similarly, mammals and hirds have three independent gene loei for compo¬

nent polypeptides of the enzyme. lactate dehydrogenase. They are known as A.

li. and C (Markert, 1964; Blanco and Zinkham, 1962; Blanco et al.. 19641.

Originally there may have been only one gene locus foi LDH, and the other

two may have heen produeed by duplication of lhe orginal one.

The gene duplication can be accomplished in two differenl ways. The lon¬

gitudinal duplication of a small segment of an individual ehromosome would

accomplish lhe purpose for a small number of genes closely linked together. In

faet, regional duplication of small ehromosomal segments appears to be occnrring

among mammals of loday. For instanee, y-globulin moleeule is made ol two

differenl kinds of polypeplide chains: lhe heavy-chain (Hl with a molecular

weight of ahout 60,000 and the light-chain (L) of ahout 20,000. In man. il is

beeoming inereasingly elear that, instead of having one gene locus eaeh for the

II- and L-chains, the so-called H-chain locus is actually made of several very

closely linked but slightly differenl genes; lhe same can be said of lhe so-called

I.-ehain locus. There apparently were longitudinal multiplication of one ancestral

gene for the H-chain and the other ancestral gene for the L-chain. In man, the

genes for ft- and 8-chains of hemoglohin are also very closely linked. The 8-

chain gene must have been derived by a regional duplication of the /?-chain gene.

While regional duplication of a small number of genes might have played

an important role in speeiation from an immediate aneestor, more draslie ehanges

must have occurred to lhe genomes during the course of vertebrate evolution.

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Simullaneous duplication of the entire set of genes ean be accomplished liy poly-

ploidization. ll can be assnmed with reasonable certainty that a series of poly-

ploidization of lhe ancestral genome have taken place somelime in the hislory

of vertebrates vvhich is. afler all. 300 million yeavs old.

I.NCOMPATIBIUTY BETWEEN POLYPLOIDY AM) THE WELL ESTARLISHED CHROMO-

SOMal sex-determining MECHAMSM — Polyploidy. however, is incompatible with

lhe well established chromosal sex-determining meehanism. When the diploid

organisms with lhe XY/XX-seheme of sex-determining meehanism become tetra-

]iloid, the male has lo maintain lhe 4AXXYY-constitution and the female,

1AXXXX. Dnring meiosis of the 4AXXYY-male, the fonr sex elements may

pair off as lhe XX-bivalent and the YY-bivalent. If such occurs, every gamete

vvould be of the 2AXY-eonstitulion. Consequently, all the offspring resulting

from the mating between a tetraploid male and a tetraploid female would emerge

as lhe intersex of the 4AXXXY-eonstilution. Even if two XY-bivalents are

formed in individual spermatocytes of lhe tetraploid male, in 50 per cent of the

cases lhe X and lhe Y would move lo the same division pole at first meiotic ana-

phase. again resulting in the production of the intersex of the 4AXXXY-cons-

titution. Polyploidy invariably disturbs the chromosal sex-determining meehanism.

Indeed, among vertebrates viable and fertile polyploid individuais have been

found among amphihians where lhe Z and the W are still largely homologous to

eaeh olher, but nol in birds and mammals where the W and the Y became a

highly specialized delerminer of the heterogametic sex. Furthermore, it was found

that even in amphihians, polyploid individuais are, as a ride, incapable of per-

petuating themselves as polyploid by bisexual mating.

ludied by Hum-

Fankhauser and

Polyploid individuais of amphihians were most lhoroughly

phrey and bis colleague (Humphrey and Fankhauser, 1956;

Humphrey, 1959) on Ambystoma mexicanum and A. tigrinum.

In the case of triploidy, males were uniformly of the 3AZZZ-eonstitution.

while three kinds of sex chromosome constilutions, ZZW, ZWW, and WWW were

found among females. Triploids of both sexes were of very poor fertility, and

males were more sterile than females; lherefore, it was not possible to perpetuate

lhe triploid race by mating of triploid males and triploid females. When mated

lo diploid males, triploid females produeed many tetraploids, revealing that these

females ovulate triploid eggs. This may account for the presence of a gynogenic

all-female triploid race found in Ambystoma jejjcrsonianum (l zzell. 1963). Tetra¬

ploid. pentaploid, hexaploid, and heptaploid individuais of A. mexicanum and

/. tigrinum also showed very poor fertility. In short. it appears that even in

amphihians with the undifferentialed sex chromosomes, lhe serious obstacle whieh

prevenis the emergence of a bisexual polyploid race exisls.

From the above, it may be deduced that various degrees of polyploidization

of lhe ancestral genome must have occurred very early in evolution of vertebrates

before the emergence of terrestial fornis.

Our study on DNA-conlents of various vertebrates confirms the above pre-

diction and reveals lhe polyphyletic origin of the genomes of various vertebrates.

UNIFORMITY OF THE DNA CONTENT OF VARIOUS PLACENTAL MAMMAI.S — All

placental mammals of today descended from a common stock of protoinsectivores

whieh emerged at the dawn of the Cezonoic era. In terms of geological time.

the history of placental mammals is brief indeed. Reflecting lliis recent origin

SciELO

10 11 12 13 14 15

EVOLUTION OF VERTEBRATE GENOMES

is the sameness of DNA content. Diverse speciation appeared to lio accomplishod

with lillle or no change in lho total genetic content. Mandol and his colloagues

(1950) were among tho first to show that each diploid nucleus of man as woll

as eallle, sheep, ])igs, and dogs oontains ahout 7.0 mg X 10 -!l of DNA, no moro,

no loss. Kocontly, we restudied lhis matter of DNA constancy by moans of

microspoctrophotomotry. Six species representing foui difforont orders were

chosen: man ( Honro sapiens, 2n = 461 representing the order PKIMATES, the dog

( Canis jamiliaris, 2n = 78) representing the order CAHNIVOKA, the horse

(Equus caballus, 2n = 64) of tho order PEKISSODACTYLA, the mouse ( Mus

musculus, 2n = 40), tho golden hamster ( Mesocricetus auratus, 2n — 44), and

the creoping vole ( Microlus oregoni, 2n = 17/18) of tho order RODENTIA. Thore

was no significant difference in DNA valuos between man, the horse, tho dog,

the golden hamster, and the mouse. A single exeoption was the creeping vole

which had a DNA value 10% lower. This species (Olmo et al., 1963) shares

with tho olher membor of tho rodont suhfamily Microtinae, Ellobius lutescens,

2n — 17 (Matlhey, 1953) the distinction of having the lowest diploid chromo-

somo niimher known among placental mammals. Such a drastio reduction in tho

number of chromosomes had lo ho accompanied by tho loss of a number of con-

tromeres with their adjacent hotorochromatic ohromosomal materiais. This loss of

gonotioally unimportant hoterochromatin would account for the 10% lower DNA

value found in tho creoping vole (Alkin et al., 1965).

It thon follows that difforont species of mammals, by and large, sliare tho

same kinds of gene looi, even if thoy belong to different orders. Allelic muta-

tions at each gene locus were mainly responsihlo for extonsive divorsification of

placental mammals.

I ho sameness of total genetic content, however, does not exclude the pos-

sihility that duplioations of a small number of genes rnay have occurred during

speciation of mammals.

Regional gene duplioations which occurred to a small number of genes of

difforont mammals, however, do not change tho over-all picture of mammalian

evolution. Extonsive speciation of placental mammals was accomplishod without

substantial change in tho total genetic content. Yet, placental mammals of today

display chromosomo constilutions of infinito varioty. The diploid number ranges

Irom a high of 80 in the primitivo primato, Tarsius bancarias (Klinger, 1963)

lo a low of 17 in two rodont species mentionod above. Nothing hut acrooontric

chromosomes aro found in tho mouse (Mus musculus, 2n = 40), and only meta-

contries in lho chinchilla (Chincilla laniger, 2n = 64) (Galton et al., 1965).

lho enormous array of karyotypes revoais tho oxtont to which lho original aulo-

somal linkage groups of a common ancestor lias boen shuffled around. An

autosomal oquivalont to lho human chromosomo 21 may only he found amona

his closest relativos, the chimpanzee ( l‘an troglodytes, 2n — 48 ) and tho gorilla

( GorUla gorilla. 2n = 18) (Hammorton et al., 1963).

I MFORMITY OF THE DNA CONTENT OF VARIOUS AVIAN SPECIES — 11 is 1)0-

liovod lhat ancestral forms of modern hirds were already in existence near the

ond of tho Jurassic period of lho Mesozoic era. Tho fóssil remains of tho toolli-

od bird, Archeopteryx lithographia found in slate deposits in Bavaria is said

lo he 150 million years old. Thus, il is olear that tho avian linoage hranchod

out frorn a reptilian linoage hefore tho olher reptilian linoage gave riso lo a com¬

mon anceslor lo placental mammals.

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Mem. Inst. Butantan

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SUSUMU OHNO

159

Reflecting this independent evolution of the Ivvo classes of warm-blooded ver-

tebrates is lhe faet lhal the male is lhe helerogamelic sex in mammals, while in

hirds il is lhe female which is llie helerogamelic sex. The avian chromosome

complements are also distinct from lhose of placental mammals in lhal they include

mimerous microchromosomes, each no larger than one micron in size.

In our experience. lhe diploid complements of present day hirds helonging

to the orders PASSERIFORMES, COLUMBIFORMES, GALLIFORMES, and ANSE-

RlFORMES followed lhe common rale in lhal nine pairs of macrochromosomcs

or ordinary chromosomes and about 60 microchromosomes constituted each diploid

complement. Members of the ordem PSITTACIFORMES were exceptional. hav-

ing more macrochromosomcs and fewer microchromosomes. For instance, twelve

pairs of macrochromosomcs and about 18 pairs of microchromosomes constituted

lhe diploid complement of lhe Australian parakeet, Melopsittacus undulatus (Olmo

et «/., 1964).

Relative DNA values were measured on the canary representing the order

PASSERIFORMES, the chicken ( Gallus gallus domesticas ) representing the order

GALLIFORMES, the pigeon ( Colnmba livia domestica ) of the order COLUMBI¬

FORMES, and the Australian parakeet of lhe order PSITTACIFORMES. As the

extreme similarity in their diploid chromosome complements already indicated the

uniformity in the total genetic content of various avian species, the ahove four

speeies representing four diverse orders were deemed sufficient.

As expected. four represcntatives of the elass AVES gave the imiíorm DNA

value. The value. however, was 44-59% lhal of placental mammals.

The ahove finding on lhe total genetic content of avian species, on one liand.

reveals lhe faet that polyploidy played no role in extensive speciation within the

elass AVES and, on lhe other liand, shows that the genome lineage which gave

rise to the elass AVES lias long been in separation from that which eventuallv

gave rise to placental mammals.

The COEXISTENCE OF THE TWO GENOME LINEAGES IN THE CLASS REPTI-

I, IA — Reptiles of lo day ean be compared with the twigs of a great tree which

flourished during lhe early Mesozoic era. About 95% of all the different kinds

of living reptiles belong to lhe order SQUAMATA, yet fóssil remains indicate that

this order never held greater importance than today. On the contrary, fóssil heds

in many parts of lhe world are strewn with shells of many kinds of lurtles. The

orders CROCODYLIA and CHELONIA had seen better days.

We have no way of directly assessing lhe geiiomes of ancient reptiles which

constituted the huge limhs of a great tree and which produeed the direct aneestor

to lhe ])lacental mammals on one liand and that to the hirds on the other. It

was fortunate that the studies on relative DNA content and chromosome constitu-

tions of a limited niimber of living reptiles enahled us to discern the presente

of two different genome lineages, one showing close affinity lo that of the

elass AVES and the other to that of the elass MAMAI AI. IA.

As stated earlier, the abundant presence of microchromosomes characterize

the avian chromosome complements. It has been known for some time that micro¬

chromosomes are also possessed by lizards and snakes which constitute the ordem

SQUAAIATA. While the exact number of microchromosomes in the diploid com¬

plement of each avian species is nearly impossible to determine, the number of

microchromosomes in each lizard or snake species can be determined with ease.

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EVOLUTION OF VERTEBRATE GENOMES

The diploid eomplement of lhe alligalor lizard ( Gcrrlionolus multicarinalus, 2n

46-48) helonging to the family ANGUIDAE of the suborder SAURIA, eontains

12 pairs of microchromosomes, while a great majorily of snakes constituting the

suborder SERPENTES, possess 10 pairs of microchromosomes.

Asidc from lhe possession of microchromosomes, thcrc is yet another common

charactcristic which rcvcals that the reptilian order SQUAMATA, suborder SER¬

PENTES in particular, belong to lhe very same genome lineage wbich gave rise

to the class AVES. The female heterogamety of lhe ZZ/ZW-type also operates

in snakes (Beçak et al., 1962: Kobel, 1962). Furthermore, the avian Z-chromo-

some and the ophidian Z-ehromosome may have hecn derived from lhe same

ancestral chromosome, as both constitute about 10% of lhe genome or haploid

set (Beçak et al., 1964).

DNA content was measured on six representatives of lhe order SQUAMATA

which were: the chameleon lizard ( Anolis caroUnensis, 2n = 86) of lhe family

IGUANIDAE and lhe alligalor lizard (Gerrhonotus miãticarinalus, 2n - 46) of

the family ANGUIDAE of the suborder SAI RIA. The suborder SERPENTES

was represented by lhe Boa constriclor (Boa constrictor amaruli, 2n = 36) of

lhe family BOIDAE, the gopher snake (Dryinarchon corais couperi, 2n = 36),

and the South American A enodoa ( Xenodon merremii, 2n — 30) of lhe

family COLUBRIDAE, and the South American jararaca (Bothrops jararaca. 2n —

- 36j of the family CROTALIDAE. These six representatives of lhe order

SQUAMATA demonslrated a DNA value of 60-67% that of placental mammals.

The value ohtained was only slightly more than that obtained for various avian

species which was 44-59% that of placental mammals (Atkin et al., 1965).

While the above finding should nol be inlerpreted to mean that lizards and

snakes of today were directly ancestral to birds, it reveals that an ancestral rep-

lile which evolved lo toothed birds belonged lo the same genome lineage which

independently gave rise to ancestral forms of modern members of lhe order

SQUAMATA: lhere was no further polyploidization of lliis genome lineage.

Among members of the order SQUAMATA the well differentiated heteromorphic

Z- and W-chromosomes are seen only in the poisonous family CROTALIDAE and

certain members of the family COLUBRIDAE of the suborder SERPENTES;

others possess lhe primitive homomorphic sex elements. Yet. there apparently

exist the effective barrier to preveni lhe evolution of a bisxual polyploid species.

Triploid species of the Teiid lizard of the genus Cnemidophorus were all

females and apparently propagated by parthenogenesis (Pennock, 1965).

While present day members of lhe reptilian order SQUAMATA demonslrated

the dose kinship lo the class AVES members of the order CKOCQDYLIA and

CHELONIA appeared to represent the pre-mammalian genome lineage.

The SouIh American alligalor (Caiman sclerops, 2n = 42) representing lhe

order CKOCQDYLIA gave the DNA value as 84% that of placental mammals.

The diploid chromosome eomplement of Ihis species is tolally different in character

from those of snakes and lizards. In fact, lhere is a striking resemblance be-

tween the diploid eomplement of Caiman and that of one species of mammals.

the ral ( Raltus norvegicus, 2n — 42). To be sure, this extreme similarity is a

pure coincidence. Neverlheless, there is little douht that among present day

members of reptiles, those helonging to the order CKOCODYLIA demonstrale the

closest kinship with placental mammals, nol only in DNA value hut in karyo-

logical characteristics as well. Although the lower diploid chromosome number

ol 32 has been reporled on the Norlh American alligalor ( Alligalor mississippien-

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SUSUMU OIINO

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(Crocodilus niloticus) , tliis reduction in chromo-

appears to be the resull of simple Kobertsonian

pairs of acrocentrics of C a i m a n are represenled

A II i gato r and Crocodilus (Hollingsworth,

sis ) and the African erocodile

some number from 42 to 32

translocalions. The 10 largest

as 5 pairs of metacentrics in

1957; van Brink, 1959).

DNA value similar to that of placental mammals was also obtained on re¬

presentativos of the order CHELONIA. The fresh-water soft-shell tnrtle ( Amyda

ferox, 2n = 66) and the desert tortoise ( Gopherus agassizi, 2n = 52) gave DNA

value 80 and 89% that of placental mammals, respeotively. While observing

Iheir metaphase figures, however, it was noted that their karyological character-

istics are not at all similar to those of placental mammals. Many small memhers

can be regarded as microchromosomes. ll appears that memhers of the order

CHELONIA demonstrate the doses t karyological affinity to lhe infraclass PtiO-

TOTHERIA, rather than lo either marsupiais or placental mammals. A rather

high diploid chromosome number of about 70 and 63 has been found in the

duck-bill platypus [Ornithorhynchus anatinus) and the spiny anteater ( Tachy -

glossus aculeatus) ol the order MONOTREMATA. Many small memhers can be

regarded as microchromosomes (Malthey. 1949; van Brink, 1959).

ll would bo of utmost interest lo find out if the male heterogamety of lhe

XY/XX-type operates in memhers of the orders CROCCDYLIA and CHELONIA

which represent the premammalian lineages. Unfortunately, the heteromorphic

sex elements have not been found in these reptiles. No sex-linked gene is known.

and the sex reversal experiments have not been performed on any of these

species.

Extremei.y high DNA values possessed by certain amphibians which

SLGGEST THE POLYPHYLETIC ORIGIN OF TERRESTRIAL VERTEBRATES — It is known

that birds, snakes, and lizards of today are the branches of one limb which

originated from the ancestral reptile O r n i t h o s u c h u s mammals emerged

from the olher limb which was started from Lycaenops. The fact that

surviving memhers of the classe REPTIL IA fell discretely into two groups

(one group belonging to the preavian genome lineage and the olher gronp be-

longing to the premammalian lineage) may be taken as an evidence that Or-

nithosuchus and Lycaenops of ancient times already belonged to the

two different genome lineages.

were derived from ancient amphibians grouped together

appears that Labyrinthodonts were of many kinds

re-

Reptiles, in lurn

as Labyrinthodonts.

presenting diverse genome lineages. Most, if not all, of the amphibians of today

belong lo the genome lineages independent from both the preavian and premam¬

malian lineages.

The most comprehensive survey on DNA values of various amphibians was

carried out by Joseph Gall of Yale l niversity; his results are quoted here witli

his kind permission. All lhe amphibian species surveyed by him demonstraled

higher DNA values than that of placental mammals. DNA values demonstraled

by tailless amphibians constituting the order SALIENTIA were still not as fan-

tastically high as those demonstrated by memhers of the order CAUDATA.

Within the order SALIENTIA, the American toad [Bufo americanus, 2n = 22)

representing the suborder PROCOELA gave the DNA value 137% that of

placental mammals. The DNA value of the Leopard frog ( liana pipiens, 2n = 26)

and lhe

frog ( Rana catesbiana, 2n = 26) of the suborder DIPLASIOCOELA

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EVOLUTION OF VERTEBRATE GENOMES

was 200% of lhe DNA value of plaeental mammals. In terms of lhe ahsolule

content, lhe íaniily RANIDAE contained I 1.6 mg X 10 ” DNA in each diploid

nucleus. Whilc thesc values are high, lhey show close enough affinity to lhe pre-

inarnmalian lineage. ll is expected lhat if a Iruly extensive survey is done on

tailless amphihians of today, lhe D\A value very similar lo thal of lhe premam-

malian lineage can he found in some of lhem.

On lhe contrary, memhers of lhe order CAUDATA showed ahsolutely no af-

finily to either lhe premammalian or lhe preavian lineages. Within this order.

lhe lowest DNA value was found on lhe newt (Triturus cristatus, 2n = 24) of

lhe suborder SALAMANDHOIDEA. Yet, it was 830% lhat of plaeental mam-

mals, and its close relative, Triturus viridescens (2n = 22 I revealed even higher

DNA value of 1300%. The Congo eel (Amphiuma means, 2n = 24) of lhe sub¬

order PROTE1DEA, had lhe fantastically high DNA value of 2700% thal of

plaeental mammals.

Anolher inleresting aspect of tailed amphihian genomes is lhat two closely

related species helonging to lhe same family often demonstrated a remarkahle

difference in lheir DNA values. For instance, Triturus cristatus and Triturus

viridescens belonged lo lhe same family SALAMANDRIDAE, yet lhe DNA value

of lhe lalter was 50% grealer than lhat of lhe former, despite lhe fael lhat holh

had lhe very similar diploid complements. Joseph Gall found lhat each lamp-

brush hivalenl of lhe latler was longer and had more loops than its counlerpart

of lhe former. On this hasis, he believes that lhe increase in DNA value is due

to regional duplicalion of chromosomal segments lhat occurred lo Triturus viri¬

descens.

It has heen shown that lhe / and lhe W or lhe X and lhe Y of amphihians

are in such a primitive stale of differentialion. The \Y or lhe Y is still a genetical

equivalent of lhe Z or lhe X. This primitive state of sex chromosomes may

permit polyploid evolution lo exeeptional memhres of present day amphihians.

One species might represent a newly arisen tetraploid stale of lhe old diploid

species. The pioneering study by Saez (1964) has indicated that polyploid

evolution may have occurred lo South American frogs helonging to lhe family

CERATOPHRYDAE. Indeed, lhe tetraploid nature of Odontophrynus americanus

has heen proveu heyond any doubt by M. L. Ileçak and her colleagues al this

symposium. The 44 chromosomes can he arranged to 11 differenl kinds of

homologues, and 11 quadrivalents rather than 22 hivalents were seen in meiosis.

Thus, among amphihians of today, lhe increase of DNA content by holh regional

duplicalion and polyploidization might still he oecurring to some extent. Never-

theless, so far as memhers of lhe order CAUDATA are concerned, it is clear that

lhey helong to the genome lineage or lineages altogether differenl from holh the

preavian and premammalian lineages.

DlVERSE GENOME I.INEAGES FOUND AMONG FISHES - The inevilahle conclusiou

to he drawn from the above survey on DNA values of lhe four classes of ter-

restial vertehrates is that lhe evolution from Crossopterygian fishes to Lahyrinlho-

dont amphihians was polyphyletic. Today, lhe suhclass ClíOSSOPTEKYGII

is represented only by the lung fish of lhe order DIPNOI and lhe coelocanth of

the order ACTINISTIA. These surviving memhers of the lobe-finned fish must

merely represent a fraetion of the diverse genome lineages which were possessed

by ancienl Crossopterygian fishes ancestral to terrestrial vertehrates. As much

as wc have no way of obtaning the information on genomes from the fossils, we

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SUSUMU OHNO

163

must turn to members of lho ray-finned fish conslituting lhe suhclass NEOPTE-

HYGII as lhe source of indircct information on ancient genome lineages.

Our study, although limited to eight species of lhe class P I S C E S , appeared

lo eonfirm the polyphyletic origin of terreslrial vertebrate genomes (Ohno and

Alkin, 1966).

It was found that surviving members of the order DIPNOI, the suhclass

CROSSOPTERVGII shovv dose kinship only to tailed amphibians (the order

CAUDATA). The DNA value, 3540% thal of mammals, was obtained on the

South American lung fish (Lepidosiren paradoxo. 2n = 38). According to

Alfrey rt al. (1955), the. absolute DNA value for the African lung fish (P r o ■

topterus, 2n = 34) was 100 mg X 10 _!) which is about 1400% thal of

mammals. The relatively low diploid chromosomes number, the absence of acro-

centrics, the enormous size of individual chromosomes, and lhe very liigh DNA

value found in the lung fish are all precise characteristics of the genomes main-

tained hy present-day memhres of lhe order CAUDATA of the class A M PH I BIA .

Although the chronology of evolution suggesls that the lung fish could nol have

been lhe direet ancestor of the tailed amphibians, it is apparent that hoth belong

lo the same particular genome lineage. This lineage is not directly related to

lhe main genome lineages which gave rise to tailless amphibians, reptiles, birds,

and mammals of today.

The DNA values which demonstrated lhe close kinshij) lo the premammalian

and preavian genome lineages were found among members of the suhclass

N E O P TERYGII.

The rainbow trout ( Salmo irideus, 2n = 58-64) is lhe anadromous species

belonging to the family SALMONIDAE of the order ISOSPONDYLI. The DNA

value, 80% that of mammals, corresponded wcll with the values possessed hy the

orders CKOCODYLIA and CHELONIA of the dass REPTILIA; thus, this

species and other members of lhe family SALMONIDAE may be regarded as be¬

longing to the premammalian lineage. It is not my intention lo imply that trouls

and mammals constilute one direet line of descenl. My view is that crocodiles,

turtles, and mammals of today descended from a particular group of ancient

Crossopterygian fish which already possessed the DNA value similar to that of

trouts.

The DNA value similar to that possessed hy lhe class AVES as a whole

and also by the order SQUAMATA of lhe class KEP TIL IA was found on the

goldfish ( Carrasius auratus, 2n = 96-104) of the family CYPRINIDAE, the order

OSTAItlOPHYSI. The DNA value obtained on this species was 52% that of mam¬

mals. Thus, members of lhe family CYPRINIDAE may be regarded as belonging

lo the preavian genome lineage.

Our study on various members of the suhclass N E O P T E R Y G 11 further re-

vealed lhe presence of DNA values mueh smaller than any of lhe values possessed

by terrestial vertebrates. Our notion that a series of polyploidization of an an¬

cestral vertebrate genome occurred while vertebrates were still in aquatic forms

appeared to be confirmed.

The DNA value of only 30% that of mammals was obtained on two members

of the order PERCIFORMES. The green sunfish ( Lepomis cyanellus, 2n = 46-48)

of the family CENTRARCHIDAE and the discus fish ( Synphysodon aequifasciata,

2n = 60) of the family CICHLIDAE.

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EVOLUTION OF VERTEBRATE GENOMES

The lowesl DNA value, only 20% that of mammals, was foimd arnong two

diverse groups of fishes. This value was obtained ou lhe swordlail ( Xyphopho-

ms hellerii, 2n - 481. hornyhead turbot 1 1‘Ieuronichthys verticalis, 2u = 18),

and fantail sole ( Xystreurys liolepis, 2n = 48).

From lhe taxonomical point of view as well as from their natural habitais,

the swordlail and lhe flatfish are as remolely relaled as tliey can he among mem-

bers of lhe suliclass NEOPTEBYGII. The swordlail. a Central American

fresh-water fisli long bred in lhe aquarium, belongs to the order MICROCYPRINI,

while two species of lhe flalfish belong lo different families of lhe order HETE-

ROSOMATA: the hornyhead lurbot lo the right-eyed flounder family, PLEURO-

NECTIDAE, and lhe fantail sole lo the left-eyed flounder family, BATHIDAE.

Their natural habitat is the ocean bottom. The swordlail and lhe flatfish ap-

parently had identieal diploid complemenls made of 48 acrocentrics gradually

declining in size and lhe lowest DNA value.

We propose lo regard these ray-finned fishes as the retainers of lhe original

diploid lineage of ancestral vertebrales. The original diploid lineage then had

the DNA value, 20% that of mammals. Jn terms of the absoluto value. this

lineage contained 1.4 mg X 10 -B DNA in eaeh nucleus.

Il then follows that the green sunfish and the discus fish belong to lhe an-

cient triploid lineage, while lhe ancient pentaploid lineage is represented bv the

goldfish and among terrestial vertehrates, by lizards, snakes, and birds.

I he rainbow troul. crocodiles, and turlles may be regarded as represenling

the oeta- and nonaploid lineages, and placental mammals, the decaploid lineage.

All ihree constituent polypeptides A, I?, and C of lhe mammalian lactate

dehydrogenase have been fdund to exist in avian species as well as in many of

the ray-finned fishes (Blanco et til.. 1961; Markert and Faulhaber, 1965). These

findings on lactate dehydrogenase are in conformily with the view that in ver-

lebrates, any DNA values above 20% that of placental mammals indicate poly-

ploid lineages; therefore, sufficient gene duplication has occurred lo these genomes.

Matfish of the order HETEROSOMATA, on the other hand, revealed the presenee

of lhe A-polypeplide oídy (Markert and Faulhaber, 1965).

Si mmary

lt appears that gene duplication played a rnosL important role in lhe evolution

of vertehrates. A new gene with a new function arose from a duplicate of the

old gene. When the same gene was represented Iwice within lhe genome. ono

redundant gene was allowed to mulale to an independent direction and acquire

a new function, while the original function was maintained by the other.

Admittedly, regional duplication of a small number of genes might slill be

occurring to individual species of higher vertebrales. A series of polyploidizalion

of the ancestral diploid lineage, however, appeared lo have occurred while ver-

tebrates were still in aquatic forms nearly 300 million years ago. Among fishes

of today, some appear to retain the ancient diploid lineage which contain 1.4 mg X

X 10“ 11 DNA per diploid nucleus. Placental mammals as a whole appear to

belong lo the ancient decaploid lineage, while birds represent lhe ancient penta¬

ploid lineage. Once the chromosomal sex-determining meehanism is well establish-

ed, no further polyploidizalion is possible.

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33 ( 1 ): 155 - 166 , 1966

SUSUMU OHNO

165

As a resull, diverse species of placental mammals contain lhe identical amount

of DNA in the diploid complement, 7.0 mg X 10 -9 . Speciation within the infra-

dass EUTHEBIA is accomplished almost exclusively I>y allelic. mutations willi

little change in the total number of gene loei. The same can lie said of various

avian species. Among reptiles of loday. snakes and lizards helong to the pre-

avian pentaploid lineage. Crocodiles and turlles, on the other hand. show dose

kinship lo the decaploid mammalian lineage.

Keferences

Allfrey, V. G., Mirsky, A. E. & Stern, H.

Adv. Enzymol., 16:411-500, 1955.

The chemistry of the cell nucleus.

Atkin, N. B., Mattinson, G., Beçak, W. & Olmo, S. — The comparative DNA

content of 19 species of placental mammals, reptiles and birds. Chromosoma

(BerU, 17:1-10, 1965.

Atkin, N. B., Mattinson, G. & Baker, M. C. — A comparison of the DNA con¬

tent and chromosome number of 50 human tumours. Brit. J. Câncer (in

press).

Beçak, W., Beçak, M. L. & Nazareth, H. R. 8. — Karyotypic studies of two species

of South American snakes (Boa constrictor amarali and Bothroys jararaca).

Cytogenetics, 1:305-313, 1962.

Beçak, W., Beçak, M. L., Nazareth, H. R. 8. & Ohno, S. — Close karyological

kinship between the reptilian suborder SERPENTES and the class A VES.

Chromosoma (Berl.), 15:606-617. 1964.

6. Blanco, A. & Zinkham, W. H. — Lactate dehydrogenases in human testes.

Science, 139:601-602, 1962.

7. Blanco, A., Zinkham, W. H. & Kupchyk, L. —• Genetic control and ontogeny

of lactate dehydrogenase in pigeon testes. J. Exptl. Zool., 156:137-152, 1964.

8. Fankhauser, G. & Humyhrey, R. R. — The origin of spontaneous hetero-

ploids in the progeny of diploid, triploid and tetraploid axolotl females. J.

Exptl, Zool., 143:379-422, 1959.

9. Galton, M., Benirschke, K. & Ohno, S. — Sex chromosomes of the chinchilta;

Allocycly and duplication sequence in somatic cells and behaviour in meiosis.

Chromosoma (Berl.), 16:668-680, 1965.

10. Hamerton, J. L., Klinger, H. P., Mutton, D. E. & Lang, E. M. — The somatic

chromosomes of HOMINOIDEA. Cytogenetics, 2:240-263, 1963.

11. Hollingsworth, M. J. — The metaphase chromosomes of Crocodilus niloticus.

Cytologia (Tokyo), 22:412-414, 1957.

12. Humyhrey, R. R. & Fankhauser, G. — Structure and functional capacity of

the ovaries of higher polyploid (4N, 5N) in the Mexican Axolotl ( Siredon or

Ambystoma mexicanum). J. Morphol., 98:161-198, 1956.

13. Ingram, V. M. — The Hemoglobin in Genetics and Evolution. New York.

Columbia University Press, 1963.

14. Klinger, H. P. — The somatic chromosomes of some primates ( Tupaia glis,

Nycticebus coucang, Tarsius bancanus, Cercocebus aterrimus, Symphalangus

syndactylus). Cytogenetics, 2:140-151, 1963.

15. Kobel, H. R, — Heterochromosomen bei Vipera beras L. (VIPERIDAE, SER¬

PENTES). Experientia, 18:173-174, 1962.

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EVOLUTION OF VERTEBRATE GENOMES

16. Makino, S. — A karyological study of gold-fish of Japan. Cytologia, 12:96-

111, 1941.

17. Mandei, P., Metais, P. & Cuny, S. — Les quantités d'acide désoxypentose-

nucléique par leucocyle chez diverse espèces de Mammifères. Comy. Rend.

Acad. Sei., 231:1172-1174, 1950.

18. Margoliash, E. ■— Primary strueture and evolution of Cytochrome C. Proc.

Nat. Acad. Sei., 50:672-679, 1963.

19. Markert, C. L. — Cellular differentiation — an expression of differential gene

function in Congenital Malformations, pp. 163-174. New York: The Inter¬

national Medicai Congress, 1964.

20. Markert, C. L. & Faulhauber, I. — Lactate dehydrogenase isozyme patterns

of fish. J. Exytl. Zool., 159:319-332, 1965.

21. Matthey, R. — Les Chromosomes des Vertébrés. Lausanne, 1949.

22. Matthey, R. — La formule chromosomique et le problème de la détermination

sexuelle chez Ellobius lutescens Thomas (KODENTIA, MURIDAE, Microtinae).

Arch. Klaus-Stift. Vererb.-Forsch., 28:65-73, 1953.

23. Olmo, S„ Jainchill, J. & Stenius, C. — The creeping vole (.Microtus oregoni)

as a gonosomic mosaic. I. The OY/XY constitution of the male. Cytogenetics,

2:232-239, 1963.

24. Olmo, S., Stenius, C., Christian, L. C., Beçak, W. & Beçak, M. L. —• Chromo-

somal uniformity in the avian subclass CARINATAE. Chromosoma,

(Berl.), 15:280-288, 1964.

25. Olmo, S., Stenius, C., Faissit, E. & Zenzes, M. T. — Post-Zygotic chromosomal

rearrangements in rainbow trout ( Salmo irideus Gibbons). Cytogentics, 4:

117-129, 1965.

26. Olmo, S. & Atkin, N. B. — Comparative DNA values and chromosome comple-

ments of eight species of fishes. Chromosoma (Berl.), 18:455-466, 1966.

27. Pennock, L. A. — Triploidy in parthenogenetic species of the Teiid lizard,

genus Cnemidophorus. Science, 149:539, 1965.

28. Saez, F. A. & Brum-Zorrilla, N. — Karyotype variation in some species of

the genus O d o nt o phr y nu s (AMPHIBIA, ANDKA). Caryologia, 19:

55-63, 1966.

29. Uzzell, T. M. — Natural triploidy in salamanders related to Ambystoma jef-

fersonianum. Science, 139:113-115, 1963.

30. Van Brink, J. M. — L'expression morphologique de la Digamétie chez les

Sauropsidés et les monotrèmes. Chromosoma, 10:1-72, 1959.

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A. FAIN

167

21. PENTASTOMIDA OF SNAKES — THEIR PARASITOLOGICAL ROLE

IN MAN AND ANIMALS

A. FAIN

Institute of Médecine Tropicale, Antwerpen, Belgique

PENTASTOMIDA constitute an old and highly aberrant group of parasites.

So far lhe systematic position of this group is not established witb certainty. In

spite of some affinities with both the arthropods and the annelids the PENTAS-

TOMIDA cannot be attached exaclly to neither of them nor to any other exisling

group of animais and it seems therefore preferable to establish for them an inde-

j)endant phylum.

PENTASTOMIDA are typically heteroxenous parasites. In the most evolved

species lhe adults live in the respiratory tract of carnivorous animais, mainly

snakes and carnivores, and the larvae develop in lhe lissues of various mammals.

The development in the intermediate hosts is very long and lakes generally

several months. Il comprises a series of molts, the term of which is the tertiar

larva of nymph which remains encysted in the lissues of the host. generally in

the peritoneal cavity. It seems that in some cases this nymph is able to leave

the cyst in which it is contained and to migrate through the lissues or the organs

of the intermediate host. The nymphs may develop in different kinds of hosts.

Many of these are accidental hosts that are not normally ealen by lhe definitive

host. This polyxenism leads to an important waste of nymphs but it ensures lo

lhe species a very wide dispersai which is finally heneficient for its conservation.

Some species of PENTASTOMIDA, mainly the most primitive ones, are probably

able lo undergo their complete life-eycle in the same host. This might be lhe

case for the species that parasitize the insectivorous lizards. So far direct develop¬

ment in the PENTASTOMIDA lias heen established only for one species (Sambo-

nia lohrmanni) that lives in the lungs of varans (Fain and Mortelmans, 19601.

PENTASTOMIDA may produce lesions in man and in animais.

Lesions produced in man by Pentastomids ok snakes

Parasitism of man by adult pentastomids is exceptional and it has been ob-

served only for Linguatula serrata, a species that normally lives as an adult in

the nasal cavity of dogs. Nymphal pentastomosis, on the contrary, has been re-

ported on many occasions. It is particularly frequent in Central África but it

is also known in other parts of lhe world. Man is not a normal host for lhe

nymphs and human parasitism is therefore always accidental. Nymphal penta¬

stomosis in man lias been reported in connexion with several species of pentasto¬

mids. 1 am dealing here only with the species that live normally in snakes.

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PENTASTOMIDA

OF SNAKES — T1IEIR PARASITOLOGICAL ROLE

IN MAN AND AN1MALS

Genus A rmilifer Sambou

milifer. It contains 3 species

tastomosis in man:

- The most important genus for man is A r -

which all are able lo produce nymphal pen-

1. Armillijer armillatus (Wyman): This species has been reported on many

occasions in man. It is known only from tropical Afriea hut in these regions

iL is very frequent. The life-cyele has been elucidated by Broden and Rodhain

(1908, Í909 and 1910) in Leopoldville. The adulls live in lhe lungs of lhe

large snakes such as lhe pythons and lhe vipers of lhe genus liilis. The

nyniphs develop in all kinds of mammals ineluding man. The degree of parasitism

in lhe natural hosls may be very high. In a small antelope of Congo I found

more llian 5000 nymphs belonging to A. armillatus. All these nymphs were en-

cysted in the peritoneal cavity. Monkeys may also be strongly parasitized. In

man lhe nymphs are generally few in number but some heavy or very heavy

infeslations have been noted. The firsl human ease has been relaled by Fruner

in 1847. Sinee then many other authors have reported new cases. In some

of lhe earlier papers the parasite was erroneously reported under the names

Porocephulus moniliformis or Armillijer moniliformis. The nymphs are general¬

ly encysted in a thin and transparent eystie memhrane. In some eireumstances

these nymphs escape from their envelope and become free. Some of them migrate

through the tissues of the bost. This nymphal migration seems to be frequent

in animais but is rare in man. Chalmeras (1899) (in Sambou, 1922) reported

a case in a negro vvho died in Acera: “Great number of parasites were observed

moving freely in the abdominal cavity over the surface of the various organs,

lo which some were also observed lo be attaehed.” Mouchel (1911) found

these non-eneapsuled nymphs on differenl occasions in natives of Congo. He

noted thal two of these nymphs were attaehed by means of their hooks lo the

head of lhe pancreas, two others were free in the peritoneal cavity and one was

free in a lymphatic vessel of lhe mesenlery. So far it is not known wilh certainty

if this escaping of lhe nymphs from their eystie memhrane occurs during the

life of the host or only after its death at the moment thal. as a rule, the parasites

pass from the intermediate lo the definitive host. The distinetion is very impor¬

tant for the migration through the tissues of the organs of the host may cause

important lesions.

In man the encysted nymphs of Armillijer armillatus are commonly loealed

in lhe peritoneal cavity. Most of them are encysted beneath the capsule of lhe

liver or embedded in the superficial layers of this organ. They may also be

found along the intestine, the mesentery or on other abdominal organs or tissues.

More rarely they are encountered in other organs such as the lungs, the brain

and under the ocular conjunctiva. In alrnost all the cases these nymphs were

perfcctly tolerated and pathological complieations were very rare. In two cases

they had produeed important lesions thal had finally produeed lhe death of the

patient. In the case deseribed by Cannon (1942), lhe nymphs were extrcmely

numerous and they had alrnost completely obstrueted the large intestine. Bou-

ekaert and Fain (1959) have observed a similar case in Congo but lhe nymphs

were loealed along the hepatic angle of the large intestine and in addition lherc

was a distinct inflammalion of the peritoneum at lhe site of the nymphal masses.

2. Armillijer moniliformis (Diesing) : The adulls of thal species are very

oommon in the lung of Asiatie pythons. Nymphal parasitism in man has seldom

been reported. This species has also been found once in the oommon P y I h'o n

(P. sebae) in Congo, but nymphs have never been found in that eountry.

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A. FAIN

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Mem. Inst. Butantan

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3. Armillifer grandis Hett.: Nymphs of that species liave been reported

from the Water-Hen (Porphyrio).

Recently I liave observed several cases of human pentastomosis produced by

nymphs that I attribute to Armillifer grandis. So far it is lhe firsl-time tfiat

the nymphs of that species have heen found in man. The nymphs were removed

on the occasion of surgical operations from mesentery and the omentum of several

natives in the Republic of Congo, province of Equatenr (region of Flandria).

They were encysted in lhese organs and apparenlly had produced no pathological

lesions. These nymphs are distinctly smaller than lhose of A. armillatus and

liave more circular thickenings. In the female nymphs the anus is closer lo lhe

vulva than in A. armillatus, but however the two apertures do not open in the

same depression as it is the case in the adults and probably also in lhe nymphs

of the genus Cubirea (see Fain and Salvo, 1966).

Genus Porocep, halus Humboldt — This genus is known only from

America and África. Up lo now human parasitism by either adults or nymphs

of lliis genus lias not observed witli certainty. The cases reported by Sambou

11922) are doubtful and probably they were misidentifications.

Lesions produced in animals by Pentastomids of snakes

Li tile is known about the palhology caused by the pentastomids of snakes

developing as larvae in the natural intermediate hosts other than man. I have

never seen any inflammatory reactions in the animais parasitized by even very

numerous nymphs of Armillifer armillatus. For instance I did not find any

lesion in the antelope from Congo that was infested by more than 5000 nymphs.

Pathological features have apparenlly not more been observed in intermediate

hosts in relation wilh the other genera of pentastomids at least in natural

cOnditions.

Il seems that in some abnormal intermediate hosts, such as experimental

hosts. pentastomids may produce inflammatory reactions. Esslinger (1962), study-

ing lhe lesions produced in rals experimentally infected with Porocephulus crutali.

found that the host reactions lo lhe immalure pentastomids follow patterns similar

to lhose occurring wilh other agents of “visceral larva migrans”. These lesions

coitld be observed in the liver of lhe rats and they were strikingly similar to

lhese which have been reported in human larval toxocariasis.

Lesions produced by Pentastomids in snakes

The lesions caused in snakes by the adult or the larval stages of pentastomids

are not well known. The adult pentastomids are generally located in the lungs

of the snakes but in some species (e.g. Kiricephalus pattoni ) sexually mature

specimens are regularly found in the dermis just under the scales. Sludies of

the tissues surrounding the parasites have shown no inflammatory reaction or

tissue proliferation on lhe part of the host (Self and Kuntz, 1966). Some species

of pentastomids are able to produce important lesions in the lungs of their hosts.

In lhe genus Cubirea al least in the females the anterior part of the body.

also called head, is distinctly separate from the rest of body by a thin neck. In

all the specimens of C. pomeroy that I have collected this head was completely

170 PENTASTOM1DA OF SNAKES

TIIEIR PARASITOLOGICAL ROLE

IN MAN AND ANIMALS

enclosed in a filtrous poueh developed on lhe externai wall of lhe lung. The-

opening of this poueh was very narrow and just large enough to give passage

for lhe neck of the parasite. The rest of lhe hody hanged freely in the lung

cavity. The removal of the parasite was very difficult and needed a careful dis-

section of the filtrous poueh containing lhe head. It is well known tliat the in-

íective nymphs arrive to lhe lung of lhe snake hy direct penetration of the gut

wall and lung tissue and not hy migration up the oesophagus and passage into

the lungs via the glotlis and the trachea. This direct migration through the

internai organs may cause perforation of hlood vessels which may lead to the

dead of the animal.

COMMENTED LIST OF PENTASTOMIDA PARASITIC SNAKES

PENTASTOMIDA are very common in snakes. This parasitism has been

observed in all the eontinents hui is particularly frequent in tropical regions.

Not less than 26 species of PENTASTOMIDA grouped in 9 genera, have been

found as adults in the lungs of snakes. They represent 5 families which belong

to lhe Iwo orders existing in the phylum (CEPHALOBAENIDA and POROCE-

PHAEIDA).

Order CEPHALOBAENIDA: This order is lhe most primitive one. It com-

prises only one family (CEPHALOBAENIDAE), with two genera:

1. Genus Cephalobaena Heymons: This genus is represented hy only

one species C. tetrapoda Heymons, which parasilizes the lung of South-American

CROTALIDAE (Bothrops and L ache sis) and COLUBRIDAE (Lepto-

phis). The life-cycle is unknown.

2. Genus Raillietiella Sambon: This cosmopolitan genus is represented

hy ahout 20 species, half of them heing parasitic in snakes, the other species

living in lizards or varans.

In África 3 species have heen descrihed in snakes. The most common is

R. boulengeri Vaney and Samhon. This species has heen found as an adult in the

lung of many kinds of snakes: BOIDAE, VIPERIDAE, ELAPIDAE and COLU-

BRIDAE. Complctely developed nymphs have heen found free in the lung of

various snakes. They represent prohably “migrating” nymphs coming from a

prey swallowed some time hefore. Encysted nymphs, some of them heing slill

in the moulling stage, have also heen found hut only in lizards. These hosts

are prohahly the intermediate hosts for R. boulengeri Ireported hy Fain, 1961).

The 2 other African species have heen encounlered only in one snake eacli.

The first is R. congolensis Fain known from the lung of an undetermined snake

in Congo, the other is R. tetrapoda (Gretillat, llrygoo and Domergue), descrihed

from a single male. apparently in the moulting stage, found in the lung of Acran-

lophis dumerili in Madagascar. In Southern Europe and in Asia there are 1

species of Raillietiella lhat parasitize snakes. The most common is R.

orientalis (Hett). Tliat species is < losely related to R. boulengeri and it has

also heen found in different families of snakes: CROTALIDAE (genus Ancis-

trodon), ELAPIDAE (genus Naja ) and COLUBRIDAE (genera Coluber

and Elaplie). A nymph prohahly belonging to that species has been found

free in the lung of an asialic snake ( Tropidonotus maculalus ) (reporled hy Fain.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 167 - 174 , 1966

A. FAIN

171

1964). The 3 other European or Asiatic species have been very seldom related

they are: R. mediterrânea (Hett) living as an adult in Coluber , R. spiralis Hett

whose liost is also a COLUBRIDAE (genus Coelopeltis) and R. agcoi Tub.

and Masil, which parasitizes an ELAPIDAE of the genus Naja. In Soulli

America tliere is only one species R. jurococerca Diesing. It has heen reported

from BOIDAE (genus Roa), from CROTALIDAE (genus Lachesis) and from

nurnerous COLUBRIDAE (genera Coluber , E I a p h e , P h r y n o n a x , S pi¬

lotes, Drymobius, Rhadinaea). The life-cycle of R. furcocerca is

still unknown. In North-America the only reported species is R. bicaudata

Heymons and Vitzhum. ll lives as an adult, in COLUBRIDAE of the genera

E l a p h e and 0 p h i b o l u s .

Order POROCEPHALIDA:

1. Genus Sebekia Sambon: All lhe species of that genus (seven, in

total) live as adults in the lungs of crocodiles. The nymphs of one species (5.

oxycephala Diesing have heen cncountered frequently in fishes and in various

snakes (genera B o t h r o p s , I) i m ades, Heterodon, E u n e c t e s)

and occasionally in lizards,

2. Genus Lei per ia Sambon: The adults live in crocodiles, the nymphs

of the South American species of the genus ( L. gracilis Diesing ) have heen found

mainly in fishes, and once in a snake.

3. Genus Sambonia Noc and Giglioti:

species (S. lohrmanni Sambon ) live in the

The adults of the

lungs of varans in África,

only known

Asia and

Australia. I have shown that species may perform its complete life-cycle in the

same host (Fain and Mortelmans, 1960). Self and Kuntz (1957) have reported

this species from the lung of a snake in Solomon Is., but this record seems

douhtful and needs confirmation.

4. Genus W a d dy c e phalus Sambon: The only one good species de-

scribed in that genus is ll . teretiusculus Baird. lt has heen found as an adult

in the hmg of several Australian elapid snakes. It has also heen reported from a

COLUBRIDAE (of the genus Elaphe) in Hong-Kong. Encysted nymphs at-

tributed to that species have heen reported from an Australian elapid snake

(genus Pseudechis). The life-cycle of that species in unknown.

5. Genus Poroce phalus Humboldt: This genus is represented hy species

all living, as adults, in the lung of snakes. The nymphs are cncountered in

mammals, in snakes and in amphihians. Three species are parasitic in American

Snakes. The first is P. crotali (Humboldt) which as an adult, is very common

in the CROTALIDAE but only in lhe genus Crotalus. Encysted nymphs have

heen reported from nurnerous mammals espeeially in Brazil hui it is not sure

if all nymphs helonged really to that species. Another American species is

P. clavatus (Wyman). The adults are met only in lhe lungs of BOIDAE of dif-

ícrent genera Roa, Eunectes and Epicrates. Nymphs attributed to that

species have heen reported from marsupiais. The third species is P. stilesi Sambon,

living as an adult in CROTALIDAE (genera L ac lies is and Bothrops)

and in a COLUBRIDAE (genus llelicops). Encysted nymphs are reported

SciELO

10 11 12 13 14 15

172 PENTASTOMIDA OF SNAKES — THEIR PARASITOLOGICAL ROLE

IN MAN AND ANIMALS

írom snakes and lizards. These three South-American species are morphologically

very close each other and some authors consider lhal lliere is prohably oídy oue

species P. crotali, and that lhe Iwo olher species are synonymous.

Sliles (1891) has worked onl lhe life-cycle of Pentastomum proboscideum

Rud., (which corresponds prohably lo Poroceplialus crotali ) from a lloa constrictor.

He descrihed 4 larval stages which were reeovered from laboratory infected mice.

The life-cycle of P. crotali has been sludied again hy Pen (1942) and Esslinger

(1962). In África two species liave been reported. The mosl frequent is P.

subiãijer (Leuckart). Il has been encountered as an adult in several genera of

snakes VIPERIDAE (Causas, Pi ti s) , ELAPIDAE [Naja] and COLUBRI-

DAE ( Mehelya). Curiously enough that species seems lo he able to hecome

completely adult only in snakes of lhe Mehelya. Encysted nymphs are com-

mon in snakes (C a u sus, Neustero p h is, Ela p s o i d e a , P s a m -

mophis) rare in mammals (monkeys and galagos). Another African species

(/'. benoiti Fain) is known from an undeterminate snake prohably a Naja.

5. Genus Kiricephalus Sambon: This genus is represented hy 3 species

living, as adults, in lhe hmg of snakes. The life-cycle of lhat genus in still iin-

knovvn. Two species are known only from America. The first is K. coarctatus

(Diesing). The adults are encountered in several genera of COLUBRIDAE (Co¬

la b e r , E l a p h e , D r y m o b i u s , T h a m n o p h i s , Tropidono-

t u s , O phibolus, Herpetodry as). Nymphs and yoimg adults

have been found encysted in lhe suhcutaneous muscles of Elaps fulvius from

Guatemala and of the North-American Elaphe melanoleucus. A young male has

also been found in a mammal Mephitis mephitis (CARNÍVORA, MUSTELIDAE).

The second American species is Kiricephalus tortas (Shipley) descrihed from a

COLUBRIDAE (Dipsadomorphiis irregularis) in Norlh-America. The third species

of that genus is Kiricephalus pattoni (Stephens) which is mdy known from Asia,

Australia and Madagascar. The adulls are found in the lung of COLUBRIDAE

(in Asia and Madagascar) and of BOIDAE (in Madagascar and Australia).

Nymphs and young adults have been found in the suhcutaneous tissues or in the

walls of the stomach of snakes. Encysted nymphs have also been reported from

frogs in Java. Self and Kuntz (1966) have found that K. pattoni may inhahil

tissues even in sexually mature stages.

7. Genus Armillifer ftambon: This genus is known from Asia, África

and Australia. It comprises 3 species, living all as adults iu the lungs of large

snakes mainly BOIDAE hui also in some VIPERIDAE: Armillifer armillatus

(Wyman) is the most common species of the genus. Il lives as an adult in

African pythons and in the large VIPERIDAE mainly Bit is. A. armillatus

may also develop in smaller snakes (Boaedon, II o t hr o p h t h a l m u s) but

it seems lhat in these liosts the parasite is not able lo reach its complete maturity.

Nymphs, encysted or free, are very common in many kinds of mammals includ-

ing mau. They have also been found, hui very rarely, in prey birds (Bubo

africanas and Pernis apivorus). Another African and less common species is

Armillifer grandis (Hett). It has been found only in large VIPERIDAE, particular-

ly in the genus Pi tis. The snake that is the most frequenlly parasitized, at

least in Congo, is Pt tis nasicornis. Other liosts less frequent are Pitis gabo nica

and Gerastes comutas. Encysted nymphs have been found in a Water-Hen of

a Zoological Garden (Fain, 1961). Similar nymphs liave been found recently

in man in Congo (Fain and Salvo, in press). The third species of the genus

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10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 167 - 174 , 1966

A. FAIN

173

is Armillijer rnoniliformis (Diesing). The adults are frequently found in Asiatic

and Australian pythons. It lias also heen reported, but very rarely, from Central

African pythons.

Encysted and free nymphs have heen found in many kinds of mammals.

8. Genus Cubirea Kishida: The two species, known in that genus are

found only in África. Self and Kuntz (1957J have reported the presence of

immature specimens of Cubirea pomeroy from a snake in Solomon Is. This

record cannot be accepted without confirmation for the young specimens of that

genus are not well known and they are very difficult to identify with certainty.

Cubirea annulata (Baird) lives, as an adult, in the lung of different species of

Naja. It has also heen recovered from Bitis gabonica and Bitis nasicornis.

Nymphs or (?) adults of C. annulata have heen found in a Demoiselle Crane

(Anthropoides virgo) and encysted nymphs attributed to that species have heen

reported from a Water-Hen ( Phorphyrio). The other species is Cubirea

pomeroyi (Woodland). It is very close to lhe former. The hosts belong to the

genus N a j a . Nymphs are unknown.

9. Genus G i g l i o l e 11 a Chabaud and Choquet: This genus is very close

to Armillijer. There is only one species, G. brumpti (Giglioli). It is para-

sitic in the lung of BOIDAE in Madagascar.

Nymphs have been found in lemur apes and in TENRECIDAE.

10. Genus Ligamifer Heymons:

which lives in Asiatic snakes.

The

re is one

■ species ( L. mazzai)

Bibliocraphy

1. Bouckaert, L: & Fain, A. — Een geval nymphale porocephalose met dodelijk

verloop. Ann. Belg. Veren. Trop. Geneesk., 39(6) :793-798, 1959.

2. Broden, A. & Rodhain, J. — Contribution à 1’étude de Porocephalus monili-

formis. Ann. Trop. Med. Parasit., 1(4) :493-504, 1908.

3. Broden, A. & Rodhain, J. — Contribution à 1’étude de Porocephalus monili-

formis. Ibidem, 2(4) :303-313, 1909.

4. Broden, 4. & Rodhain, J. — Contribution à 1’étude de Porocephalus armilla-

tus. Ibidem, 4(2) :167-176, 1910.

5. Esslinger, J. H. — Development of Porocephalus crotali (Humboldt, 1808)

(PENTASTOMIDA) in experimental intermediate hosts. J. Parasitol., 48(3):

452-456, 1962.

6. Esslinger, J. H. — Hepatic lesions in rats experimentally infected with Poro¬

cephalus crotali (PENTASTOMIDA). J. Parasitol., 48(4) :631-638, 1962.

7. Fain, A. — La Pentastomose chez l’homme. Buli. Acad. Roy. Méd. Belgique,

série VI, 25(7):516-532, 1960.

8. Fain, A. — Les Pentastomidés de 1'Afrique Centrale. Ann. Mus. Roy. Afr.

Centr., série 8, Sei. Zool, 92:1-115, 1961.

9. Fain, A. — Observations sur le cycle évolutif du genre Raillietiella

(PENTASTOMIDA). Acad. Roy. Belgique Buli., Classe des Sciences, série 5,

(9): 1036-1060, 1964.

SciELO

10 11 12 13 14 15

PENTASTOMIDA OF SNAKES

THEIR PARASITOLOGICAL ROLE

IN MAN AND ANIMALS

10. Fain, A. & Mortelmans, J. — Observations sur le cycle évolutif de Sambonia

Lohrmanni chez le Varan. Preuve d’un development direct chez les PENTAS-

TOMIDA. Acad. Roy. Belgique, 5e. série, 4<>(6):518-531, 1960.

11. Fain, A. & Salvo, G. — In press. 1966.

12. Hett, M. L. •—■ On the family LINGUATULIDAE. Proc. Zool. Sco. London

(1) :107-159, 1924.

13. Heymons, R. — PENTASTOMIDA In: Bronns Klassen u. Ordnungen des

Tierreichs, 5:1-268, 1935.

14. Sambon, L. — A synopsis of the Family LINGUATULIDAE. Journ. Trop.

Med. Hyg., 188-208 and 391-428, 1922.

15. Self, J. T. & Kuntz, R. E. — Pentastomids from African reptiles and mam-

mals and from reptiles of Florida Island, British Salomon Islands (South Pa¬

cific). J. Parasit., 43(1) :194-200, 1957.

16. Self, J. & Kuntz, R. — The PENTASTOMIDA. Review Proc. lst. Internat.

Congr. Parasitology, 1:620-621, 1966.

Discussion

L. D. Brongersma: “Is Armillifer moniliformis also present in Central África?”

A. Fain: “Yes, I found it in an African python in Leopoldville, but it is pos-

sible that that species has been introduced in África by means of Asiatic pythons

of Zoological Gardens.”

2 3

5 6

11 12 13 14 15

PATOLOGIA DO ENVENENAMENTO

E PREVENÇÃO DE ACIDENTES

PATHOLOGY OF ENVENOMATION

AND PREVENTION OF ACCIDENTS

Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 175 - 178 , 1966

NEWTON C. McCOLLOUGII and JOSEPH G. GENNARO

175

22. THE DIAGNOSIS, SYMPTOMS, THEATMENT AND SEQUELLA OF

ENVENOMATION BY CROTALUS ADAMANTEUS AND GENUS

AGKISTRODON

NEWTON C. McCOLLOUGH and JOSEPH G. GENNARO

(U.S.A.)

For lhe past len years the authors have had an inlense interest in the effect

of the Crotalus adamanteus and lhe Agkistrodon piseivorus venom on the sofl

tissues of the extremities of patients hitten in Florida. Dr. Gennaro carried oul

a large amoitnt of experimental work and I. interested in the clinicai portion of

the study, look careful case histories of all patients wh o were involved in loss of

major tissue in either amputation or slough. Dr. Gennaro’s studies, largely through

the tagging of venom and antivenom, carne lo some conclusions long before 1 did

in the clinicai study. The clinicai and lahoratory findings were parallel to a

rather remarkahle degree. This involved not only lhe envenomation and the

symptomatology but also lhe residis of treatment. The number of amputations

in the clinicai series amounted to 39 and the serious sloughs which led lo severe

disahility amounted to 20 additional cases. By far lhe majority of the lutes

trealed were those of Crotalus adamanteus and only a few of the Agkistrodon

piscivorus were seen.

Epidemiology of the situation in Florida was taken up very seriously January

l st , 1962, by the Department of Health and a Venomous Snake Bite Commiltee

appointed l>y lhe Florida Medicai Association. The Department then began a

Begistry of Venomous Dites by snakes and these have been tahulated statistically

eaeli year. Al the present time, in Florida, we have registered annually ap-

proximately 270 to 300 snake lutes, all of which have not been definilely identified

as lo the snake; but the large majority of lhem were definitely venomous accord-

ing to fang mark description and sequelae. The number of deaths lias averaged

about three a year. We have been fortunate insofar as a number of treating

physicians look colored photographs of the patient consecutively until either lhe

extremity was lost or recovery of partial function occurred.

We have concluded of eourse lhat lhe character of lhe venom, i.e. L.D.,- l0 or

the amounl of lhe venom injected into the patient by the bile of either one of

these snakes can only be eslimaled by analizing the gravity of the clinicai picture.

Then, and only then, can the proper treatment be carried out. For instance. the

bite of a snake which delivers a relatively small aniounl of venom in a small child

may be an extremely serious matter when in an adult it would not. So, the

weighl of the victim has a great deal lo do with the therapeutic considerations

as does lhe number of fang marks. The most important, however, are those signs

derived from the clinicai picture of the patient and lhe rapidily in the sequence

of symploms which leads one to evaluate the amoiint of lhe envenomation. Wood,

Hohack, & Green began a elassification of envenomation in their original article:

Grade I encompassing only local symptoms. Grade II. local symptoms plus some

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176 THE DIAGNOSIS, SYMPTOMS, TREATMENT AND SEQUELLA OF ENVENOMATION

BY CROTALUS! ADAMANTEUS AND GENUS AGKISTRODON

mild systemic symj)toms. Grade III, severe local symploms with moderate systemic

symptoms. These gradations werc drawn from their observation of bites of Cro¬

talus horridus of the easlern and northeastcrn United States.

Dr. H. M. Parrish, who has done a great deal of work with tliis subjecl,

added a very important elassificalion, namely Grade 0, ín which lhe palient lias

the fang marks but exhibits no local or systemic symploms of envenomation. This

simply is applied lo the patient bitlen by a venomous snake which lias fixed bis

fangs in the sofl tissue but bas not delivered any venom. We have found tbis

in our statewide studies to be a not uncommon situation. The accnracy of lhe

snake, as far as injecting the venom at lhe moment of fang penelration, is often

questionable. Dr. Gennaro and this aulhor, in consideration of the severe local

symptoms and systemic collapse of individuais bitlen by Crotalus adamanleus en-

larged this elassification to inelude Class IV. We felt this necessary insofar as

lhe Crotalus horridus vvliieh Wood. Hobaek & Green were studying does not, by

any stretch of tbe imagination, except in very rare cases, provide the severe clinicai

picture which appears in the slides we will show you today of Crotalus adaman-

teus bites in man.

From lhe clinicai standpoint lhere is a curious varialion in lhe venoms of

Crotalus adamanleus bites from individual to individual. Whether this is tbe re-

action of the patient to lhe venom, or whether lhe venom of the snake varies in

these regards, we do not understand. We have seen the hemorrhagic featnres

develop to the point thal they resulted in death from bowel. bladder, subcutaneous,

intraperitoneal and intrathoracic hemorrhage and yel, in others, the neurotoxic

featnres are dominant: i.e. faseieulation, immediate severe weakness which is usual-

Iy generalizei! and marked painfnl muscular eramping. In these particular patients

who exhibil these neurotoxic symptoms, lhere may be very little evidence of tbe

hemolytic picture and even the local swelling may not be as marked. These are

some of the characteristies of lhe Crotalus adamanteus which seem to be akin to

lhose found in tbe terrificus and lo a lesser extent in the Crotalus durissus.

From reading case re[iorts from the western pari of lhe United States, and study¬

ing the number of amputees which have been reportei! in these arcas, it is our feeling

llial lhere may be a tcndeney in tbe Crotalus atrox and others in the western and

soulli western States for the neurotoxic factors lo be more prominent than tbe

hemolytic and proteolylic factors. The typieal bile of lhe Crotalus adamanteus

has a mixture of both, the loeally deslruetive predominating. The onset is im¬

mediate and progression rapid. I will not allempl to discuss the fact that tbe

secondary tissue products of the hemolytic and proteolytie factors magnify and

add lo both the systemic and local clinicai situations.

In trealment of Grade III and IV bites, I will point oul lo you in lhe very

beginning, that the intravenous roule utilizing the polyvalent antivenin is lhe only

way that antivenin should be administered. The four hours or more required

for intramuscular absorption is a waste of time. Both experimentally and clinically

we agree with Jackson, who did the original work, in 1926, that incision and

suclion for 30 minutes is beneficiai if properly earried oul. The use of a complete

tourniquet we have never been able lo prove is of any value unless you are intent

ou saving the palient at lhe risk of damaging the extremity. The incisions should

be very small, one lo two millimelers in length, and can be cruciate or single

incisions aeross the orifiee of lhe fang rnark. They merely slightly enlarge the

fang puncture, should only go through the skin itself, and can be classified as

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 175 - 178 , 1966

NEWTON C. McCOLLOUGH and JOSEPM G. GENNARO

177

mark to the point Iliat suction

makc suction less effectivc

is classified as a Grade I liite, there is a question as

necessary at all. If no sensitivity is demonstrable, the

perculaneous. They open lhe orifice of lhe fang

is more effectivc. A larger or deeper incision vvi

because of bleeding and may result in deeper spread of the venom.

lí lhe local swellin.

lo whether antivenin is

local and systemic evidence of envenomation of Grades 111 and IV are hest abolished

by intravenous injection of antivenin in 250 to 500 c.c. of saline. If an anaphy-

lactie reaction occurs il wi 11 be as prompt and severe as if serurn is administered

íntramuscularly. In fact, if such a reaction occurs after one cubic centimeter is

given by vein, it can be stopped and proper lherapy administered by the same

vein. To the contrary 10-15 c.c. buried in muscle cannot be restrieved and the

treatment of anaphylactic shock rendered more difficidt.

We have collected a series of cases, which does not exceed more than four

or five. in which the intramuscular or local injection of antivenin apparently had

no effect on the precipitous downward course in botli the bitten extremity and

the vital signs of the patient. When large amounts of antivenin are given intra-

venously, and we have used up twelve to sixteen ampides in a single instance over

a period of two hours, with a total of twenty, the change in the patient is noted

in a matter of two or three hours. The pulse settles to a reasonable levei, the

blood |>ressure rises, and the patient becomes alert and cooperative. No patients

treated in this manner have failed to respond. We are firmly convinced that the

amount of antivenin administered should be in excess of that needed to precipitate

lhe venom injecled by the snake and one can never estimate this until he recognizes

the improvement previously mentioned. At this time there can be a reasonable

waiting period before further antivenin is given. We have always administered

rather massive doses of antivenin when confronted with what we considered to

be a criticai situation and we, apparently in all of the cases, have covered the

venom injecled to the point that there was never any sign of reversal of lhe

clinicai picture, i.e. dominanee of lhe venom over the antivenin injecled.

You will note lhe tvpe of incision that we have recommended for lhe relief

of tension which is bloeking circulation lo lhe distai portions of the extremity.

These leave intact most of the skin and the subcutaneous fatty layer of faseia

with its network of blood vessels. and permit il lo act as a dressing over the deep

longitudinal fascial incisions which release the inner tension of the muscles. It

is the deep faseia which is the harmful constrietive agent in these instantes, not

the skin. If longitudinal incisions of the skin correspond anatomically to the

longitudinal incisions of the deep faseia, the muscle would burst through the wound

in a massive herniation and more tissue would be lost. There is some

compression which results from the skin as it holds hack the herniation

muscle in the method deseribed.

can t be eontroversial. We have

sinall transverso incisions, in lhe

release. of a profuse amount of

certainly ])lays a major pari also

ing lhe efferent circulation

blood etc. is understood.

In conclusion, I would like to reporl that the epidemiological studies by the

Snake Hite Commitlee of lhe Florida Medicai Association, and the Florida Public

Health Department, have reported annually in lhe past three years 250 to 300

biles per year. Two-hundred of these bites have been due lo venomous snakes.

gentle

of the

I his is really beneficiai control of edema and

also noted that there is an escape through these

skin and subcutaneous faseia, after deep fascial

serosanguinous fluid from the extremity, which

in dimishing the internai tension which is block-

lo the liand or foot digits. Supportive therapy i.e.

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178 THE DIAGNOSIS, SYMPTOMS, TREATMENT AND SEQUELLA OF ENVENOMATION

BY CROTALUS ADAMANTEUS AND GENUS AGKISTROnON

By far the majority are due lo lhe C rotulas snakes. About one per cent of

lhese biles have proveu fatal. There are usually eighl lo ten coral snake biles

per year and lhe remainder of the venomous snake biles are due lo the Agkistro-

don piscivorus and contortrix. We had one death lasl year from Agkistrodon pis-

civorus envenomation. The rcporls which have accompanied lhe biles of lliis

snake show ralhei marked hemolysis and marked fibrinolysis wilh the clotling

mechanism of the blood almost reduced lo zero in severe cases. Fewer and fewer

doctors in Florida are using cryolherapy and more are using intravenous poly-

valenl antivenin, wilh rather aslonishing success. There have been no cases of

anaphylaxis reported. Lasl year an AK (above knee) and then laler a hip dis-

articulation was done on one patient who was treated wilh cryotherapy for eigli-

teen successive days due a bile of a Crotalus adamanteus iu the lower extremity.

Il is our mutual conclusion lhat cooling or ieing has no place in treatment. Six

coral snake biles were treated with lhe antivenin from the Instituto Butanlau

with success. In each instance the antiserum was used intravenously. I he re-

ports of the program initiated by the Snake Bite Committee serves as a guideposl

lo the treatment and is also a source available to the practicing in management.

Most of the bites in the pasl year reached hospital care within one-half hour of

the time of lhe bite.

Il has been my pleasure and a great honor to bring you this small bit of

information from Florida and the Southern part of the United States; hui 1 must

say that 98% of our statistics and case colleetions have originated within the

State of Florida.

Discussjon

A. Delgado: “Please, I would like to know which should be the precise indica-

tions and advantages for making transversal incisions in the arm or leg bitten, (if

even doing so the patient will probabiy lose the member according to the evolution

shown by the slides).”

N. C. McCollough: “The transverse skin incisions through which longitudinal

deep íascial reieases can be easily accomplished may readily save all or a large

part of the limb. This is really vascular surgery in so far as it opens collapsed

arteries.”

//. Bicher: "Did the speaker observe cardiovascular faiture in his patients?

And if so, did the transversal section help to this situation?”

N. C. McCollough: “I did not understand this question fully, but stated we felt

there was a definite cardioto.xin.”

F. Kornalík: “Have you any evidence about the blood-coagulation changes in

patients bitten by Agkistrodon contortrix or A. piscivorus.”

N. C. McCollough: "Yes, in one or two cases the coagulations properties of

the blood in patients bitten by this snake were markedly reduced.”

P. J. Deoras: “It is necessary to make an incision at the site of bite when

this kind of measure may not be useful as a first aid?”

N. C. McCollough: “Yes, we feel it increases the efficiency of suction. As

described, it is not harmful.”

A. do Amaral: “In your opinion (or Dr. Gennaro’s) which is the maximum

(record) amount of venom secured from a full-grown specimen of C. adamanteus ?”

N. C. McCollough: “About 600 mg dried weight.”

A. do Amaral: “Has Dr. Gennaro ever used enzymic medication (hyaluronidase)

against the oedema producing effect of the venom hyaluronic acid, when used in

connection with specific antivenin.”

N. C. McCollough: “Not that I know of.”

SciELO

10 11 12 13 14 15

Mem. Inst. Butuntan

Simp. Internac.

33 ( 1 ): 179 - 188 , 1966

F. KORNALIK

179

23. THE INFLUENCE OF SNAKE VEM)MS OE FIBRINOGEN

CONVERSION AND FIBRINOLYSIS

F. KORNALÍK

University Karlovy, Praha, C.S.S.R.

The studies ou the influence of snake venoms seem at the |)resenl time to be

a special liranch of lhe coagulation theme. Most of the work has heen devotei!

lo the procoagulant action of the venoms. Far less numerous are the papers deal-

ing with anticoagulant properties of snake venoms.

In some of our previous works we were able to show. that the in vilro anti-

coagulant action of this second group is at least partially due to the destruetion

of prothrombin. However the role of lhe fibrinolytic properties of these venoms

still remain to be elucidated. In this respect the most active proved to l»e those

from Agkistrodon piscivorus, Agkistrodon contortrix and surprisingly also the

venom of Vipera lebetina. The results obtained indicated that the fibrinolytic

compound of these three toxins are mntually very similar as regards the quality

of their action and differences are merely of quantitative nature, the venom of

Agkistrodon piscivorus being the most potent.

The first approach to the eslimation of fibrinolytic properties of any substance

is the classical method of the native and heated fihrin plates, where plasminogen

has heen destroyed hy the heat. We can see the lytic areas produced hy 0.1%

solution of lhe toxins (Fig. 1). N = native plates, H — heated. In the center

trypsin in 0.01 c /c jor comparison.

At first one wonld conclude that the difference indicates the presence of the

plasminogen activating system in the toxins. That was also our interpretation of

these findings till we had lhe opportunity lo work with pnrified plasminogen.

Plasminogen has heen added both to the fibrinogen solution of which the

plates have heen made and/or it had heen incubated with lhe venoms before drop-

ping them on the plates. In neilher case there was any significant difference in

the lytic areas produced hy venoms with or without plasminogen, i.e. the latter

was not activated hy lhe venoms (Fig. 2).

FIIG + l'LG — plasminogen added to the fibrinogen solution.

TOX + BLG = plasminogen incubated with toxins, X native plate.

If the bovine fibrinogen solution is incubated with venoms it gradually loses

its ability lo be converted into fihrin by thrombin, i.e. it is eilher denatured or

split-off. There is no significant difference in the course of this action if plas¬

minogen incubated for 10 min at 37°C either with loxin or toxin alone is added

to the fibrinogen solution. From all these results it can he concluded, that lhe

venoms have practically no plasminogen activating properties (F ig. 3).

SciELO

10 11 12 13 14 15

180

THE INFLUENCE OF SNAKE VENOMS ON FIBRINOGEN CONVERSION

AND FIBRINOLYSIS

By addition of 0.01% solution of epsilon amino kaproic acid (EAK) fihriu

clol lysis from lhe test tuhe wall is prolonged from 5 to 8 min. Without EAK

the venom acts synergically with plasmin. This is reflected in the shortening of

the lysis lime lo 4 lo 3 min. Toxins alone would cause the clot lysis only after

a considerably longer period (over 20 min.), which corresponds lo Iheir own

filirinolytic activily. On the olher liand toxins added to the test tuhe simultaneous-

ly with EAK are ahle, to a certain extent, to paralyse lhe inhibitory effect of a

specific fibrinolysis inhihilor which EAK is known to he (Fig. 4).

CORRELATION BETWEEN THE VENOMS, PLASMIN AND EPSILON AMINO KAPROIC ACID

IN LYSIS TIME OE A FIBRIN CLOT

The dynamism of clot formation and its lysis can he very well ohserved

through the method described by Gruedlinger in which the clol formation and

lysis is assessed by ploting changes of the turbidity (measured ])holomelrically at

350 (xm) of the tested system against lime. This method is definitely more ac-

curate and therefore we have used il lo ascertain lhe fihrinolytic properties of

the venoms — plasmin mixtures and their hlocking by various inhihitors of lhe

proteolytic enzymes. We can observe lhat lhe venoms are ahle lo paralyse lhe

inhibitory effect of soya-hean inhihilor (SB1). Trasylol (THA) and EAK (Fig. 5).

SyNERGIC ACTION OF VENOM WITH PLASMIN AND RESTRICTION OF THE INHIBITORY

EFFECT OF VARIOUS INHIBITORS BY VENOM

To gel an idea ahout the quantitative relations hetween the lylic activities

of toxins, plasmin and trypsin and ahout the influence of the different inhibitors

exerted on lhese enzymes an arhitrary unit has to he established. For this purpose

the lurhidimetric method was rather time consuming and therefore we have used

the fibrinogenolytic properties of lhese active substances.

From lhe curves in Fig. 6 we can see lhat lhe fibrinogenolytic aclivity of

300 U/cc of plasmin can he compared in case of Agkistrodon piscivorus venom

with 0.1 rng/cc and in case of A. contortrix venom with 0.25 mg/cc. Adhering

to lhese quantitative relations, we have added Io the tested system various inhihi¬

tors in different concentrations. Both plasmin and trypsin are inhibited by

0.01% EAK, 0.001 SBI, 250 U/TRA, whereas even hundred limes stronger

concentration of lhese inhihitors had practically no effect upon lhe fihrinogeno-

lytic activity of venoms (Fig. 6).

Quantitative relation between plasmin & venoms and different inhibitors

We were furlher interested if the splil products which result from the aetion

of venoms upon íihrinogen are of a nature similar to those produced by the lytic

aetion of plasmin, at leasl if lhere is an antithrombin VI activily which is at-

trihuted to the polypeptide D.

As can he seen in Fig. 7 fibrinogen split products of both lytic agents have

been added lo fibrinogen and thrombin solution and the increasing turbidity

indicaled the course of fihriu formation, i.e. the activity of thrombin. From the

curves we can see lhat in case of snake venoms a considerahle activity of anti¬

thrombin VI is present within splil products of toxin fibrinogenolytic aetion

(Fig. 7).

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33 U): 179-188, 1966

F. KORNALÍK

181

AnTITHROMBIN VI ACTIVITY OF SPL1T PRODUCTS PRODUCED BY ACTION OF SNAKE

VENOMS UPON FIBRINOGEN

Beside the fibrinolytic properties the tested venoms are known to possess a

fair amount of proteases. It was of interest to compare the proteolytic activity

of the venoms with the lytic activities of plasmin and trypsin and to find out

how lhe toxin proteases are affected by inhibitors. Proteolysis has been assessed

l>y a slightly modified method of Anson Mirsky using casein as substrate. We

can observe that lhe caseinolytie proteases of the venoms are not inhibited by

inhibitors, imlike trypsin, which is (Fig. 8).

AcTION OF DIFFERENT INHIBITORS UPON VENOM PROTEASES AND TRYPSIN

To be at least partially sure that both the activities, fibrinolytic and proteo¬

lytic could be attributed to the same enzyme we had to isolate the active substances

from llie venorn. After several attempts, using different separative procedures the

most convenient method proved to be starch gel electrophoresis. From the curves

showed in Fig. 9, we can see that both these activities always go along. The

lytic fractions had no other enzymic activities. They were roughly 50 times less

loxic than the whole venorn and the lytic enzymes are 150 times more concentrated

lhan in the whole venorn, as could be computed from the protein content. These

results are similar in all lhe three venoms. We were nevertheless not able to

separate completely the haemorrhagins from these lytic fractions. Besides lhe

proteolytic enzymes there is a different enzyme — the esterase spliting TAME —

present in lhe venoms. This activity does not go parallel with the protease. Both

these results are in full agreement with lhe findings obtained recently by Japanese

authors for fractions of Habu snake venoms (Fig. 9).

Starch gei. electrophoretic patterns of Agkistrodon piscivorus venom and

THE ENZYMIC ACTIVITIES OF THE FRACTIONS

In the experiments in vivo we have injected sublethal doses of Agkistrodon

piscivorus venom (400 p.g/100 gr) subcutaneously into white rats. In these

animais the routine blood coagulalion check-up has been performed in time in-

tervals of 30 min. 2 h. and 24 h., respectively. Except for a slight hyper-

coagulability of lhe whole blood in the first 30 min. there were no significant

changes in cloting time of plasma in experimental animais compared with the

control group.

The only pronounced difference was in the activity of euglobulin fraction in

animais 30 min. after toxin application. Surprisingly the fibrinogen content was

not altered in the sense of a decrease. On the contrary we could observe a

significant rise in fibrinogen content in animais 24 h. after application. Both

these findings can, in our opinion, be attributed to the stress reaction caused by

toxin (Fig. 10 and 11).

By means of a 10% solulion of formaldehyde in 60% alcohol applied in

rats jugular veiu an artificial thrombosis can be produced. 24 h. after operation

on lhe average in 60% of so treated animais a thrombus can be found. The

same amount o( venom (400 /tg/100 gr) has been injected in the animais 10 h.,

before or 10 h. after the operation. The toxin increased the amount of incidence

SciELO

10 11 12 13 14 15

182

THE INFLUENCE OF SNAKE VENOMS ON FIBRINOGEN CONVERSION

AND FIBRINOLYSIS

of thrombosis from 60% lo 100%. This is j)rol>al)ly caused by the action of

lhe haemorrhagins exerted ou the vessel wall rather lhan hy the rise in the

fibrinogen contenl.

lt was surprising that venoms, wliich have in vitro a remarkahle fibrinolytic

activity are in animais unahle lo produce any change in lheir fibrinogen content.

this being so even in amounts far exceeding those which have been used in experi-

ments in vitro. ít indicated the presence of a specific inhibitor in the blood

which could inactivate the venom fibrinolysins. From the fig. 12 we can see

that this seems to be the case. Fibrinogenolytic activity of both the most potent

venoms i.e. Agkistrodon piscivorus and A. contortrix practically ceases in the

presence of sera from different animais, human being included I Fig. 12).

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):179-188, 1966

F. KORNALtK

183

Fig. 1

V LE BE TINA

V LEBE TINA

A CONTOBTBlX

A PtSOVQQuS

A CONTOPTfHX

* ^SCIVQBUS

rBr ps iN

TffrPsiN

A CONTQRTQtX

A PISCIVORUS

TRTPSlN

Fig. 2

SciELO

10 11 12 13

134 THE INFLUENCE OF SNAKE VENOMS ON FIBRINOGEN CONVERSION

AND FIBRINOLYSIS

Fibrinogenolysis by toxins and plasminogen

COAGUL

TIME

■ TOXIN (BUFFER) ♦ BUFFER

- TOXIN (BUFFER) * PLASMINOGEN

Fig. 3

FBG 1%

TOXIN 0,1%

PLASMINOGEN 02%

LYSIS TIME

70-

8 -

2 *

TOXINS BUFF.

ALLDNE

EAK

APISC.

ACON.

APISC.

*EAK

ACON.

♦EAK

2% FBG 0,2 CO

0,2% PLM 0,1 cc

IOOXcSTK 0.1 cc

lOXcc TH RB O.lcc

0,01% EAK O.lcc

0,1% TOXIN O.lcc

Fig. 4

SciELO

10 11 12 13 14 15

Agkistrodon contortrix

rQG ‘ PIM • TOX (BUFn •INHIBIBUFF)

■ TQXN

• BUFFEff

• iNHlBITOfí

.INWBITOR ♦ TOXIN

Fig. 5

PLASMIN 300 -cc

TRYPSIN 0.001%

A PISCIVUPUS 001 %

A CONTORTRIX 0.025%

IccFBG >

IccE tíZYME > 02cc * 0.2cc T RB

0.5cc irjHiBI TOR )

Fig. 6

2 3 4 5 6

SciELO

10 11 12 13 14

- Blank 1,5 cc Fibrinogen 1%

—. —.— PLM + FBG (= Antithrombin VI) 1,0 cc Antithrombin VI

Toxln + FBG ( — Antithrombin VI) 0,2 cc Thrombin (10 fí/cc)

Fig. 7

PROTEOLYTIC ACTIVITY AFTER INIIIBITORS

Substrate caseine

0,4

0 . 2 -

BL SOI EAK 7 BA

a. ptsavoRus

0 . 1 %

BL SBI EAK TRA

A. CONTORTRIX

0 . 1 %

Fig. 8

Bi. SBI EAK ISA

V LEBETINA

0 . 1 %

BL SBI EAK TRA

TRYPSIN

Q 1 %

2 3 4

SciELO

10 11 12 13 14 15

Euglobulin fraction in rats after 4 mg/g of Akgistrodon contortrix venom

* 180 '

CON. 1/2 2 24 H

Fig. 10

SciELO

10 11 12 13 14

PROTEIN CONTENT

FBG after Agk. piscivorus toxin

mc]FBG/1cc

CON. 1/2 2 24 H.

Fig. 11

Serum inhibitors

fcc TOXIN *0.5cc PLASMAtBUFFERi* Icc FBG

\ - \/- - /

O.lcc ♦ O.lcc thrb

Fig. 12

Mem. Inst. Butantan

Simp. Internac.

33(1) :189-191, 1966

E. EFRATI

189

24. CLINICAL MANIFESTATIONS OF SNAKE B1TE BY VI PERA XANTHINA

PALESTINAE (WERNER) AND THE IR PATHOPHYSIOLOGICAL BASIS

P. EFRATI

Department of Medicine, Kaplan Hospital, Rehovot, Israel

Vipera xanthina palestinae is practically the only poisonous snake in Israel.

Bites caused by other snakes, including Echis colorata (Guenther), have been

extremely rare.

The aclual annual incidence of snake bites in onr country is not known, as

no compulsory notification of cases is reqnired. As a rough estimate, I would

assume the incidence lo be from 150 to 250 cases per year. For the last 25

years I lias under my personal observation 21 cases of viper bite admitted to

two hospitais. Of lliis material 1 have investigated 15 severe cases for the clinicai

manifestations which correspond, as a matter of fact, to lhe natural course of

poisoning, as no specific antivenin was given. Specific antiserum produced by

Institut Pasteur, Paris, lias been on the market since 1956 only. The treatment

used was mainly anti-shock treatment.

Soon after having been bitten, the viclim experiences local pain (15)*.

Shortly afterwards he becomes overwhelmed by weakness (11) and restlessness

(10). 15 to 30 minutes after the accident the patients vomits repeatedly (14),

perspires profusely and complains of abdominal pains (14) and diarrhoea (12).

Sometimes lhe diarrhoea Iasts several liours and the íaeces becomes sanguinolent.

Al this stage lhe viclim usually reaches the hospital. He is pale, restless and

covered by cold perspiration. Two patients were admitted in a State of clouded

consciousness. Physical examination revealed in 11 of the 15 patients a conspicu-

ous swelling of lhe tongue, regardless of the localization of the bite. Sometimes

the speech becomes slurred. fn 7 out of 15 patients angioneurotic oedema (Quin-

cke) of the lips was observed and one (not included in the present series) lost

consciousness for several hours, apparently because of oedema of the brain.

Seven of lhe patients had hypotension on admission and in 7 others the

blood pressure could not be obtained. 1 he pulse was usually weak, rapid or im-

perceptible. Rarely bradycardia was found. Fang marks could be detected in

11 patients, and they were usually not characteristic.

The affected extremity is often swollen on admission. Later lhe oedema

advance centripetally, reaching the niost proximal joint in 4 to 5 days. Often

the oedema crosses the trunk and spreads iijj and down on lhe opposite side.

Number in parenthesis indicate the number of cases out of the 15 investigated ones,

which manifest the symptoms.

SciELO

10 11 12 13 14 15

190 CLINICA L MANIFESTATIONS OF SNAKE BITE BY VIPERA XANTHINA

PALESTINAE (WERNER) AND THEIR PATIIOPHYSIOLOGICAL BASIS

Comparing the circumference of the affectcd extremity vvhich lliat of tlir normal

one, ono can finei differences of as much as 1 1 cm (al lhe proximal part of the

ihigh!).

Several hours after the liite hacl been inflicted, lhe swollen skin reddens in

many places, representing subcutaneous haemorrhages and/or ascending lymphan-

gitis. Later, hlisters make their appearance differing in size and colour, con-

taining plasma or blood. L sually they ahound vvhen the swelling becomes cons-

picuous. Some of lhem contain as much as 30-80 ml of fluid. On one occasion

we removed 250 ml of blood from a single blister. Laboratory investigalions

often reveal signs of haemoeoncentralion, haemoglobin reaching 16-20 Gm% (II).

Severe leucocytosis was found in 8 patients 115,000-34, 000/cumm) and a sliglit

one in olhers. In two cases of the series, thrombocytopenia (10,000; 90,000)

was observed, and in two olhers fibrinolysis occurrcd. Half of the patients had

albuminúria, disappearing subsequenlly.

Pathocenesis and pathoi.ogical iuiysioi.ogy

According lo the literature, lhe viper venom, having a molecular weight

higher than 20,000 is absorbed from lhe site of lhe bile through the lympb ves-

sels into lhe circulation. The slow flow of the lymph enahles lhe inactivalion

of a certain quantity of lhe venom "en route”. On lhe other hand, a “spreading

factor possessing hyalonuridase-like activity favours lhe absorption of the venom.

There is some clinicai evidence supporting these assumptions. One can see for

instance, lymphangitis and/or haemorrhages advancing along lymph lo the regional

lymph nodes. On post mortem examination lhere is sometiines a peculiar dis-

tribution of haemorrhages in lhe urinary hladder or in the uterus: they remain

on one side of the mid-linc.

Evcn small quantities of venom enlering the circulation may cause generalizcd

anaphylactoid rcaclion manifested by gaslro-intestinal disturbances (vomiling, ab¬

dominal pains, diarrhoea), peripheral shock and angioneurolic ocdema (type Quin-

cke) of tongue, lips, glottis, hrain, etc. Obviously, grealer quantities of venom

cause a more severe reaclion. There is some experimental evidence suggesling

lhat release of hislamin from the tissues, initiated by the venom, tniglil be lhe

pathogenclic mechanisrn of lliis anaphylactoid reaclion.

The primary anaphylactoid shock, is left untreated, passos readily over lo

secondary shock, owing to lranssudalion of plasma and bleeding into lhe tissues.

Proteolytic enzymes — called haemorrhagins — in lhe venom increase lhe per-

meability of small vessels resulting in exlravasation of fluid. Contraction of the

blood volume perpetuates lhe failure of lhe circulation (shock). In addition,

bleeding into vital organs (beart, lung, hrain) may kill the patienl.

The morbidity is also prolonged due to severe secondary anaemia. We have

never encountered massive haemolysis causing anaemia. Complications in kidney

function — shock kidney — occurrcd very rarely indeed. Because of lhe increased

permeability of lhe small vessels, loxic suhstances may be resorbed from the

intestines, causing liver darnage.

No cases of disturbances in the function of lhe central nervous system were

observed vvhich eould be ascribed to neurotoxic factors in the venom. Clouded

consciousness, coma, restlessness, occurrcd togcther wilh manifestations of the

anaphylactoid syndrome and cleared by treatment wilh gluco-corticoid hormones.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Intornac.

33(1):189-191, 1966

E. EFRATI

191

afíirming — in lliis way — their anaphylactoid origin. In some cases there were

(listurliances in peripheral nerves located near to the site of the bile, apparently

caused by direcl local action of the venom. In a recent case transient aphasia

occurred with positive Babinsky’s sign. probably due to haemorrhage.

In a patient treated by lis and in another one of a nearby hospital, radiculitis

occurred as a pari of a severe serinn reaction.

Accordingly, vve suggest the following essential treatment:

1. First-aid: complete immobilizalion of the affected extremity.

2. Adequate quantities of specific serinn injected intravenously as earlv as pos-

sible.

d. Anti-shock treatment for shock and haemorrhages.

4. Hydrocortison for the anaphylactoid syndrome.

Treatment carried onl as suggested lias changed completely the clinicai course,

as well as lhe jirognosis of viper bile.

Literature

1. Efrati, P. & Reif, L. — Am. J. Trop. Med. & Hyg., 2:1805, 1953.

2. Efrati, P. — Harefuah, 63:315, 1962.

3 . Barness, J. M. & Trueta, J. — Lancet, p. 623, 1941.

4. Duran-Reynals, F. — J. of Exp. Med., 69:69, 1939.

5. Feldbercj, XV. & Kellaway, C. H. — J. Exp. Biol. Med. Sc., 15:80, 1937.

Discussion

E. R. Trethewie: “If the anaphylactoid shock is due to histamine one might

expect this effect to be less in children, because the amount of histamine in tissue

in children is less. Histamine release is proportional to the histamine content of

tissue.”

P. Efrati: “Nothing to answer."

II. I. Bicher: “Did the speaker consider a rôle for the neurotoxin, described

in this venom, in the pathogenesis of his syndrome?”

P. Efrati: “The neurotoxic action of the venom was revealed only in mice

poisoned by multiple lethal doses of venom and protected by antiserum. In huraan

beings such a dose would cause death before neurotoxic symptoms could appear.”

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan AKIRA OIISAKA, TAMOTSU OMORI-SATOH, HISASHI

Simp. Jnternac. KONDO, SATORU KONDO and RYOSUKE MURATA

33(1) :193-205, 1966

193

25. BIOCHEMICAL AND PATHOLOGICAL ASPECTS OF HEMORRHAGIC

PRINCIPLES IN SNAKE VENOMS WITH SPECIAL REFERENCE TO

HABU (TRIMERESURUS FLAVOV1RIDIS ) VENOM

AKIRA OHSAKA, TAMOTSU OMORI-SATOH, HISASHI KONDO,

SATORU KONDO and RYOSUKE MURATA

2 nd Department of Bacteriology, National Institute of Health, Shinagawa-ku,

Tokyo, Japan

Introduction

Hemorrhage is one of lhe mosl prominent symptoms following the biles by

Grotalinae or Viperinae snakes (1,2). It was thought that certain proteolytic

enzymes in snake venoins cause lhe hemorrhage (1-7).

We estahlished a quantilative method for determining the hemorrhagic activity

of lhe venom of Trimeresurus Jlavoviridis (8). We initiated systematic studies on

the principies responsihle for lhe hemorrhage by this method.

Evidences from our (9-12) and olher (13-15) laboratories suggested the pre-

sence of more than one hemorrhagic principie in snake venoms. We fractionated

the venom of Trimeresurus jlavoviridis by zone electrophoresis and reported lhe

presence of Iwo hemorrhagic principies, that we designated as HR1 and HR2 (9).

They are distinct imnnmologically from each olher (16). Attempts were made

lo correlate the hemorrhagic activity with proteolytic activity, lethal toxicity, and

other pathological aelivilies (9-1 1, 17, 18).

The purpose of this presentation is to review our data and the data by

olhers on the biochemical and pathological aspects of the hemorrhagic principies

in lhe snake venoms.

SpECIFICATION AND QUANTITATIVE DETERMINATION OF THE HEMORRHAGIC LESION

In order lo approach hiochemically the mechanism of lhe local actions

caused by lhe venom il is essential (1) to specify the experimental conditions

to reprodnce a separate pathological change involved in the whole local pathological

lesion and (2) to establish a method for determining quantitatively the specific

principie responsihle for each specified pathological change on the basis of the

principie of bioassay.

We wish to express our gratitude to the Division of Public Health, Kagoshima

Prefecture, Japan, for their generous gtfts of Habu venom.

1, | SciELO

194

BIOCHEMICAL AND PATIIOLOGICAL ASPECTS OF HEMORRIIAGIC

PRINCIPLES IN SNAKE VENOMS WITII SPECIAL REFERENCE TO HABU

(TRIMERESURU8 FLAVOV1RIDI8) VENOM

As for thi‘ hemorrhagic principies, lhese requirements were salisfied since we

proposed the quanlitative method for delermination (8). The method consisls of

intracutaneous injection of 0.1 ml of venom inlo lhe depilated hack skin of rah-

hits. measurement of size of the hemorrhagic spot afler 24 hr from lhe inside of

the retnoved skin and the estimation of lhe activily hy the parallel line assay

method.

As shown in Fig. 1. the hemorrhagic spot observed from lhe inncr snrface

is sharply demarcated. A definite hemorrhagic spot visihle from the inner snr¬

face was produced vvilh such a small amount of Hahu venom as 0.1 lo 0.3 pg;

while ahont 100 lo 300 times as much dose was required lo produce a feehle re-

action, not clearly discernihle from lhe onter snrface (Fig. 21. The hemorrhage

following the injection with such a large amount of venom as 300 pg spreads

throughoul the dermis and muscular layer. and necrosis of the muscle fihers was

also ohserved (9). With such a large amount of venom, so-called “necrosis” (19)

nr “hemorrhagic necrosis” (20) was ohservahle (8, 18).

When lhe residis ohtained hy our procedures were analysed statistically, lhe

log-dosage response curves ohtained with a large numher of crude venoins and

their fractions were proved lo he linear and parallel lo each other(8). Some

of the results are shown in Fig. 3. By measuring the potencies relative to lhat

of a standard venom using lhe parallel line assay method, we succeeded in quan-

tilative determinations of the hemorrhagic activities of various preparations of

llalni venom. We eoncluded, therefore, lhat lhe size of lhe hemorrhagic spol

in lhe skin as detennined from the inside, but not from the outside, is an exact

measure for lhe intensity of the hemorrhage under the specified condilions (8).

We demonstrated lhat lhe method was applicable to venoms of different species

of snakes.

Distributjon of hemorrhagic, eethai. and proteolytic \ctivities in

SNAKES VENOMS

We carried out comparative studies on hemorrhagic, lethal and proteolytic

aclivilies of venoms from various species. Lethal toxicity (9,18) was assayed hy

intravenous injection inlo mice of an inhred slrain weighing 14-17 g with four

to fivc doses graded with 1.25-fold intervals. Death wilhin 4- days was ascrihed

to lethal toxicity of the venom.

Il is noled from Tahle 1 lhat hemorrhagic activily is distrihuled in all lhe

venoms of both Crotaunae and Viperinae, although the ratios of LD.-,,, lo

minimnm hemorrhagic dose (MHD) cspecially for samples No. 5 ( Bothrops alrox),

No. 9 (Crotalus durissus terrificus) and No. 15 (Vipera russellii) are very small;

almost none of lhe venoms of the ELAPIDAE manifest hemorrhagic activity and

therefore the ratios for such venoms are much smaller. The only exception is

Ophiophagus hannah venom (sample No. 21 I whose hemorrhagic activity is as

high as lhat of Crotalinae or Viperinae venoms.

Macroscopie observation indicated the similarily of hemorrhagic lesions caused

hy venoms from different species of snakes. The common slope (h) of log-dosage

response curves lor lhe hemorrhage is 4.72 with lhe venom of Trimercxurus //«-

voviridis. Values for b ohtained with all the other venoms were approximately

the same as that for Trimeresitrus jhmniridis.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butanian

Simp. Internac.

33(1): 193-205, 1966

AKIRA OHSAKA, TAMOTSU OMORI-SATOH, HISASHI ^95

KONDO, SATORU KONDO and RYOSUKE MURATA

The results (Table ]) may he an indication that the hemorrhagic activity

is not directly assoeiated with the proteolytic aelivity or the lethal toxicity in lhe

crude venoms.

TABLE 1

DISTRIBUTION OF THE HEMORRHAGIC, LETHAL AND PROTEOLYTIC

ACTIVITIES IN SNAKE VENOMS

Snake venom

Hemorrhagic activity

MHD and its fiducial

limits (/tg)

Lethal activity

LD,„ and its fiducial

limits (/ig)

Proteolytic

activity

(units/mg)

1. A. contortrix con¬

tortrix

1.90 (1.20-2.92)

200 (163-245)

33.7

105

2. A. contortrix mo-

kasen

1.20 (0.76-1.90)

125 (102-153)

48.1

104

3. A. piscivorus pis-

civorus

0.80 (0.52-1.20)

60.0 (48.0-75.0)

41.5

4. A. halys

0.14 (0.09-0.22)

16.0 (13.0-20.0)

35.8

114

5. Bothrops atrox

2.11 (1.40-3.20)

5.6 (4.7-6.7)

44.5

2.7

6. Bothrops jararaca

0.75 (0.48-1.20)

18.5 (16.0-22.0)

74.0

7. C. adamanteus

0.04 (0.03-0.06)

18.5 (16.0-22.0)

9.76

462

8. C. atrox

0.43 (0.28-0.67)

45.0 (38.0-54.0)

83.6

105

9. C. durissus terri-

ficus

10. C. viridis viridis

18.0

0.56 (0.37-0.85)

3.6 (3.0-4.3)

21.0 (17.0-26.0)

39.2

39.5

0.2

11. T. flavoviridis

0.20 (0.14-0.30)

54.0 (46.0-63.0)

33.0

270

12. T. flavoviridis to-

k are n sis

1.15 (0.78-1.80)

160 (130-196)

37.8

139

13. T. elegans

0.30 (0.20-0.46)

71.0 (59.0-85.0)

13.0

237

14. T. okinavensis

1.38 (0.91-2.10)

140 (117-167)

69.0

102

15. Vipera russellii

21.0

2.2 (1.8-2.6)

5.56

0.1

16. Vipera ammodytes

0.47 (0.31-0.72)

7.4 (6.3-8.7)

41.7

17. Vipera palestinae

0.54 (0.34-0.92)

7.1 (5.9-8.5)

4.96

18. Causus rhombea-

tus

19. Bitis arietans

0.81 (0.53-1.30)

0.15 (0.10-0.22)

> 250

15.0 (13.0-18.0)

0.26

18.7

> 300

100

20. Bitis gabonica

0.04 (0.03-0.06)

13.5 (11.5-16.0)

11.7

338

21. Ophiophagus han-

nah

22. Bungarus fasciatus

0.84

» 100*

54.0 (46.0-63.0)

18.5 (16.0-21.0)

12.0

0.06

0.18

23. Naja melanoleuca

» 100

6.0 (4.8-7.5)

7.00

« 0.06

24. Naja naja atra

» 100*

8.0 (6.5-9.8)

0.12

« 0.08

25. Naja naja

» 100

5.6 (4.4-7.2)

2.85

« 0.06

A.: Agkistrodon CCrotalus T.: Trimer es urus

* A pinkish macule vvas observed at the site of injection.

EvIDEXCE FOR THE PRESENCE OF TIVO HEMORRHAGIC PRINCIPLES I.Y CERTAIN

SNAKE VENOMS AND FOR RELATIONSHIPS OF THESE PRINCIPLES TO PROTEOLYTIC.

LETHAL AND OTHER PATHOLOGICAI. ACTIVITIES

The presence of more than one hemorrhagic principie in certain snake venoms

has heen suggested (9-16); attempts have been made lo correlate the hemorrhagic

activity lo proteolytic aelivity (9-11. 14, 15, 18,21).

SciELO

10 11 12 13 14 15

-lOC BIOCHEMICAL AND PATHOLOGICAL ASPECTS OF HEMORRHAGIC

DD TMPTDI fC! TXT CM A \ 7 TPXTrNA/T O D/TTII CDDPTU TO T/ V 1 T.' IJ T,’ M T7 1 TH II A i;

1 ^ PRINCIPLES IN SNAKE VENOMS WITH SPECIAL REFERENCE TO HABU

(TRIMERESURUS FLAVOVIRIDIS) VENOM

We fractionated the venom of Trimeresurus flavoviridis by zone electro-

phoresis and demonstratcd the presence of two hemorrhagic principies, URI and

HR2 (9. 10), which are distinct immiinologically from each other(16). Both of

lhe hemorrhagic principies contained proteolytic activity (9. 10). CM-cellulose

chromatography also indicated the presence of two hemorrhagic principies; one

was separated from the main part of proteolytic activity, while the other associated

with it (11,14). Iwanaga and his associates (21) purified "proteinase b”, one

of the tliree proteinases present in the venom of Agkistrodon halys and stated

that it is one of the two hemorrhagic principies in this venom.

ll would he of much interest lo clarify whether or not the principies responsible

for hemorrhage also manifest lethal toxieity. We separated one of the hemor¬

rhagic principies (HR2) from the main part of lethal toxieity but failed lo

separate the other principie (HR1) (9). Imnmnological studies of HR1 not yel

published suggested that separate entities are responsible for the hemorrhagic

activity and the main part of lethal toxieity. Separation of hemorrhagic activity

from lethal toxieity was indicated also by Gitler and his associates (15) with the

venom of W'alterinnesia aegyptia. Qn the other hand, Omori and his associa¬

tes (13) fractionated the venom of Agkistrodon halys by DEAE-cellulose chromato¬

graphy and reported a dose association of the major part of the lethal toxieity

with the main hemorrhagic fraction.

It would be also of much interest to know whether or not one and the same

principie is responsible for necrosis and hemorrhage (7, 18). Histological ob-

servations done by us (9) indicated that lhe muscle degeneration, which led lo

necrosis or dealh of the muscle fiber in its severer forms, did not run parallel to

either hemorrhagic activity or proteolytic activity.

Further purification of the hemorrhagic principies is needed to correlate

hemorrhagic activity with proteolytic, lethal and other pathological activities.

Purification of the hemorrhagic principles in the venom of

Trimeresurus flavoviridis

We attempted purification of the hemorrhagic principles of Trimeresurus

flavoviridis (Batch No. 64-A). The two hemorrhagic principles HR1 and HR2

(Step 1 in Table 2), separated by zone eleetrophoresis (9), wcre further purified

by different procedures shown in Fig. 4.

Purification of URI: Step 2 — A pooled fraction of HR1 (corresponding

to 6 g of lhe crude venom) separated by zone eleetrophoresis was concentrated

by lyophilization and dialysed against 0.005 M Tris-HCl buffer, pbl 8.5. The

solution at a concentration of 50 mg protein per ml was treated with solid am-

monium sulfate to 60% saturation (369 g per liter) (22) and left lo stand at 0"

for several hours for partial settling. The precipitate was collected by centrifuga-

tion at 8,000 r.p.m. for 15 min. The precipitate was dissolved in 30 ml of

0.005 M Tris-HCl buffer, pH 8.5 at a concentration of approximately 65 mg

protein per ml. Step 3 — The solution was passed through a Sephadex G-100

column (4X97 cm) previously equilibrated with lhe same buffer. Step 4 — The

hemorrhagic fractions from Step 3 were eombined and concentrated to about 14 ml

by lyophilization. The solution was passed through a Sephadex G-200 column

(5X115 cm) previously treated with lhe same buffer. Step 5 — The hemor¬

rhagic fractions from Step 4 were eombined, concentrated by lyophilization and

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 193-205, 1966

AKIRA OHSAKA, TAMOTSU OMORI-SATOH, HISASHI 797

KONDO, SATORU KONDO and RYOSUKE MURATA

(lialysccl against 0.005 M Tris-HCl buffer, pH 8.5. tlie final volume being about

7 ml. The dialysate was plaeed ou a CM-celluIose column (3X50 cm). The

break-through fractions containing the hemorrhagic activity were combined. Step

0 — The combined fraction was concentrated by lyophilization and dialysed

against 0.005 M Tris-HCl buffer pH 8.5. The dialysate of 7 ml containing

120 mg of protein was applied to a DEAE-cellulose column (1.5X30 cm). A

linear gradient elution with 100 ml of the buffer and 400 ml of the buffer con-

taining 0.5 M NaCl was started.

Punfication oj HR2: Step 2 — A pooled fraction of HR2 (corresponding

to 6 g of the crude venom) separated by zone electrophoresis was concentrated

by lyophilization and a final volume adjusted to 30 ml. The solution at a con-

centration of 37.5 mg protein per ml was passed through a Sephadex G-100

column (5X100 cm) previously equilibrated with 0.005 M Tris-HCl buffer, pH

8.5, containing 0.15 M NaCl. Step 3 — The hemorrhagic fractions from Step 2

were combined and concentrated to 40 nd by lyophilization. The concentrated

solution containing about 15 mg protein per ml was passed through a Sephadex

G-75 column (5X86 cm) previously equilibrated with 0.005 M Tris-HCl buffer,

pH 8.5. containing 0.15 M NaCl. The hemorrhagic activity was eluted in the

void volume. Step 4 — The hemorrhagic fractions were combined, concentrated

by lyophilization and dialysed against 0.005 M Tris-HCl buffer, pH 8.5 for

several hours. Twenty-six ml of the dialysate containing 467 mg of protein was

applied lo a DEAE-cellulose column (4.6X12 cm). After a break-through peak

had been collected, elution was carried out with 500 ml of lhe buffer containing

0.3 M NaCl. The break-through fraction contained the hemorrhagic activity.

TABLE 2 — SUMMARY OF PURIFICATION PROCEDURES FOR HR1 AND HR2

Step

Protein

(mg)

Hemorrhagic activity

Proteolytic activity

MHD

(pg)

relative

specific act

recovery

( %)

specific act. relative

. ... . . . . specific act.

(units/mg of protem)

Startmg material

6,000

0.220

1.00

100

2 4.80

10 0

HRI

1. Electrophoresis

3.670

0.255

0.94

5 5.0

6.55

0.2 6

2. Ammonium sulfate

1.89 0

0.259

0.86

2 7. 2

8 26

0.33

( 0 - 60%)

3. Sephadex G-100

5 00

0.0825

2.69

2 2.2

14.41

0.5 8

4. Sephadex G-200

1 62

0.0367

5.95

1 6.3

16 20

0.6 5

5. CM-cellulose

1 20

—

6. DEAE-cellulose

a fraction

1 4 4

0.0193

11.41

\ 6.6

7 . 66

0.31

b fraction

22.5

0.0214

10.29

17.65

0.71

HR 2

1. Electrophoresis

1,125

0.220

1.00

1 8.8

7 4.10

2.99

2. Sephadex G-100

590

0.31 1

0.7 1

7.0

9 6.20

3 88

3. Sephadex G-75

467

0.31 2

0.7 1

5.5

1 2 9.00

5.20

4. DEAE-cellulose

333

0.294

0.75

4.2

12 8.00

5. 17

SciELO

10 11 12 13 14 15

inc BIOCHEMICAL AND PATHOLOGICAL ASPECTS OF HEMORRHAGIC

-Li/o DDTXTPrnl T.-tí TM ^NTAt/17 TnTVíTTTO UMTMI CTTTTPT A I lí IP L'T'TT T’\in,' -m I T A I

PRINCIPI.ES IN SNAKE VENOMS WITII SPECIAL REFERENCE TO IIABF

(TRIMERESURUS FLAVOVIRIDIS) VENOM

The yield and lhe exlent of purificalion at each slep are summarized in

Tahle 2. The hemorrhagic activity of HH1 was eluted Irom DEAE-celhilose inlo

Iwo peaks. The firsl peak of hemorrhagic activity (a-fraction in Slep 6) eontain-

ing less proleolytic activity than lhe second one served as the lest sample of HRI

in the following experirnent. The specifie hemorrhagic activity of HH2 did not

increase after Step 2 and lhe preparation in this step served as lhe test sample

of HI!2 in the following experirnent.

The action of the hemorrhagic principles on animal cells cultivated

in vilro

The crude venoin of Trimeresurus jlavoviridis and of the partially pnrified

hemorrhagic principies, HI11 and H112, were tesled for loxie action on animal

cells cultivated in vilro (171. The eell strains employed ineluded HeLa cells,

MLg cells originated from the lung of new-horn miee of ddY strain and T5

cells originated Irom human emhryonic fihroblasls.

The change of the cells observed in the earliest stage was rounding. When

the rounded cells predominated. the cells hecame detached from the glass surface

resulting in disruption of the cell monolayer. We designated lhe activity responsi-

lilc for this change as the cell monolayer-disrupting activity (17).

Tahle 3 shows the effcct of the crude venom and lhe partially purificd he¬

morrhagic principies on the monolayer of T5 or HeLa cell. Il is noted that 11112

with a high proteolytic activity was more potent in the cell monolayer-disrupting

activity than II111 with a low proleolytic activity.

As shown also in Tahle 3, HI11 at 30 gg protein per ml did not show any

cell monolayer-disrupting effcct within 24 hr but did show some effect after 48 hr.

The cell monolayer-disrupting activity of II111 on T5 cells is roughly onc sixlli

that of lhe crude venom on lhe basis of protein eontenl (Tahle 3). The specifie

hemorrhagic activity of HUI is about 11 limes highcr lhan that of lhe crude

venom (Tahle 3). Therefore, the cell monolayer-disrupting activity of H111 per

unit hemorrhagic activity is calculated as about one seventieth that of the crude

venom. We conclude, therefore, that lhe hemorrhagic activity of lllll is virtually

iudependcnl of lhe cell monolayer-disrupting (or cylopalhie) activity (17).

Thcse results on one harid confirmcd and on the olher hand contradicled

lhe suggestion made hy Caertner and her associates (23) that there is a elose

association of cytopathic with hemorrhagic and proteolytic activities.

Fig. 5 demonstrates lhe ahsence of parallelism hetwccn disruption of the MLg

cell monolayer and eyloeidal action hy lhe venom preparations. The MLg cell

monolayer was disrupted hy lhe crude venom and hy the preparation of II112

hui the disruption did not run parallel lo the viahility of the cells. It is, lliere-

forc. very likely that lhe venom preparations primarily acl on the cell surface

causing detachrnent of the cells from the glass surface hui not causing serions

damage lo the vital function of lhe cells (17).

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):193-205, 1966

AKIRA OHSAKA, TAMOTSU OlMORI-SATOH, HISASHI

KONDO, SATORU KONDO and RYOSUKE MURATA

199

TABEE 3 — EFFECT OF THE CRUDE VENOM AND THE PARTIALLY PURIFIED

HEMORRHAGIC PRINCIPLES ON THE MONOLAYER OF T5 OR HeLa CELL

Cell strciin

used

Incubation

periodt hr)

Ver.om

preparations

/Hr

Crude l v p f

-'•0)

= 1.0''

HR 1

/ Hr = 11.4\

'■Pr = 0.3'

/ Hr=0 .7-,

HR 2 ( Pr=3 .9^

30jig

lOjjg

5jig

30 jjg

10 jig 5pg

30pg lOpg 5pg

1.5

444

44 4

3.0

444

44 4

444

T 5

4.5

4 44

44 4

444

24.0

44 +

4 4

+ + -*

444 4

4 8.0

444

4 4 4

444 444

1 .5

44 4

- NO

444

- NO

3.0

444

- NO

4 44

* + + NO

HeLa

4.5

444

- NO

444

+ + + NO

24.0

444

- NO

444

+ + + NO

48.0

444

44 4

- NO

44 4

♦ 4+ NO

No change (identical to the control cell culture)

Slight morphological changes of the cells vvithout detachment from the glass surfaee

Rounding of the cells without detachment from the surfaee

Partlal disruption of the cell monolayer

+ + : Complete disruption of the cell monolayer

Not tested

Specific hemorrhaglc activity relative to the crude venom

Speelfic proteolytlc activity on casein relative to the crude venom

Per ml of culture médium

COYCLUSION

We succeeded in reproducing experimentally the hemorrhage under our specified

conditions. We also succeeded in establishing a quantitative method for determin-

ing the hemorrhagic activity. By this method we initiated systematic studies on

the principies responsible for the hemorrhage.

We demonstrated that hemorrhagic activity is widely distributed in all the

venoms of Crotalinae and Virerinae snakes. Evidenees were accumulated to sug-

gest the presence of at ieast two or more hemorrhagic principies in venoms of

certain snakes including Trimeresurus flavoriridis. The venom of Trimeresurus

flavoriridis was fractioned to correlate the hemorrhagic activity with proteolytic

activity, lethal toxicity or other pathological activities.

However, further experiments will be necessary to characterize more precisely

the principies responsibles for the hemorrhage.

SciELO

10 11 12 13 14 15

200

BIOCHEMICAL AND PATHOLOGICAL ASPECTS OF HEMORRIIAGIC

PRINCIPLES IN SNAKE VENOMS WITII SPECIAL REFERENCE TO IIABU

(TRIMERESURUS FLAVOV1RWIS) VENOM

ADDITIONAL LEGEND TO TABLE 1

Hemorrhagic activity was determined by the method previously described (8). The

minimum hemorrhagic dose (MHD) was defined as the least quantity of venom causing

a hemorrhagic spot oí 10 mm in diameter 24 hr after the intracutaneous injection. Lethal

activity was assayed by intravenous injection into mice of an inbred strain weighing

14-17 g with four to five doses graded with 1.25-fold intervals. The LD r „, was calculated

by the Reed-Muench method (24). The standard error of the LD.„ was calculated accord-

ing to Pizzi (25). Proteoiytic activity was estimated at pH 8.5 with casein as substrate.

One unit of the activity was defined as the amount of enzyme hydrolyzing casein at

such an initial rate that the amount of TCA-soluble Products formed per minute gives

the same optical density as that of 1 ^ig of tyrosine with the Folin reagent.

The venom of Crotalus adamanteus was purchased from Ross Allen’s Reptile Institute,

Silver Springs, Florida, U.S.A. The venom of Bothrops atrox was obtained from a sup-

plier in Brazil. Naja naja atra venom of Formosan origin and Bungarus fasciatus venom

were supplied by Dr. T. Suzuki of the Institute for Protein Research, Osaka University,

Osaka, Japan and Naja naja venom by Dr. B. N. Ghosh of the University College of

Science, Calcutta, índia. Venoms of Trimeresurus okinavensis and Agkistrodon halys

were obtained from a supplier in Tokyo. Trimeresurus flavoviridis venom was supplied

by the Division of Public Health, Kagoshima Prefecture, Japan and Trimeresurus elegans

venom by the Institute of Hygiene of the Ryukyu, Japan. The venom of Trimeresurus

flavoviridis tokarensis was supplied by Dr. H. Fukushima of Kagoshima University, Kago¬

shima, Japan. AH the other venoms were purchased from the Califórnia Corporation for

Biochemical Research, Los Angeles, Califórnia, U.S.A.

S .jararaca

HR 2

I UQ

HR I

T flavoviridis

lOmm

Fig. 1 — Patterns of hemorrhage observed from the inside of the removed skin. An

aliquot (0.1 ml) from each of 3-foId dilutions of the venom of Trimeresurus flavoviridis

or the partially purified hemorrhagic principies (HR1 and HR2) was injeeted intra-

cutaneously into a rabblt and the reactions were observed after 24 hr. The venom of

Bothrops jararaca was also injeeted for comparison.

2 3

5 6

10 11 12 13 14 15

0.3

Doses of venom in ug ( scole in log. )

Fig. 3 — Dosage response curve for a crude venom (Batch

n" 48) and its electrophoretic fractions (F13 and F26).

Fig. 2 — Patterns of hemorrhage observed from the outside. The skin is the same vvhich

is demonstrated in fig. 1. A: The venom of Trimeresurus flavoviridis ; B: IIR1; C: IIR2;

D: The venom of Bothrops jararaca.

2 3 4

SciELO

10 11 12 13 14 15

909 BIOCHEMICAL AND PATIIOLOGICAL ASPECTS OF HEMORRHAGIC

PIÍINCrPEES IN SNAKE VENOMS WITII SPECIAL REFERENCE TO HABU

(TRIMERESURUS FLAVOVIRIDIS ) VENOM

Storch column

electrophoresis

Gel filtration on

SephadexG- 100

Gel filtration on

Sephadex G - 75

Precipitation with ammonium

sulfate ut 60% soturation

DEAE-cellulose

chromatography

Protein I E2 ao I

Hemorrhagic activity

Proteolytic activity

DEAE-cellulose

chromatography

Fig. 4 — Purification procedures for HR1 and HR2.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Slmp. Internac.

33(11:193-205, 1966

AKIRA OHSAKA, TAMOTSU OMORI-SATOH, HISASIII 203

KONDO, SATORU KONDO and RYOSUKE MURATA

+ + +

° ô

Duration of the treatment inhr

Fig. 5 — Disruption of the MLg cell monolayer without cyto-

cidal action by the venom preparations. (The concentration

of the venom preparations was 20 microgram/mi. See also

the legend to Table 3 for the grades of disruption of the cell

monolayer).

Rekerences

1. van Heyningen, W. E. — Toxic proteins. The Proteins, lst Ed., Academic

Press, New York, 1954, pp. 345-387.

2. Slotta, K. H. — Chemistry and biochemistry of snake venoms. Progress in

the Chemistry of Organic Natural Products, 12:406-465, 1955.

3. Houssay, B. A. — Classification des actions des venins de serpents sur 1’orga-

nisme animal. Compt. rend. soc. biol., 105:308-310, 1930.

4. Kellaway, C. H. — Animal poisons. Ann. Rev. Biochem., 8:541-556, 1939.

5. Zeller, E. A. — Enzymes of snake venoms and their biological significance.

Adv. in Enzymol., 8:459-495, 1948.

6. Porges, N. — Snake venoms, their biochemistry and mode of action. Science,

117:47-51, 1953.

7. Kaiser, E. & Miclil, H. — Die Biochemie der tierischen Gifte, lst Ed., Franz

Deuticke, Wien, 1958, pp. 134-222.

8. Kondo, H., Kondo, S., Ikezawa, H., Murata, R. & Ohsaka, A. — Studies on

the quantitative method for the determination of hemorrhagic activity of Habu

snake venom. Japan. J. Med, Sc. & Biol., 13:43-51, 1960.

SciELO

10 11 12 13 14 15

10 .

11 .

12 .

13.

14.

15.

1G.

17.

18.

19.

29.

21 .

22 .

23.

24.

25.

BIOCÍIEMICAL AND PATHOLOGICAL ASPECTS OF HEMORRHAGIC

PRINCIPLES IN SNAKE VENOMS WITH SPECIAI. REFERENCE TO IIABU

( TMMERESURUS FLAVOVIRIDIS) VENOM

Ohsaka, A., Ikezawa, H., Kondo, H„ Kondo. S. & Uchida. N. — Haemorrhagic

activities of Habu snake venom, and their relations to lethal toxicity, proteo-

lytic activies and other pathological activities. Brit. J. Exper. Path., 41:478-

486, 1960.

Ohsaka, A., Ikezawa, H., Kondo, H. & Kondo, S. — Two hemorrhagic principies

derived from Habu snake venom and their difference in zone electrophoretical

mobility. Japan. J. Med. Sc. & Biol., 13:73-76, 1960.

Ohsaka, A. — Fractionation of Habu snake venom by chromatography on

CM-cellulose with special reference to biological activities. Japan. J. Med.

Sc. & Biol., 13:199-205, 1960.

Omori-Satoh, T., Ohsaka, A., Kondo, S. & Kondo, H. — A simple and rapid

method for separating two hemorrhagic principies in the venom of Trimere-

surus flavoviridis. Toxicon, 5, in press, 1967.

Omori, T„ Iwanaga, S. & Suzuki, T. — The relationship between the hemor¬

rhagic and lethal activities of Japanese Mamushi ( Agkistrodon halys blomhof-

fii) venom. Toxicon, 3:1-4, 1964.

Maeno, H. — Biochemical anaiysis of pathological lesions caused by Habu

snake venom with special reference to hemorrhage. J. Biochem. ( Tokyo ), 52:

343-350, 1962.

Gitter, S., Moroz-Perlmutter, C., Boss, J. H., Livni, E., Rechnic, J., Goldblum,

N. & de Vries, A. — Studies on the snake venoms of the Near East: Wal-

terinnesia aegyptia and Pseudocerastes fieldii. Amer. J. Troy. Med. & Hyg.,

11:861-868, 1962.

Kondo, H., Kondo, S., Sadahiro, S., Yamauchi, K., Ohsaka, A. & Murata, R.

— Standardization of antivenine. II. A method for determination of anti-

hemorrhagic potency of Habu antivenine in the presence of two hemorrhagic

principies and their antibodies. Japan. J. Med. Sc. & Biol., 18:127-141, 1965.

Yoshikura, H., Oguwa, H., Ohsaka, A. & Omori-Satoh, T. — Action of Tri-

meresurus flavoviridis venom and the partiaily purified hemorrhagic principies

on animal cells cultivated in vitro. Toxicon, in press, 1966.

Ohsaka, A. & Kondo, H. — Biochemistry of snake venoms. Recent Advances

in Medicai Science and Biology, 1:269-322, 1960.

Minton, S. A., Jr. ■ — Some properties of North American pit viper venorm

and their correlation with phylogeny. Venoms, lst Ed., American Association

for the Advancement of Science, Washington, 1956, pp. 145-151.

Mitsuhashi, S., Maeno, II., Kawakami, M., Hashimoto, II., Sawai, Y„ Miyazaki,

S., Makino, M., Kobayashi, M., Okonogi, T. & Yamaguchi, K. — Studies on

Habu snake venom. 1. Comparison of several biological activities of fresh

and dried Habu snake venom. Japan. J. Microbiol., 3:95-103, 1959.

Iwanaga, S., Omori, T„ Oshima, G. & Suzuki, T. — Studies on snake venoms.

XVI. Demonstration of a proteinase with hemorrhagic activity in the venom

of Agkistrodon halys blomhoffii. J. Biochem. (Tokyo), 57:392-401, 1965.

Laskowski, M., Sr., Kassell, B., Peanasky, R. J. & Laskowski, M„ Jr. — Hoppe-

Seyler Thiefelder Handbuch der physiologishen Chemie, Ed. 10, Vol. VI/c, in

press.

Gaertner, C., Goldblum, N., Gitter, S. & de Vries, A. — The action of various

snake venoms and their chromatographic fractions on animal cells in culture.

,/. Immunol., 88:526-534, 1962.

Reed, L. J. & Muench, H. — A simple method of estimating 50% end points.

Amer. J. Hyg., 27:493-497, 1938.

Pizzi, M. — Sampling variation of the fifty per cent end-point. determined by

the Reed-Mueneh (Behrens) method. Humun Biology, 22:151-190, 1950.

10 .

11 .

12 .

13.

14.

15.

1G.

17.

18.

19.

29.

21 .

22 .

23.

24.

25.

2 3

5 6

11 12 13 14 15

SciELO ^

Mem. Inst. Butantan

Stmp. Internar.

33(1) :207-211, 1966

H. SCHENONE

207

26. LATRODECTISMO Y LOXOSCELISMO EN CHILE. INCIDÊNCIA,

CARACTERÍSTICAS clinicas, pronostico, tratamiento y

PREVENCION

H. SCHENONE

Departamento de Parasitología, Universidad de Chile, Santiago, Chile

El Latrodectismo es producido exclusivamente por Lalrodectus mactans, araria

de hábitos exlradomésticos que se encuentra de preferencia en campos de cultivo

de alfalfa y trigo. Sólo por excepción ha sido encontrada en la vivienda humana

como resultado de un transporte pasivo junto con productos agrícolas. Los acci-

dentes ocurren en su grau mayoría en las áreas rurales, durante el día en el pe¬

ríodo comprendido entre los meses de Diciemhre y Ahril inclusives (Verano y pri-

mera mitad dei Olono). Los indivíduos afectados son en su mayor parte ohreros

agrícolas, aunque en algunas oportunidades hemos lenido ocasión de observar

casos de Latrodectismo en mujeres y ninos(lO).

El cuadro clínico, que ha sido descrito en Chile a partir de 1852 (1-2-9-11),

se caracteriza por dolor urente en el sitio de inyección dei veneno, seguido por

dolores generalizados intensos, sensación de angustia, espasmos y temblores mus¬

culares, y aumento de las secreciones sudoral. salivai, lagrimai y nasal. No se

observa lesión local. Un signo clínico muy importante es cl marcado aumento de

la presión arterial, que en ocasiones puede ser de gran valor para orientar el

diagnóstico (14). En algunos casos se presentan dolores abdominales intensos,

que sumados a Ia rigidez de la pared dei abdômen, pueden inducir al diagnóstico

de abdômen agudo (2-14). En un grupo de 6 pacientes que en 1957 estudiamos

desde el punlo de vista electrocardiográfico, en 5 observamos alteraciones dei seg¬

mento S-T y de la onda T que se normalizaron en un lapso aproximado de 2 se¬

manas (16). Toda la sintomatologia descrita va atenuándose en forma progresiva

y termina por desaparecer al cabo de una semana, aunque el paciente queda

muehas veces en una condición de adinamia y de hipersensihilidad músculo-cutá-

nea que persiste por un tiempo más prolongado (2-11). De acuerdo con la lite¬

ratura y con nuestra propia casuística, la mortalidad es de aproximadamente

2.4% (3-11-15).

Sin pretender desconocer cl valor dei tratamiento específico a base de suero

anti-latrodectus (4), queremos destacar el êxito espectacular que se obtiene me¬

diante el empleo de neostigmina (prostigmina) inyectable, opinión que es compar¬

tida por numerosos colegas que ejercen en las áreas rurales de Chile (14). La

neostigmina. de la que se puede disponer fácilmente en cualquier servicio médico,

por su acción parasimpático-mimética actuaría neutralizando los efectos farmaco¬

lógicos dei veneno de Lalrodectus, cuya actividad estimuladora dei sistema

SciELO

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LATRODECTISMO Y LOXOSCELISMO EN CHILE. INCIDÊNCIA,

CARACTERÍSTICAS CLINICAS, PRONOSTICO, TRATAMIENTO Y PREVENCIÕN

simpático cs particularmcnle acentuada a partir de la cuarla a sexta horas después

de ocurrido el acidente, plazos mínimos en que el promedio de los pacientes acu-

den a solicitar atención médica.

La prevención de los accidentes produeidos por la mordedura de Latrodeclus

maclan.s debe hasarse en una adecuada información sanitária, aunque es dei co-

nocimiento popular el peligro que esta araria representa. En algunas oportunida¬

des hemos oblenido buenos resultados en la eliminación de este aracnido en te¬

rrenos altamente infestados, mediante rociamientos aéreos con emulsión de Lindano

al 0.25'% en la proporción de 125 miligramos por metro cuadrado; o mediante

la incineración de hierbas y malezas que liabían sido invadidos.

El Loxoscelismo es causado por Loxosceles rufipes, araria predominantemente

doméstica que se encuentra de preferencia Iras de los cuadros y muebles. Al

igual que Latrodeclus mactuns no es espontaneamente agresiva y sólo ataca in-

yectando su veneno, cirando se siente agredida al ser aplastada en forma inad¬

vertida contra la superficie cutânea. Loxosceles , que desarrolla grau parte

de su aetividad durante la noebe, ha sido encontrada en numerosas áreas urbanas

dei país comprendidas entre los paralelos 18 y 39 de latitud Sur. En un estúdio

realizado en la ciudad de Santiago en 1963, se pudo comprobar su existência

err el 38.4'% de 395 viviendas escogidas al azar (12).

Lesiones necróticas causadas, al parecer, por ararias Loxosceles han sido

descritas ya a fines dcl siglo pasado (11), aunque fué Macchiavello en 1937 quien

demostro el papel etiológico de Loxosceles en lo que denomino aracnoidis-

rno cutâneo o mancha gangrenosa de Chile (6-7).

La mayoría de los accidentes ocurre durante la época de calor, aunque larn-

bién pueden presentarse en plena época invernal, no hahiendo predominio por sexo

ui por edad. En el 85% de los casos el aecidente Uivo lugar en el âmbito do¬

méstico, ya sea durante cl suerio nocturno, o cuando cl paciente estaba vistién-

dose (12-17-21).

En líneas generales el cuadro clínico puede adoptar dos modalidades evolu¬

tivas: la cutânea o benigna y la eutáneo-viseeral, grave o sistémica. En el mo¬

mento inicial de ambas modalidades hay localmente scnsación de clavadura que-

mante, la que es seguida posteriormente por dolor intenso que se irradia a las

vecindades dei sitio de penetración dcl veneno. Focas horas más tarde, sobre

una zona de edema duro, doloroso y a veces acentuadamente progresivo, aparece

una placa violácea pálida al comienzo, que se transforma en una mancha equimó-

lica que se va haciendo cada vez más oscura, la que puede alcanzar hasta 25

centímetros de diâmetro. Esta mancha, que ha reeibido cl nombre de placa live-

doide, suele aparecer rodeada de un balo eritematoso y presentar en su superficie

ampollas de diferente tamano y de contenido seroso o serohemorrágico. A medi¬

da que transcurre cl liempo prosigue el oscurecimienlo de la placa, la que termina

por transformarse en una escara negruzea, la que finalmente se desprende en un

lapso aproximado de 4 semanas, dando lugar a una úlcera en algunas ocasiones.

Esta lesión cutânea normalmente no se acompana de adenopalía regional (17-20-

21). Algunas veces hemos tenido oportunidad de observar casos de Loxoscelismo.

en que la mordedura había ocurrido en la cara, y en los cuales el paciente pre-

sentó un edema gigante que compromelió todo el rosto, extendiéndose hasta cl

cuero cabelludo y cl cuello, sin aparecer lesión equimótica ni necrótica en todo

el transcurso de la evolución. Este es lo que constituye clínicamente la forma

cutânea benigna de predominio edematoso (13).

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

38(1):207-211, 1966

JI. SCHENONE

209

Como ya Io hemos manifestado, Ia sintomatologia que hemos descrito co¬

rresponde al Loxoscelismo cutâneo Iienigno, pero en el curso de las primeras 24

a 48 horas después de ocurrido el accidente, puede agregarsele uua serie de ma-

nifestaciones tales como fiebre elevada, hematúria, hemoglubinuria, anemia e icte¬

rícia, que earaclerizan a la forma cutáneo-visceral, que puede terminar en coma

y muerte (8-12-17-20-21). No hemos observado ningun tipo de relación entre la

aparición dei compromiso visceral y la ubicaeión o tamaho de la lesión local, ya

que hemos visto pacientes que se recuperaron y que habían presentado una lesión

local mínima, a veces puntiforme, conjuntamente con un grave compromiso sisté¬

mico, al mismo tiempo que oiros presentaron una extensa y severa lesión local

que se acompanó también de grave compromiso visceral. Por otra parte en las

formas cutâneas benignas, los pacientes han presentado lesiones de la piei que han

ido desde las lesiones petequiales hasta extensas zonas de necrosis (8-13-17-20-21).

En el tratamienlo de la forma cutânea de Loxoscelismo el empleo oportuno

y mantenido de antihistamínicos inyeetables lia dado buenos resultados, especial-

mente en lo que se refiere a la eliminación o reducción dei dolor y dei edema (12-

13-17). En casos de necrosis cutânea extensa ha sido necesaria la reparación

quirúrgica (12-17). En la forma cutáneo-visceral. que hasta 1952, época en que

empezaron a usarse los corticoides para su tratamiento, era de pronóstico fatal,

el uso de estas sustancias por via inyectable ha dado resultados satisfactorios (5-

8-12-17-20). Recomendamos Ia via parenteral debido a que en los casos fatales,

además de las lesiones renales y de otros parenquimas, se han observado feno-

menos de congestión, edema y hemorragia en diferentes segmentos de la mucosa

dei tubo digestivo que impedirían la absorción adecuada de cualquier fármaco

que se administre (20). Reconocemos el valor dei uso oportuno y en dosis ade-

cuadas de los sueros específicos anliloxosceles, y aunque los hemos usado en al-

giinos pacientes, nuestra experiencia es limitada para poder dar un juicio defini¬

tivo al respecto.

En general, el 10% de los casos de Loxoscelismo corresponde a la forma

cutáneo-visceral, de los cuales en la aetualidad, sólo un 10 a 20% tiene una

evolución fatal, siendo la mayoría de estos últimos, pacientes que solicitaron atcn-

ción médica en forma rnuy tardia.

La prevención de los accidenles causados ]ior las aranas Lo xos ceies se

hasa fundamentalmente en acciones educativas: conocimiento de la existência de

estas aranas, de sus hábitos y de su peligrosidad. Sobre estas bases se debe pro¬

piciar la adopción de medidas tendientes a evitar que ocurra el accidente, tales

como mantener las camas alejadas de las paredes y examinar y sacudir las ropas

que pueden servirles de refugio momentâneo, en el momento en que se las va a

usar, y medidas de aseo cuidadoso y periódico de la vivienda con el objeto de

destruir las aranas y sus telas, y eliminar las condiciones que hagan posibles su

superviveneia y mulliplicación (19).

Referencias

1. Cruzat, D. — La picadura dei Latrodectus formidábilis. Boi. Med., 1:108-110,

1884.

2. Gajardo-Tobar, R. — La picadura de la arana dei trigo (Latrodectus mactans).

Rev. Med. Chile, 69:707-713, 1941.

3. Gajardo-Tobar, R. — La anatomia patológica dei latrodectismo. Boi. Inf.

Parasit. Chilenas, 6:20-21, 1951.

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210

LATRODECTISMO Y LOXOSCELISMO EN CHILE. INCIDÊNCIA,

CARACTERÍSTICAS clinicas, pronóstico, tratamiento y PREVENCIÓN

4. Gajarclo-Tobar, R. & Vildósola, E. — Anotaciones acerca dei tratamiento dei

latrodectismo. Los seis primeros casos curados con suero anti-L at r o d e c t u s.

Rev. Med. Valparaiso, Chile, 17:328-335, 1944.

5. Kirberg, M., González, I. & Bauzü, J. — Loxoscelismo cutâneo visceral y cor-

tisona. Arch, Hosp. Clin. Roberto dei Rio, Santiago, 14:112-113, 1952.

6. Mncclúavello, A. — La Loxosceles laeta, causa dei aracnoidismo cutâneo o

mancha gangrenosa de Chile. Rev. Ch. Hist. Nat., 41:11-19, 1937.

7. Macchiavello, A. — Aracnoidismo cutâneo o mancha gangrenosa de Chile. Pub.

Hlth. Trop. Med., 22:467-505, 1947.

8. Meneghello, J. & Emparanza, E. — Loxoscelismo cutâneo-visceral y cortisona.

Boi. Inf. Parasit. Chilenas, 7:11-12, 1952.

Miquel, J.

1852.

Sobre la araria venenosa de Chile. An. Univ. Chile, 9:332-334,

10 .

Olcese, A. & Schenone, H. — Latrodectismo en nihos. Pediatria, 7:135-140,

1964.

11. Puga, F. — El Latrodectus formidabilis de Chile. Impr. Draeger et Lesieur,

Paris, 663 pp. — Notes et Mem. Soc. Sei. Chili, 1892.

12. Reyes, H. & Schenone, H. — Algunos aspectos clínicos y epidemiológicos dei

loxoscelismo en Chile. Proc. 7th Cong. Internat. Trop. Med. & Mal., 4:194-

195, 1963.

13 Schenone, H. — Estúdio de 27 casos de loxoscelismo. Boi. Chil. Parasit., 14:

7-13, 1959.

14. Schenone, H. — Aspectos prácticos en ia clínica dei síndrome dei latrodectis¬

mo y su tratamiento con neostigmina. Boi. Chile. Parasit., 14:80-82, 1959.

15. Schenone, H. — Aracnidismo en ei mundo. Proc. 7th Cong. Internet. Trop.

Med. & Mal., 4:199-202, 1963.

16. Schenone, H., Niedmann, G., Bahamonde, L. & Bonnefoy, J. — Alteraciones

cardiovasculares observadas en el latrodectismo. Boi. Chile. Parasit., 12:29-30,

1957.

17. Schenone, H. & Prats, F. — Arachnidism by Loxosceles laeta. Report of 40

cases of necrotic arachnidism. Arch. Derm., 83:139-142, 1961.

18. Schenone, H., Reyes, H. & Carrasco, J. — Ensayo de eliminación de Latroc-

tus mactans en campos cultivados. Boi. Chile. Parasit.,, 17:52-53, 1962.

19. Schenone, H. & Reyes, H. — Loxoscelismo. Nociones sobre su epidemiología

y profilaxis. Boi. Chile. Parasit., 18:38-39, 1963.

20. Schenone, H., Semprevivo, L. & Schirmer, E. — Consideraciones a proposito

de dos casos de loxoscelismo cutâneo visceral. Boi. Chile. Parasit., 14:19-21,

1959.

21. Schenone, H., Fanta, E. & Zamora, A. — Viscero-cutaneous loxoscelism in

children in Chile. Proc. llth Internat. Cong. Pediatrias, Tokyo, Nov., 1965.

In press.

Discusion

H. Pesce: “Latrodectismo: En la fase vago-paralítica da excelente resultado

en los casos dramáticos adrenalina — 1 ml.” Loxoscelismo: Indicación de dial isis

extra corporea (artificial) en los casos de nefrosis dei nefrón distai por hemolisis

y hemoglobinuria.”

SciELO

10 11 12 13 14 15

Mom. Inst. Butantan

H. SCHENONE

211

Slmp. Internac.

83(1):207-211, 1966

F. Saliba: "Suas observações sôbre inoculação em animais de veneno loxoscé-

lico são iguais ao quadro humano?”

H. Schenone: “En animales susceptibles como ei conejo, se puede reproducir

un cuadro local y general muy similar al que se observa en el hombre.”

Gajardo-Tobar: “1 — En chile no he observado casos de Latrodectismo por

picadura de Latrodectus curacaviensis. Todos han sido por veneno de Latrodectus

mactans.

2 — Sobre uso de suero: Excelente resultado tanto para Latrodectismo como

para Loxoscelismo. Naturalmente, para cada caso, el suero específico debe em-

plearse precozmente y con cantidad adecuada.

3 — El nombre justo de Loxosceles es, como dice Dr. Biicherl, L. rufipes,

pero todavia resulta un poco aventurado hacer cambiar a los médicos la nomencla¬

tura porque recien han incluído en la patologia el Loxoscelismo.”

H. I. Bicher: “Did the speaker find clinicai correlation to the cardiotoxic action

ascribed by Dr. Maretic to the Latrodectus ?”

H. Schenone: “En un grupo de enfermos que presentaban un cuadro de La¬

trodectismo observamos alteraciones dei electrocardiograma que eran similares a

un trazado correspondiente a isquemia.”

H. Pesce: 1» — Acerca dei Latrodectismo, deseo poner de relieve que tanto

en Perú como en Chile, raramente el paciente llega donde el médico en las pri-

meras horas después dei accidente. La primera fase es caracterizada por un sín-

drome simpático-tónico, que dura de 2 a 4 horas. En esta fase, el Dr. Couric, de

Miami, obtuvo resultados espectaculares con el uso de % a 1 miligramo de cloruro

de adrenalina, por via subcutânea, con desaparición pronta dei dolor que no cedia

ni a la morfina. En la segunda fase. la vagotónica, es conocido el buen resultado

de la prostigmina (neostigmina).

2 V — Acerca de los casos víscero-hemolíticos de Loxoscelismo, coincidimos con

su menor frecuencia y su gravedad. En la serie de 31 casos peruanos de Izú, hubo

13 (42%) víscero-hemolíticos; dentro de estos hubo 5 fallecidos (16%) correspon-

diendo todos a ninos menores de 5 anos, en los cuales por lo tanto Ia mortalidad

fue dei 100%. Un estúdio reciente de Maya (1963) en el Perú relata 3 casos de

Loxoscelismo hemolítico grave en los que fue aplicada la hemodiálisis con rinón

artificial. El primer caso fue en un obrero de 30 anos, que trabajaba a 3900 metros

de altitud, com gr. 4,7 de urea por litro; al 8" dia, después de la diálisis, murió

por bronco-neumonia bilateral. El segundo caso, 25 anos, llegó a gr. 7,2 de urea

por litro y estuvo 33 dias en anuria; con 3 diálisis curó en 4 meses; controlado

sano a los 2 anos y medio. El tercer caso, 9 anos, tuvo gr. 7 de urea por litro y

estuvo en anuria 20 dias; curó con 1 diálisis en 3 meses y medio.

Debemos recordar que, según varias esladísticas, Ia sobrevivência máxima en

anuria por nefrcsis dei nefrón distai, varia de 1 a 4 semanas; y según Baslow la

sobrevivência media en una serie de 500 casos fue de 9 dias.

En cuanto al prognóstico, el mismo Baslow dice que con urea inferior a 3

gramos hay mortalidad dei 34% y con urea mayor de 5 gramos la mortalidad llega

al 78%.

Por lo tanto la indicación de la diálisis es importante: antes se hacia cuando

el grado de uremia se iba elevando; en Lima se efectúa de inmediato, sin esperar

la uremia alta.

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):213-226, 1966

D. S. CHAPMAN

213

27. THE CLINICO-PATHOLOGY AND TREATMENT OF SNAKEBITE IN

SOUTHERN AND CENTRAL AFRICA

D. S. CHAPMAN

University of Natal, Faculty of Medicine, Durban, South África

Introduction

lí there are no fairies at lhe bottom of one’s garden, in Soulh África there

niay be a snake or Iwo. África shares with other world areas a high attainment

of its herpetologists and a paucity of detailed medicai observations. Indeed.

ihere is seldom opportunity lo study thc early and often urgent symptoms of

snake bile as those exposed are mainly primitives and rural dwellers.

A local difficulty is apparent. Elsewhere, a certain family or even species

is outstanding as the cause. In Southern África, by lhe many varieties available,

these wilh a wide varialion in colour and markings within the species (e.g. the

cobras), by the frequent mimicking of venomous by harmless snakes, and by the

fact lhat the snake usually flits after an altack. it is not surprising that snakes

are rarely identified.

In our hospital experience also, it has not been possihle to differentiate the

elapine snakes on clinicai grounds but from the scanty evidente previously avail¬

able, the very rapidity and severity of a venom action has tended to point to a

particular species, the black mamba. However, lhe puff adder has often been

identified by its sluggish grossness and we have assumed that our most serious

cases of local tissue death were caused by it when the snake was not identified.

Similarly the night adder has been identified often enough to apportion blame.

Elapine bile victims have tended to report early enough (lig. 1) for the clinician

to give a fair description of the clinicai features but where adder bile victims

have reported late (and only because of persistence of swelling or because of

gangrene), there have been few competent early observations.

Though it is often claimed that amateurs can easily recognise our cobras

and adders, most victims do not qualify, are bitten in their ignorance, often in

the dark, and are often children to whorn all snakes are large.

The common venomous snakes

Southern and Central África have the dubious distinction of harbouring some

137 venomous snakes comprised in three families, COLUBRIDAE, ELAPIDAE and

VIPERIDAE but not more than 25 species however, are capable of causing death

(Table 1). Of all areas Natal, my home province, has the greatest variety of

snakes and highest incidence of bite.

SciELO

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214

THE CLINICO-PATHOLOGY AND TREATMENT OF SNAKEBITE IN

SOUTHERN AND CENTRAL AFRICA

TABLE I — COMMON POISONOUS SNAKES OF SOUTHERN AFRICA

COLUBRIDAE

ELAPIDAE

VIPERIDAE

Skaapstekers

Boomslang

Bird snake

True Cobras (4)

Rinkals "Cobra"

Mambas (3)

Puff Adcier

Nlght Adder

Gaboon Viper

Berg Adder *

Mole Viper **

Carpet Viper *

Iiaemotoxic

Neurotoxic

Local cytotoxic

* and ** Also has special elapine effects.

Has dominant coiubrid effects.

The three COLUBRIDAE shown in Table 1 are all peculiar lo Southern

África. They are back-hiters with a limited capacity for a successful strike hut

lhe boomslang and lhe bird snake have at least extremely potent vcnoms which

have a marked effect on hlood coagulation. Our peculiar experience is fortunate-

ly tempered hy lhe docilily of these snakes.

The ELAPIDAE have small permanently erect fangs and are all dangerously

venomous with a serious neuro-toxic effect. Many are small inoffensive burrowers

hut included are 4 mambas and 4 true cobras. The Rinkals “cobra” and the

black-necked cobra also spit accurately to cause conjunctivitis; hut ihis can he

adequately deall with hy simple lavage.

The vipers show the highest development of fangs which are mobile, large

and hollow. Undoubtedly the Puff Adder is rightly feared for ubiquity, the

potency of its cytotoxic venom and is productive of the majority of our serious

bites. The Gaboon viper is our largest viper and very venomous. A fearsome

creature hut reputedly good natured, it is reluctant to strike except when trodden

on. Found in rain forest areas, it is fortunately rare. There are two species of

night adder which frequently cause the Iess serious bites. The berg adder has

also a special elapine effect. Mole vipers abound and cause many of our lesser

biles. The carpet viper, found in the more Northern areas, appears to have a

special coiubrid effect.

The highest incidence of snakes is in the more populous areas where Iand

has been cleared for cultivation and where habitation has inviled rodents.

Epidemiology

I present here evidence of a 7-year study of over 1,000 cases of snake bile,

892 of which were admitted to our hospital. Table II presents lhe cases accord-

ing to each family, together with an analysis of lhose with serious effects, and

the fatal cases. It is seen lhat overall there was a high incidence of morhidity

and mortality though relative to the size of the population at risk the incidence

of bites was low. Noted too is lhat in 484 instances the snake was not identified.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):213-226, 1966

D. S. CIIAPMAN

215

TABLE II — SNAKE BITE — 7 years 1957-1963 — Natal

Species

Total Cases

COLUBRIDAE

Skaapsteker .

Natal Black Snake

Ilerald Snake .

ELAPIDAE

True cobra .

Rinkals cobra .

Black Mamba .

Green Mamba .

VIPERIDAE

Berg Adder .

Pulí Adder .

Nlght Adder .

Puff or Night Adder

Unknovvn

venomous

harmless

1068

538

Died

Severe

effects

Sotne human altitudes to snakes

Let lis consider some importanl luiman attitudes which infltience lhe incidence

and severity of snake Idte.

Whether as some would claim we are not bom to fear snakes but are con-

ditioned lo il, fear is there and fascination too. Both no doubt lead to trouble.

In our first aid we advise calm and immobilisation, yet the emotional distress is

real and if only to get to a snake-bite outfit, the impulse is to rim. In my series,

some adults and many children were frightened long hours after the attack.

Only a fevv of tis surely think snakes are beautiful but many are attracted

and mainly perhaps by the possible danger.

So boys beeome amateur herpetologists and as they grow they become in-

different to the possible dangers. Ignorance reaches its zenith in the case of

snake-handlers even to their ignoring bites when working in snake parks because

they think they “know” their snakes and falsely believe in immunity from previous

bites. Nor can snakes be tamed and an usually docile one can take offense at

any lime. Thns lias one experienced herpetologist and at least 4 snake catchers

lost their lives in Southern África. Snake catching and handling are obviously

hazardous and il does not seem tliat enougb care is taken.

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Exeept when dangerously close, Identification appears quite difficnlt. Our

local snake park received its prize black mamlia, sent in error as a rather hand-

some specimen of a mole snake.

The wliite population is snake conscious al least with regard to the facililies

of protection and lliose such as nature lovers and fishcrmen carry snakediite out-

fils, no doubt lhough, many with out-of-date antivenoms and dirty instruments.

I nfortunately the main population at risk are the rural Baniu and Asiatics who

in fact. do seem ready enough to seek hel|) from mission hosjiitais, agricultural

hospitais and farms, all of which are equipped.

Age incidence oj bitc

Children run the greatest risk in their play (usually barefooted) and through

their curiosity of moving ihings (Fig. 2). Though the smaller children have no

fear. they are bitten in large numhers. The older children also molest snakes.

One, a Bantu herd-hoy tried to slrike a more agile mamba with his hand and

died by his audacity.

Some snakes such as the rinkals ‘‘cobra" can sham death and some bite

when they are dead. A Bantu male adult claimed the severed head of a black

mamba which bad just been shot for the purpose of fortifying a native remedy.

He placed il in his pocket and taking it out a few minutes later, he was promptly

bitten and was immediately very ill with neurotoxic symptoms. He survived.

A snake census is meritous but not entirely satisfactory. Numbers can be

readily increased by the reward — conscious deliberately rearing less harmful

snakes — even to enterprising schoolboys selling back to our snake park, snakes

stolen from it the day before.

No doubt to allay our fears, herpetologists stress that only some 300 of lhe

world’s 3,000 species are dangerous to man and few will attack unless molested.

This is cold comfort to the 1,000 victims of this series.

Seasonal incidence

Snakes may be more shy than lhose they bite but there is ample evidence

of aggression in the breeding season when it is warmer and more humid (Fig.

3) and when they are cul off from their lairs. Whatever the basis for attack,

snake bites do occur in large numbers wherever their ])alhs cross with othcr

animais. Yet lhe overall risk is small (Table III). In South África one lias

nine times the chance of injury travelling by car lo a picnic than from lhe

snakes there; and 40 times the chance of dying.

TABLE

III

Injury

Death

0.33

Roa d

211 . 3

14 . 4

X 9

X 40

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D. S. CHAPMAN

217

Situation of the bite

The very great majority of bites (Fig. 4) occiir on the feet and lower legs,

84%. The principal remainder are on the hand (7.7%). The risk is witli ter-

restial snakes, often through treading on them. vvell camouflaged as they are,

and most commonly with lhe more lazy adders which strike lower down than do

lhe cobras (and especially the mamhas) which rear to strike. We share with

other areas, alarm over head and trunk hites and even lhose of the upper limb,

situations where a tourniquet cannot he applied, and where better absorption is

likely I >y more ahnndant vascular connections.

The moral is plain — when in lhe country lo wear hools or at least shoes

and never open sandals; also thick long trousers wherever the vegetation is

ahnndant. To tread warily is not enough. Such precautions would reduce the

risk hy at least one half. One wonders whether snake catchers and herpetologists

in the ir field w'ork so protect themselves, and do lhey always wear gaontlets?

The clinicai, effects of bite

Table IV considers the general highlights of clinicai action for both elapine

and viperine snakes in our experience, and nsing the evidence of the fatal cases.

TABLE IV — SNAKE BITE DEATHS

1 — Long delay in treatment 2 — Profound effect in children

3 — Antivenom — too littie, too late. unsuitable site, wrong type

4 — Negative distant necropsy findings

Hospital aclmission

ELAPINE (8)

VIPERINE (9)

quite early

late

Clinicai, onset

rapid

late

march

rapid

slovv .

Dramatlc slgns

consistent

lacking

Local, paín

littie

often severe

swelling

littie

rapid, massive extravasation

A. H i t e s b y C o l a b r i d s n a k e s ,

Altack by these snakes is rare in any Southern African experience. All 5

cases in my series of such bite were almost symptom free and did not show lhe

special haemotoxic effect.

B. H i I es b y E I a p i n e s n a k e s (Table V)

The essential effect is distant from the bite, neurotoxic and rapidly produced

hy venous spread. We relate our first aid lo lhis — the tourniquet (the vital

first step) and the antivenom given intravenously.

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THE CLINICO-PATHOLOGY AND TREATMENT OF SNAKEBITE IN

.SOUTHERN AND CENTRAL AFRICA

TABLE V — THE CLINICAL EFFECTS OF SOUTHERN AFRICAN ELAPINE SNAKES *

Clinicai

No.

Fatal

Clinicai

No.

Fatal

Respiratory distress .

Breathless .

Shailovv respiration .

Lovv blood pressure .

Cardiac arrest .

Increased svveating .

Hyperpyrexia .

Nausea .

Severe vomiting .

Pallor .

Nose bleed .

Severe pain .

Swelllng, none .

little .

moderate . .

severe .

Vertigo .

Drovvsy .

Restless .

Unconseious .

Convulsions .

Headache .

Hallucinations .

Difficult swallowing *

Difficult speech .

Throat pain .

Increased saiivation .

Ptosis .

* Hospital group of 35, of which 25

identified and 10 iikely.

** Both associated with long applied

tourniquet.

*** Excludes those unconseious.

Experimentally all South African cobras have more potenl vciioms than have

the mambas but lhe mamba gains ils rightful dreaded reputation on accoimt oí

lhe greater volume of venom il can more rapidly inject.

Dealh from a cobra or a mamba bile is iu anything from minutes lo an

average of 8 hours and lhe majorily of lhose alive at 24 hours will survive and

wilhout ill effects.

There is a paralysis of molor action by a curare-like effeet peripherally on

molor nerve-endings or by a central brain-stem action, probably both. And there

is plenty of evidence of widespread effects on the cerebrum — vertigo, convulsions

and unconsciousness often occurring before respiratory failure is developed well

enough to give cerebral anoxia. The noticeable eyelid plosis, strabismus and

speech inco-ordination are useftil diagnostic features but not harmful.

The outstanding effeet is a paralysis of respiration and of deglutition so that

a victim embarrassed by the former can drown in his own saliva. Saiivation is

not increased but only seems to be; jusl as in other instances of interference of

deglutition (e.g. oesophageal câncer, head injuries).

Once commenced, respiratory palsy is rapid in progress bui there is probably

no march of palsy from one muscle group lo another. That diaphragmatic action

appears lo last the longest is probably beeause it is seen besl and especially when

SciELO

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Mem. Inst. Butantan

D. S. CHAPMAN

219

Simp. Internac.

33(1):213-226, 19(56

it is aided I>y vigorous struggles of abdominal muscles. In our cases il was not

easy to test lhe tone of limli muscles but it was seen that these also share in lhe

increasing flaccid palsy from lassitude lo inertia. Muscles which need to over-

come the effect of gravity are rnost easily seen to fail — in many cases the neck

muscles tire and the head falis. Plosis of the eyelids may well represent merely

such a failure. Finally the basic sphincters fail. to give incontinence of urine

and faeces.

Respiratory failure seems to take different forms depending ou the venom

dose. Most usually. the failure is quiet, breathing becomes increasingly shallow,

the subject also lying quietly unconscious, as seen in many of our cases. But

there can be more of a fight and an increased labour of respiration.

It has often been reported that for the less rapidly effective cobra venoms,

the heart can continue to beat strongly long after respiration has ceased, which

of course, is common to olher forms of respiratory failure. Such, at leasl would

mean that if respiration could be maintained artificially, the outlook need not be

so gloomy especially if the venom could be neutralised, which for cobras we have

proof that it can. We had one such case who in fact “died 4 hours after a

mamha bile (one hour after admission), was revived by exposed cardiac massage

to what we thought was normal heart action and was maintained for two days

on a respirator before he succumbed. Two others survived after 5 days of res¬

piratory support and I can now report three other successes from the past two

years.

More lhan half our cases had some disturbance of deglutilion or speech and

nearly all of these had profuse salivation. General cerebral effects were also com-

mon. One-third were unconscious on or after admission, 4 for very long periods

and yet survived.

Restlessness was seen most often with conscious subjects and lhe usual de-

meanour to death was of quiet unconsciousness and shallow respiration.

Many had profuse sweating which, with a low blood pressure, constituted

for some observers the picture of “shock”, but most of these cases also had fever.

Eyelid plosis was seen only in 8 cases. Pupillary changes were very variable

or absent and were not considered of diagnostic importance.

Half the cases had respiratory distress, all severe, and half of these survived.

In this elapine series, the local effects of hite were minimal or not existent.

Slighl swelling was occasional and pain was rare.

Though all South African venoms are essentially anticoagulant in vitro, ex-

cept in the case of the COLUBRIDAE, the haemorrhagic action is not important.

No elapine cases ever had haemorrhages nor jaundice and in fact the haemor¬

rhagic action in viperine hites was always local and not, I think, related to a

coagulation defect.

The value of antivenom-

1 consider from my series that we now have abundant proof of the great

value of the antivenom available (Table VI). Fourteen of 25 serious cases of

elapid bile responded to antivenom, some very dramatically. One noted too the

pleasant freedom from sensitivity reactions (1%) as contrasted with figures as

high as 30% in some world areas.

2 3

5 6

10 11 12 13 14 15

•220

THE CLINICO-PATHOLOGY AND TREATMENT OF SNAKEBITE IN

SOUTHERN AND CENTRAL AFRICA

TABLE VI — DETAILS OF ANTIVENOM THERAPY IN 892 CASES (Polyvalent

B. lachesis — H. haemachatus — N. nívea antivenom. S.A.I.M.R.)

Gtven

Not given *

No record

Antivenom .

712

142

Not required ** .

186

Requlred .

> 6 hours after bite .

* Includes 26

reporting 24 +

30 mts or more .

nours aner Diie ana ± runKai

Cobra spitting.

•* These cases had few or no

signs. No observation period.

Intravenous .

Sensitivity 7

Early

Late

Fatal

B. ELAPINE SERIES . 35

Severe . 25

Died . 9

Good response . 14

AIso other treatment . 3

II is also seen llial lhe doctors of Southern África seem shy of giving anti-

venoms intravenously, yel it is hy tliis route that success will come in elapine

]>ite cases. They also tend to ])rescribe doses which are lovv. We can see in

Table VI how few were given 30 mis of lhe antivenom or more, yet this is lhe

advised minimal dose for serious cases. Children also must be given at least an

adnlt dose, a dose dependent on their sizc relativo to the size of the snake.

I would never withhold this polyvalent serum from cases of puff adder bite

but it is difficult to evaluate the benefit. It was problematic whether il was ever

of any help in this series. Antivenom never appeared to resolve or indeed limit

any moderate sized or severe swellings. Many injections were ohviously of course,

given too late.

In Southern África we now have a polyvalent anti-mamba serum, but il will

be barder still to prove its value in the face of lhe very rapid fatal action of the

mamba venoms. It was only used in one case in this series and did not alter

the march of events.

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D. S. CHAPMAN

221

Fig. 5, records in pictorial forni, lhe march to death or recovery in severe

cases iii both the elapine and viperine series. Of 25 identified elapine bites, 8

died rapidly while 14 were resolved dramatically by the use of antivenom, 2 more

succeeding by lhe aid of assisted respiration; and one, it vvas thought, by the

exhibition of steroids.

C. Bites b y Viperine S n a k e s (Table VII)

The dominant clinicai effect of a viper venom is local, cytotoxic and self-

limiting. There is no general bleeding diasthesis. General effects are uncommon,

usually late and not neurotoxic (except in the case of 2 species). Spread of

toxin is by lymphatics, which ivas shown well in 43 cases where lymphangitis

was the main effect. This is a very important fact in management and immobil-

isation must be encouraged both of the part bitten and of the subject as a whole.

The effect is a destruction of all tissues especially blood vessels and their

contents hut the cytolysis with its tissue necrosis, the coagulation and thrombosis

of the blood produces its own barrier to spread. Bleeding adds lo the internai

pressure lo increase the ischaemia. So produced are the swelling, induration,

haemorrhages as ecchymoses and blisters, and necrosis.

TABLE VII — LOCAL EFFECTS OF SNAKE BITE

(7 years: 1957-1963 — Natal)

Cases

1 892

Swelling .

726 (slight, 290; moderate, 300; severe, 136)

Extravasation ...

Abcess .

20 (initial, 12; later, 8)

Necrosis .

23 (local, 11; extensive, 12)

Oligaemic shock .

25 (died, 9; dramatic response, 9)

Lymphangitis .

“Cellulitis” .

104 (by snake, 100; by bactéria, 4)

Thrombophlebitis .

4 (superficial, 1; deep, 3)

Tissue necrosis and oligaemic shock

Though an overwhelming envenomation is possible, for instance by an intra-

venous injection, with consequent convulsions and unconsciousness, each death in

this series had a rapidly expanding extravasation of a limb. None had frank

gangrene hut one had marked ischaemia distai to the main blood collection.

Nearly all took some time to die as contrasted with the elapine series and

in my opinion death was due to decompensated oligaemic shock caused by the

rapid massive blood loss, either not treated at all or in those who carne too late

for help.

There is a strong inclination however, in cases who have apparently been

doing well and who many hours after a bite have a sudden and fatal collapse.

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THE CLINICO-PATIIOLOGY AND TREATMENT OF SNAKEBITE IN

SOUTHERN AND CENTRAL AFRICA

íor clinicians to invoke a new speeial factor of venom action now with a dislant

as against a previous local effect. This is unlikely and no evidence is in fact

available. Three of the 9 fatal cases collapsed in hospital and others were rushed

in when it had occurred at home.

If enough cases could be shown lo develop collapse following rcmoval of a

long applied tourniquet, we might irnply a release of breakdown products of

muscle metabolism bul in thc inany instanccs we released tourniquets, collapse

did not follow.

It lias secnied thal the role of such breakdown products is no greater in lhe

production of general effects in viperine bite than with other examples of in-

faretion when, during the body’s struggle lo reject particularly frank gangrene,

the patient can hecome very ill — but he does so gradually and does not die

rapidly.

As I have indicated, if it occurs, any coagulation defect rnust lie local, per-

haps localised hy lhe barriers of necrosis etc. and no cases had jaundice. At

necropsy no distant effects were found and there was never evidence of pyogenic

invasion. Yet many had severe anaemia when first reporling.

Consider a limb ra[iidly filling with blood from the general circulation while il.~

own contents are immobilised by what amounts to an enclosed catastrophe of

tissue death. If a surgeon acquainted with crush injuries was to wilness this,

he woidd not hesitate to replace the blood lost and he would do it quickly. ll

does not surprise that the volume increase of a part can represent fully half the

original blood volume of the patient. In my series some extravasations reached

well into the trunk aflcr involving a whole limb.

The majority of victims are fortunately young adults who can adequately

compensate for the sudden oligaemia and at first, will appear quite normal. Bul

compensation cannot be kept up indefinitely and when it breaks, “shock” is then

often irreversible. The picture of collapse in viperine bites is identical with such

decompensation after crush injuries and extensive hurns. The clinician should

he aware that the signs usually described for oligaemic shock are either those of

intenso compensation or of decompensation — but shock is actually present from

the onset of swelling, building as it goes. We should treat the situalion and not

wail for lhe signs. Those who survive on their own (many must hover on the

brink), when lhey are admitted late, often appear toxic and their condition is

not unlike the “illness of trauma” following crush injuries.

Except where the swelling is due to the inflammatory oedema consequent on

lhe tissue insult by venom, when improvement will come from infusions of other

fluids such as plasma, only blood will suffice. T have indicated that lhe infusions

inay have to be very large. Care of course is required with late arrivals — for

these, packed cells should be used.

Vasopressor substances or steroids cannot restore blood volume depleted of

blood and in fact the former are dangerous. They do not increase cardiac out¬

put but do increase the work of lhe heart and most important. produce profound

renal ischaemia. Antivenom neither. can replace lost blood.

Thc sudling

This is two tnain lypes:

2 3

5 6

11 12 13 14 15

D. S. CHAPMAN

223

Mem. Inst. Butantan

Slmp. Internac.

33 ( 1 ): 213 - 226 , 1906

(1) Lillle or no surface extravasation with lhe limb sofl or hard.

(2) Haemorrhages evident as ecchymoses when the swelling is usually

more massive and solid.

Necrosis is induced in eilher type hy the very concentration of venom but

íound more often in the second type. It is then often extensive, superficial

or deep.

The lower limb has often been seen markedly flexed at lhe knee and by lhe

hardness of the swelling, we feared serious muscle involvement hy haemorrhage

or coagulation but in most of these cases, residual induration was rare.

The resolulioii of swelling

Many of lhose admitted with severe and extensive swellings had persistence

of these for over 10 days. The possihle importance of venous occlusion as lhe

basis of slow resolution is suggested hy the outcome in 4 illustrative cases. One

child had a thrombosis of the long saphenous vein of a lower limb as lhe solitary

clinicai feature. Three women who were hitten on the feet had swelling of the

limbs which had not been considered severe at the onset, still present at the end

of 3 months (Table VII). Solid swelling involved the greater part of a lower

limb with a tendency lo subside after rest and elevation. In all 3, venography

showed irregularity of and evidence of recanalisation in the deep venous system

(as though after thrombosis). Two of these women in the earlier phase of their

venous oedema had severe pain and marked tenderness on palpation of the popliteal

vein behind the knee. All 3 adults were hack to normal after a further 4

months.

Fourteen cases had an extremely rapidly developed soft swelling of much of

a limb, with almost as rapid a subsidence over some 12 hours. There were

judged to have had lymphatic oedema caused hy bites of the lesser adders.

Necrosis

Occlusion of a main vessel was only rarely the cause of the infarction seen,

which even when large, lended to be patchy. The action is mainly on smaller

vessels, sparing at least some tissue. Some of the deleterious effect is perhaps

due to the explosive effects of disruption. Twelve cases of necrosis were extensive,

4 involving the whole lower limb below the knee.

The role of bactéria

Bactéria come no doubt on snake fangs and by dirty incisions but are they

important? Though abscesses were found in 20 cases, only 8 had had an incision

and some of lhe others almost certainly represented lysis of necrotic tissue.

Local temperature rise and redness were seen frequently. Diagnosed by some

observers as “cellulitis” they occurred far too early after the snake attack to in-

criminate bactéria. Why not an inflammatory response to snake toxin?

Including lhose cases coming to necropsy, septicaemia or pyaemia were never

encountered. Gas gangrene and tetanus never occurred either.

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THE CLINICO-PATHOLOGY AND TREATMENT OF SNAKEBITE IN

SOUTHERN AND CENTRAL AFRICA

Only 9 cases of viperine snake lute had general effects separate from any

(listant influence of lhe local lesion. These vvere considered sensitivity reactions

to lhe venom hefore any antivenom had heen given. Six had vomiting, 2 had

abdominal colic and there was one inslance each of dizziness, headache, sweating,

urticaria, hyperpyrexia, drowsiness.

Fig. 5 shows graphically lhe outcome of 18 severe viperine hites when the

snake was identified. Though 9 died, some were admitted only after 24 hours

when they had collapsed at home, presumably from the decompensation of hlood

loss. Nine others responded well lo hlood infusion.

THEATMENT

A summary of “A System of Management of Snake Rite in Southern and

Central África”, whieh includes a “Simple Regime of Early Trealmenl”, is ap-

pended.

My criticisms of past and presenl modes of trealmenl is given in the light

of my experience of snake habits, lhe probable routes of snake venom action, and

the facilities available in our areas. From all the work, the simple regime was

devised.

Discussion

A. Barrio: “ln envenomation by ELAPIDAE neostigmine is useful, as de-

monstrated in neuro-muscular preparations, in 1949, in collaboration with O. Vital

Brazil?”

D. Chapman: “I have no experience with M i c r a r u s-envenomation. Pro-

stigmine has been used in some of our cases of elapid snakes, but not with any

measurabte effect.”

F. Kornalík: “Corroborate your opinion about the cause of death in viper-bites

being oligaemic shock. We had the same results in experimental animais in which,

even when a sublethal dose of venoms has been applied, anemia occurs with a rise

of the haematocrit from 45% to even 80%.”

D. Chapman: “The oligaemic shock is of great importance and is the primary

cause of the illness and death which follows our African viper bites.”

P. J. Deoras: “Have you any data about the areas the tourniquel has been

applied by various workers?”

P. J. Deoras: “Have you any data about the areas the tourniquet has been ap-

ing our African viper bites.”

Table J.EGENDS

Table I — The common poisonous snakes of Southern and Central África.

Table II — Snake bite in Natal in a 7-year period showing the morbidity and mortality

tncidence, relative to each snake species.

Table III — Contrast the risk. A contrast of the chance of snake bite and its mortality

with road accidents in South África.

Table IV — The outstanding events seen in those dying from elapine and viperine

snake bites.

Table V — The clinicai effects of Southern African elapine snake bite.

Table VI — I-Iow the available antivenom was used or abused. Its success in Identified

severe elapine bites is given. Note the low sensitivity incidence.

Table VII — The local effects of snake bite. Though this analysis includes all cases of

bite, swelling was not a feature of elapid bite and the figures given here

virtually represent the viper bite experience.

2 3

5 6

10 11 12 13 14 15

NUMBER OF VICTIMS

Mem. Inst. Butantan

Simp. Internac.

33 ( 1 ): 213 - 226 , 1966

D. S. CHAPMAN

225

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THE CLINICO-PATHOLOGY AND TREATMENT OF SNAKEBITE IN

SOUTHERN AND CENTRAL AFRICA

SITE OF SNAKE BITE

7 years 1957-63 Natal

1-5

* 7 -

DEATH

Fig. 4 — Situation of the Bite. The

figures represent percentages.

SNAKE BITE — THE MARCH & RECOVERY OF SEVERE CASES

ELAPINE VIPERINE

NORMAL

TIME IN HOURS

Fír. 5 — A graphic record oll the march to death or recovery in severe cases of identified

elapine and viperine snakebite.

2 3 4

6 SciELO 10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

S3(1):227-233, 1966

HERBERT LIESKE

227

28. POISONOUS SNAKE BITES IN GERMANY

HERBERT LIESKE

( Germany )

Poisonous snake biles are only of lillle importance in Germany, [hus most

doctors are nol familiar with a proper treatment, and there are only a very few

puMications dealing vvilli lhe problem of snake bite in Germany.

The only native poisonous snake lo be found in Germany is the adder or

common viper (Kreuzotter, Vipera berus, as it is called in Latin) which belongs

lo the viper family, and in addition, we find lhe Vipera aspis (Aspis viper ) in

the Southern part of the Black Forest (Schwarzwald). Biles caused hy these two

kinds of snakes do not produce grave symptoms and it is mostly the children

wh o are bitten hy snakes while strolling across the moors or through the meadows.

lhe board of statistics (Statistisches Bundesamt) at Wiesbaden cannot provide

wilh any recent statistics concerning either adder — or poisonous snake bites in

general or the deaths resulting from lhem. A partia! statistical collection of snake

bite accidents during the years 1883 and 1892 — this is before lhe introduction

of the serumtherapy — indicates 14 deaths (lhat is 6.4%) out of 216 adder bites.

Between 1907 and 1912, 265 adder bites were reported officially and only 6

deaths (lhat is 2.3%) were recorded. During the last 20 or 30 years the number

of bites and deaths resulting from them has been decreasing, the reasons being

lioth the introduction and the use of the serumtherapy and the housing develop-

ment which nalurally caused a further decrease in the number of the adder

family. (Part of the decrease, by the way, is eertainly due to offering rewards

lo people who caught adders.)

Notes on bite accidents are now oídy to be found from time to time in some

publications. Between 1951 and 1956, for example, only 19 adder bites were

treated in lhe hospital department of lhe Inslitute for Tropical Medicine in Ham-

bourg, where snake bite accidents occurring in the northern part of Germany are

looked after, and none of lhe patients died.

lf, however, you compare the number of killed and injured persons due to

traffic accidents in Hambourg, wilh its population of two million people, e.g. in

April 1966 1,010 people were injured and 25 died, while in December 1965,

134 deaths caused by various accidents were registered, and now compare to these

figures those of the snake bites, you will eertainly realize lhe little importance

of adder bites for lhe doctor in general.

Fatal adder bites are nowadays seldom heard of in Germany. In 1930, Fock

reported the death of a girl harvest worker, who was bilten in an ankle vein

and died 3 hours later. In 1952, Kirsch reported 2 deaths out of 20 cases treated

for adder biles. Only lately it was made known to me that in 1959 a weak-

hearted 60 year-old lady died of an adder bite. The case has never been published

1, | SciELO

228

POISONOUS SNAKE B1TES IN GERMANY

officially. In comparison to the hites of lhe rather harmless German adders those

of the imporled tropical snakes are more serious and often prove fatal. Sueli

accidents occasionally happen in lhe dock arcas during unloading of imporled

fruits (e.g. bananas), when lhe poisonous snakes travelling as stowaways are

disturbed. Furlhermore similar accidents are reported frorn zoos, snake farms.

pet shops, fairs, and snake fanciers. From 1951 until 1966, for example, 4 tropical

snake hites were treated in the hospital department of lhe Institute for Tropical

Medicine in Hambourg, 2 heing from a Bothrops schlegelii which, concealed in

a cluster of bananas, bit 2 stevedors, the other tvvo being biles of a Crotalus

viridis and a Bi tis arietans which bit 2 pet handlers. In all cases the biles could

be treated successfully.

The falalilies caused hy tropical snakes in Germany are far more frequent

than those resulting from adder hites. Since 1956 three fatal Cobra bites were

reported, which, because of their rarily and dramatical sirigularity, were widely

eovered by the press, and in onc case resulted in courl proceedings: A showman

in Konigswinter on the Rhine suffered the first of tliese bites. In 1956 he was

bitten in the tip of bis right forefinger by a 4 1/2 year-old Naja naja which he

had reared from an egg. He made an incision himself and applied a tourniquet

to bis arm. Il was U/y hours later when he first called for a doctor and then

was injected with a Cobra anti-serum (5 ml locally and 5 ml intramuscularily)

which was already outdated for one year. Further anti-serum, which had been

ordered immediately aflerwards, was injected 3% hours after lhe bite occurred.

However, although it was only a 10 ml polyvalent serum dose, produced by the

Rehring Werke for treatment of bites from European and Mediterranean vipers

and not effective against Cobra bites, it was nevertheless injected. 6 hours and

10 minutes after lhe accident the patient died from respiratory paralysis. In

December 1962 an experienced 55 year-old animal keeper was bitten by a 120 em

long Indian cobra in lhe tip of bis right forefinger, while working in an animal

compound in the Bremen Zoo. Mardly 10 minutes later the patient, a tourniquet

already applied, was in hospital, where the specific antivenom not being available,

he was injected locally and intravenously with 20 ml of an anti-serum for the

treatment of Furopean an Mediterranean snake hites. U /2 hours after the bite

30 ml of a polyvalent serum, produced by the Behring Werke for treatment of

bites from Norlh Afriean vipers and some kinds of Cobra snakes, which had

been ordered in the meanwhile, was injected. Only 5 hours after the bite 60 ml

of a polyvalent serum for Cobra venorn intoxicalion which had been flown in

by helicopter could be injected intravenously. In addition, a Ringer drop in-

fusion, hlood transfusions, gammaglobulin, and a 4 hourly injected dose of 40 mg

prednisolone (Urbason) were given, and, at intervals, artificial respiration was

carried out. The patient at first responded well to the treatment and the siluaiion

seemed less grave, however he died suddenly 17 hours after the bite from res¬

piratory paralysis. In April 1963 another bile from a Cobra (Naja naja) proved

fatal. The 37 year-old owner of a small snake farrn in the Danube region was

bitten in lhe left forefinger while removing lhe poison from the snake s vonom-

tooth. He tied off his forefinger himself and only an hour later he went lo lhe

hospital where he hrought with hirn 3 different ampules of a polyvalent anti¬

venom for lhe treatment of hites from Mediterranean vipers and the poisonous

snakes of África, Central and Soulli America. Before removing the tourniquet

and after a thorough exeision of the wound was carried out, he was injected inlra-

muscularily with a serum against poisonous snakes of the Afriean conlinent.

After th is treatment the patient left the hospital against the strong advice of lhe

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 227-233, 196(5

I1ERBERT LIESKE

229

dorlor. But 21/í> hours he was admitted lo lhe hospital again liecause of an

acute deterioration of li is State. He was given a drop infusion and artificial

respiration was carried out. After 12 and about 18 hours after lhe bite, 10 ml

of African snake serum were injected again besides the daily injection of 5 mg

Decortin H which was given intravenously. 28 hours after the bite the patient

suddenly died from heart failure and respiratory paralysis.

lhe 3 cases reported have the following in coramon:

1. The patients bitten though handling poisonous snakes every day

were nol sufficienlly informed about lhe danger of a snake bite.

2. I hey did not possess a specific antivenom.

3. l.ven lhe doctors who had lo carry out the treatment couhl only

apply the antivenom — if at all — too late.

After this exposition of the snake bile situation in Germany kindly aliou me

to give you some details about the treatment of snake bites usually carried out

in Germany.

I he treatment is divided into first aid measures and the definite therapy

including the application of antivenom.

rirst aid measures are fairly well known lo doctors and laypeople. The ap¬

plication of a tourniquet and absolute immobilization of the bitten limb is a

measure everyhody seems to know. Incision, suction, and somelimes cooling the

bitten area is done in most cases. Sometimes bleeding from lhe bitten area is

allowed. In order to stimulate the patienfs hlood circulation coffee or tea is

administered to the bitten person. Whenever a person is bitten by an adder in

Germany specific antivenom is readily available al many chemist’s, usually as

polyvalent serum of the “Europe”-type, produCed by lhe Behring Werke. This

antivenom may he injected by the doctor administering first aid when immediale

transportation lo a hospital is not lhought necessary. It is interesting to note

lhat surgeons usually make incisions in order lo open the bitten area, whereas

internai specialists commonly prefer the more conservative treatment without the

surgical knife.

From my own experience with tropical snake biles as well as adder biles in

Germany 1 must eonfess that 1 have never seen advantages of the incision therapy.

Ou the eontrary, there were more disadvantages because of delayed healing and

heavy sears in the incision area.

Chemical neutralization of the snake venom or burning out the wounds is

no longer part of lhe snake bite treatment in Germany. Unfortunately there is

still lhe pracliee of spraying potassium permanganate solulion into the fang marks,

though this treatment is ineffective or even harmful. Cryotherapy is of no im¬

portante in Germany. Besides lhe nonspecific therapy the administration of anti-

venoms is the treatment generally ehosen even in cases of adder bites. According

to the seriousness of the case — concerning age and health of the patient, and

taking the locality and the symptoms of the bite into consideration — 10 to 20 ml

are injected, parlly locally, partly intramuscularily or even intravenously. In

order lo avoid allergic reactions serum tests are always recomrnended. 20 up to

30 ml, al the inosl, of antivenom have proved to he sufficient in cases of adder

bites. There is not one case report in the literature available to me, where larger

doses were used. Since the first publicalions about using eorticosteroids in the

SciELO

10 11 12 13 14 15

230

POISONOUS SNAKE BITES IN GERMANY

trealment of snake bites (Mells, 1951; lloback & Green, 1953) providing good

resnlls lliis Ireatment lias found ils way into Germany lieing Iried increasingly.

Bcsides lhe specific therapy wilh antivenom and anlihistamines Ireatment

vvith corticosteroids is regarded lo lie lhe most efíective one. In lhe German

medicai literature Haas. Hartmann íi Wündisch, and Lieske have puhlished suc-

cessful Ireatment of snake hites using corticosteroids. These aulhors slrongly

suggest a combined Ireatment of snake hites administering antivenom and corli-

steroids, although lhey have not had a chance, so far, lo use lhe combined treat-

ment vvith more than half a dozen patients. Haas lias given a review of the

literature dealing vvith lhe use of corticosteroids and afler detailed research comes

to the conclusion lhal lhe trealment vvith corticosteroids can only he efficient. if

the ilrug is administered in lime and in sufficient quantities. 100 up to 100 mg

intravenously or intramuscularily injected seem to be helpful in the Ireatment of

biles from Elapides, Viperides, and Crotalides.

While Benyajati and col. in Thailand observed. in 1901, lhal even afler

application of 100 mg of hydrocortisone only patients already seemed lo be

responding to lhe trealment, whereas patients who had been given little or no

antivenom were in a deep coma wilh respiration difficulties, the injection of

5 mg Decorlin H daily or 40 mg of Urbason four times a day did not reseue

lhe patients in Germany, who were hitten by a cobra.

In Scandinavia Tallqvist and col., in 1961. out of 163 adder bites treated

11 c /f vvith antivenom and cortisone, and 8% with corlisone only and even lhen

saw favourahle resnlls.

The fatality of the bites from tropical snakes in Germany during lhe lasl

few years only residis from lhe total lack, or lhe delayed injection, of a sufficient

flose of antivenom, as lhe 3 cases of falai cobra bites clearly demonstrate. This

is partly due to commercial reasons: The antivenoms are expensive, they have a

1 i mi teci dnrability, and bites are rare.

The fatal snake bile cases in mind il has been discussed in Germany that

ovvners or keepers of exotic snakes shoiild be forced by law to have the specific

antivenom at hand, but lhe government has not reacted to these suggestions yel.

Nowadays there are only a few leaflets issued by the Behring Werke or

notifications in the medicai press from time to lime making knovvn to lhe public

where snake bile antivenoms are available in Germany, when snake bile ac-

cidents occur.

Of course lhere is a nonspecifie trealment besides lhe combined antivenom-

corticosteroid therapy. Salt Solutions, plasma and blood infusions form a helpful

measure in fighting the collapse or shock syndrome. Prophylactic measures against

sccondary infection, such as tetanus and gas-gangrene, are usually carried out.

To avoid the fatal respiratory paralysis in cases where neurotoxic venoms have

been inoculated, internai specialists, surgeons, and specialists for anaesthesia should

cooperate in applying artificial respiration if necessary. In Germany iron lung

therapy has not been used yel.

May I draw these final conclusions:

1. Bites by lhe native German adder do not bring forlh serious problems.

Trealment is easy and even wilhout antivenom there are hardly fatal cases.

2. Bites from imporled exotic poisonous snakes are highly dangerous and should

be treated immedintely wilh specific antivenom and corticosteroids.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):227-233, 1966

HERBERT LIESKE

231

3. Lethality after exolic snake lutes is significantly higher than after native

adder bites, the reasons being l)olli the ignorance about the dangerousness

of exolic snakes and the impossibility to get hold of the specific antivenom

in cases of emergency.

4. Increasing imports of exolic snakes and more frequent travelling to the

tropics are compelling German doctors to look into the problems of poisonous

snake bites and develop a keen inlerest in the best treament.

TREATMENT OF ADDER BITES IN GERMANY

Author

Number of cases

Tourniquet

First aid

Suction

measures

Incision

Kellner

Lieske

Tropenkranken-

haus *

Incision

£ locally

£ intramusc.

Medicai 5

^ intraven.

treatment and

Corticosteroids

therapy during

hospitaliza tton Antihistamines

Antibiotics

Tet.-prophyl.

Additional therapy

cognac, splint

glucose

sympatol, splint

Periston, splint,

Stroph.

* Not yet published.

DISTRIBUTION OF ADDER BITES IN GERMANY

Author

Number of cases

Children up to 16 years of age

Localization of h.iml

b *te foot

Kellner

Lieske

Tropenkranken-

haus *

May

•f

Time of bite June

(month) j u iy

August

* Not yet pubtished.

SciELO

10 11 12 13 14 15

232

POISONOUS SNAKE BITES IN GERMANY

FATAL COBRA (Naja naja) BITES IN GERMANY

Year

1956

1962

1963

Profession

showman

animal keeper

snake farm owner

Localization of bite

right forefinger

left forefinger

1) outdate Cobra

1) Europ.-Medit.

1) African anti-

antivenom 10

antivenom

venom 10 ml

ml i.m. loe.

20 ml i.v.

i.m.

Type and quantity of anti-

venom injected

2) poliv. Europ.-

Medit. antive¬

nom 10 ml i.m.

2) poliv.-Afric.

antivenom

30 ml i.v.

2) African anti¬

venom 10 ml

i.m.

3) poliv. Cobra

3) African anti-

antivenom

venom

60 ml i.v.

40 mi i.m.

1) 1% h

1) 15 min.

1) 1 h

Time between bite and anti-

venom treatment

2) 3 li h

2) 1 h

2) 12 h

—

3) 5 h

3) 18 h

Corticosteroids

every 4 h 40 mg =

5 mg daily = 10

160 mg

Antihistamines

Yes

Additional therap. measures

Oxygen, vasopres-

artif. respiration,

artif. resp., infus.

sors

gamma globulin,

Ringer sol., blood

Hospitalization

Yes

Time between bite and death

6 h 10 m

17 h

28 h

Cause of death

respirat. paralys

respirat. paralysis

References

1. Benyajati, C., M. Keoplung, R. Sribhibhadh — Experimental and clinicai studies

on glucocorlicoids in cobra envenomation. J. troy. Med. Hycj., (14:46-49, 1961.

2. Esch, G.

1965.

Tõdliche Kobra-Bissverletzung. Dtsch. med. Wschr., 90(6) :261-264,

3. Fock, K. — Kreuzotterbiss. Med. Welt, 3:257, 1930.

4. Haas. J. — Die Corticoidtherapie nach Intoxikation mit Schlangengiften, sowie

eigene Beobachtungen bei der Behandlung eines Bisses von Bothrops nasutus.

Z. Tropenmed., 17(1): 26-35, 1966.

5. Hartmann, G., E. Wündisch — Cortison und Cortison-Derivate in der Therapie

der Schlangenbisse. Landarzt, 41:384-386, 1965.

6. Hoback, W. W., T. W. Green — Treatment of snake venom poisoning with

cortisone and corticotropin. J. Avier. med. Ass., 152:236-237, 1953.

7. Kellner, H.

Kreuzotterbisse. Med. Welt, 22:1197-1203, 1965.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

IIERBERT LIESKE

Simp. Internac. ^oo

33(1): 227-233, 1966

8. Kirsch, R. — über Kreuzotterbissverletzungen. Zentralbl. f. Chir., 5:198, 1952.

9. Lieske, H. — Symptomatik und Therapie von Giftschlangenbissen. Sonderdruck

Behringwerk-Mitteilungen. Die Gifschlangen der Erde, pp. 121-160, 1963.'

10. Lieske, H. — Klinik und Therapie des Kreuzotterbisses. Landarzt, 35:549-553,

1959.

11. Metts, J. C. — Proceedings of the 2nd Clinicai ACTH Conference. Editor, J.

H. Note, Bd. II: Tlwrapeutics. Blakiston Co., Philadelphia, 1951.

12. Probst, H. — Kobrabiss mit letalem Ausgang. DMW, 82:2031-2032, 1957.

13. Schüssler — Persõnliche Mitteilungen.

14. Tallqvist, H., K. õsterlund — Huggormsbett. Nord. Med., 68:1073-1077, 1962.

Discussiox

P. J. Deoras: “Kõnnen bei Bissverletzungen in Deutchland mit tropischen Gift-

schlangen nicht polyvalente Seren verwendet werden?”

H. Lieske: “Da es sich bei tropischen Giftschlangenbissen immer um dem Opfer

bekannte Giftschlangen handelt, ist natürlich monovalentes dem polyvalenten Serum

vorzuziehen, falis solches vorhanden ist. Hinsichtlich der deutschen Antiseren kann

Ihnen auch Dr. Zwisler von den Behringswerken Auskunft geben.”

O. Zwisler: “Antisera produced by the Behringswerke AG, Germany, are poly-

specific and direct against C r o t alu s and Bothrops species.”

H. Pesce: "En los casos en que no se dispone de suero antiofídico especifico, se

ha aplicado con êxito en Iran el “Periston-N” polivinílico de la Bayer, en perfusión

intravenosa, 500 cm.? iQue experiencia hay en Alemania?”

H. Lieske: “Periston-N (Bayer) ist in Deutschland bei Giftschlangenbissen bisher

wenig verwendet worden. Über gute Erfahrungen zur Behandlung haemolytischer

Vorgange berichtet jedoch Hentsch aus Indonesien, der bis zu 2 Mal tgl. 500 ml.

intravenõs ais Schnelltropf (45-60 Tropfen pro Min.) gab.”

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1) :235-240, 1966

E. R. TRETHEWIE and P. RAWLINSON

235

29. DIAGNOSIS OF SNAKE BITE

E. R. TRETHEWIE and P. RAWLINSON

Department of Physiology and Zoology , University of Melbourne, Australia

Introduction

Micro techniques are now available for estimating precipitin antibody (Crowle,

1961). This lias lieen usecl for Iabelling lizard serum (Rawlinson, 1964) and

antibody to albumin in guinea-pig anaphylaxis (Trethewie, 1964). In view of

the difficulty often experienced concerning tbe diagnosis of snake bite this techriique

has been applied to simplify tbis.

Method

The technique emjiloys slides coaled with agar in which small wells are cut

— one centrally and a number cireularly around at a distance of 0.5 cm. This

is illustrated in figure 1. The slides are coated with a preparation of agar 1%

Jonagar and 1% NaCl to which is added a 1/10,000 solulion of aqueous Mer-

thiolate and distilled water to a volume of 100 ml. This is allowed to soak for

30 minutes and then heated to 98°C in a water bath. 12 ml of this solution

is thoroughly mixed and poured into a 10 cm siliconized petri dish, to make a

layer 2 to 3 mm thick. Once the agar is set, holes are punched to lhe desired

pattern. This agar “skin” is removed from the petri dish under water with a

spatula. It is placed on a slide where it is trimmed to size and lightly touched

or còvered with filter paper which draws off excess saline. It is left for 24 hours

at roorn temperature to dry. It is sometimes necessary to replace the filter paper

after the first seven minutes. When the agar on the slide is dry the filter paper

is removed, using distilled water. The stain is prepared from 1.5 g Azocarmine B

(red), 500 rnl methanol, 100 ml acetic acid, and 400 ml distilled water. It is

put in a large petri dish into which lhe slide is placed and left for about five

minutes. Excess stain is wiped off the glass. A washing solution of 1,800 ml

ethanol and 200 ml glacial acetic acid is placed in a second petri dish into

which the slides are placed for destaining for about seven minutes. The use of

tweezers makes the handling of the slides less difficult but care must be taken

not to scratch lhe agar.

Principle

It appeared lo one of us (E.K.T.) that in view of the difficulty in assessing:

1. Whether a child has been bitten by a snake at all (say in long grass) or

2 3

5 6

11 12 13 14 15

236

DIAGNOSIS OF SNAKE BITE

2. The type of snake liiling which:

(i)

may

not

bave been seen

(ii)

seen

brie

fly and insufficiently to

diagnose or

1 iii)

seen

someone unintiated in

knowing the type when seen

adequately;

that this technique could lie applied to the diagnosis of snake bite.

Australian snakes present a well rnarked feature of cross and multi-antigenicity.

This is so well developed lhat originally polyvalent tiger antivenene was regarded

as lhe treatment of choice for snake hite hy any snake in Australia. Most snakes

in Australia are venomous. They are Notechis scutatis (Tiger snake), Oxyura-

nus scutellatus (Taipan), Acanthopis antarcticus (Death-adder), Pseudechis por-

phyriacus (Black snake), Denisonia superba (Copperhead), and Demansia lextilis

(Brown snake). Publishing recently on snake bite 1 referred to regional distribu-

tion in Australia as a guide to treatment (Trethewie, 1966) where the snake hiting

is not definitely known. This affords additional information as regards administer-

ing the speeific antivenene.

As regards antivenene treatment and excluding for the monienl other local

and general

measures, we recognize that sera from the Tiger snake, Taipan,

Brown snake and Death adder are sufficiently distinct to make them individually

useful for treatment rather than lhe universal use of tiger antivenene.

Experimental

When we place a known venom in the centre well of our preparation and

differing antivenenes in the five circular wells we finei the following distribution

of precipitin reactions (Table 1). This we find is adequate to make a diagnosis.

TABLE 1

Snake or venom

Antivenene

N.S.

o.s.

p.p.

A.A.

D.T.

Notechis scutatus .

Acanthopis antarcticus .

111

Pseudechis porphyriacus .

111

Demansia textilis .

111

We have compared the use of saline extract of the bitten arca, serum from

whole blood, and the expression of serum from the bite or injection for diagnosis.

Expressed serum is the besl material.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

E. R. TRETIIEWIE and P. RAWLINSON

237

Simp. Internar.

33(1):235-240, 196fi

In the case of Tiger snake venom placed in a well, or expressed serurn froin

lhe “bite” (injected) area of the animal (in this case a guinea-pig), marked

preeipitin reaction occurs againsl the Tiger antivenene and moderate precipitation

against lhe remaining fotir (Fig. 1).

In the case of Deatli adder venom injected into a guinea-pig leg (20 mgm)

extract shows marked precipitation against Death adder antivenene, moderate

against Tiger, slight against Taipan and Black snake and no signifieant reaction

vvith Brown antivenene. These appearanees are far clearer on the original glass

slide preparation (Fig. 2).

With Black snake venom (5 mgm injected into a guinea-pig) likevvise marked

precipitation occurs vvith Black snake, moderate with Tiger and Taipan and slight

reaction with Brown antivenene (Fig. 3).

In one instance a Brown snake was place against a guinea-pig and encouraged

to bite and in this instance seriirn expressed from lhe bite on the guinea-pig’s leg

(Fig. 4) showed marked reaction with Brown antivenene, moderate with Taipan.

slight with Tiger, and none with Black and Death adder. When live snakes are

used. as is projected, reactions may be expected to be more clear cut because

dried eommercial serum may be faulty.

These findings are summarized in lhe Tahle I and Fig. 5. Il can be seen

that if one considers only the two latter columns there is an almost signifieant

separation of the venom designation. When all five antivenenes are used the

distinetion is confirmed by maximal precipitant forms against the homologous

antivenene.

In this way we are able to separate serum obtained from different types of

bite or injection in the experimental animal. The reaction time is five or more

hours except with Brown snake which is two hours and we are endeavouring lo

speed up this reaction. We suggest in the first instance lo give antivenene ac-

cording lo the information of the snake if available and the geographic region

bitten (with reference lo lhe colour of the snake) and in the absence of this

information to use Tiger antivenene in the first instance and subsequent injections

to follow the diagnostic patlern. Il is suggested that development of this technique

may ohviate the confusion arising from the two prohlems, 1. was the subjecl

bitten at all? and 2. what was the nature of the snake? We are proceeding

with these experiments.

Discusstox

The above technique shows that material obtained from the site of the bite-

either saline injected espeeially where there is severe thromhosis, or bled material

from incision, affords satisfactory diagnosis of the snake-bite when set up in

double diffusion.

The advantages of this proeedure are obvious especially where specific anti¬

venene is essential for treatment and this is preferable in the case of Australian

snake bite as regards Tiger, Brown snake, Taipan and Death adder.

The time required for a positive lest — 2 hours in the case of Brown snake

and longer with the olhers, al room temperature — is a practical difficulty, bui

we have been able to obtain a positive result more quickly at 37"C and this

technique is now being standardized. This shonld give an adequate answer in

approximately one hour which is quite suitable for treatment. In Australia an

initial injection of Tiger polyvalent antivenene is advised.

2 3

5 6

10 11 12 13 14 15

238

DIAGNOSIS OF SNAKE BITE

SUMMARY

1. Diffusion techniques with agar plates give a set pattern of reaction for eacli

Australian snake venom against lhe series of individual anlivenenes.

2. Venom injected into a guinea-pig allows the collection of material froni the

region of lhe hilo for similar analysis.

3. Material from the region of the actual hite of one snake on a guinea-pig

also provided adequate diagnostic material.

4. ít is considered this technique tnay he employed in hospitais lo ensure ac-

curate snake hite diagnosis.

Rekerences

1. Crowle, A. ./. & Lueker, D. C. — Antigen antibocly precipitation contrasted in

gels of high and physiological salt concentrations. Nature, 192:50, 1961.

2. Rawlinson, P. — Immunological Studies of Lizard Serum. Thesis. Department

of Zoology, University of Melbourne, 1964.

3. Trethewie, S. R. — Allergic antibody to crystalline egg-albumin. Unpublished

experiments (1964).

4. Trethewie, E. R. — Symptomatology, pathology and treatment of bites of

venomous snakes of Australia. In: Venomous Animais and Their Venoms.

Academic Press. In print.

Fig. 1 — Reaction to Tiger venom (Centre

well) against Death Adder (Aa), Brown

Snake (Dt), Black Snake (Pp), and Taipan

(Os) antivenene. (Room Temperature).

Fig. 2 —■ Reaction to material from the

region of injected Death Adder venom

from a guinea-pig (Centre Well). Outer

wells contain Brown Snake (Dt), Tiger

Snake (Ns), Taipan (Os), and Black Snake

(Pp). (Room Temperature).

2 3

5 6

10 11 12 13 14 15

D.t.

Ns — Dt

A a. Q s -

Nls.

■P-

D.t.

Fig. 3 — Reaction to material from the

region oí injected Black Snake venom

írom a guinea-pig (Centre well). Outer

wells contalns — Death Adder (Aa),

Brown Snake (Dt), Tlger Snake (Ns), and

Taipan (Os). (Room Temperature).

Fig. 4 — Reaction to material from the

region of a bite of a Brown Snake on a

guinea-pig (Centre well). Outer wells

contain — Death Adder (Aa), Tiger (Ns),

Black Snake (Pp), and Taipan (Os).

(Room Temperature).

VENOMS

N S

^Tig«r Snak«.^

A A

P P

(õlack Snokff^

SciELO

10 11 12 13 14 15

240

E. R. TRETHEWIE and P. RAWLINSON

Discussion

S. Minton: “How long might a precipitin test be obtained after death?

otfier words, how long does lhe venom persist in lhe body?”

E. R. Trethewie: “We had a case of Tiger snake bile where death ocurred

'after four days and at autopsy extract of the skin area bitten killed 100 mice with

typical symptoms of Tiger snake envenomation. Allergic antigen stays in the tissue

of the skin for several days and may produce a delayed reaction of seven days.

Therefore I consider much venom would be left in the bitten area to give a

diagnosis by this technique of medico-iegal importance for several days.”

10 11 12 13 14 15

GASTAO ROSENFKLD

241

Mem. Inst. Butantan

Simp. Internac.

33(1):241-244, 1966

30. COMMENTS OF THE MODERATOR

GASTAO ROSENFELD

Instituto Butantan, São Paulo, Brasil

It is a role of the moderator to comment and to make a summary of papers

presented to the Symposium. I will not withdraw from this obligation, all the

more so that I and the collaborators of the Department of Physiopathology and

the Hospital Vital Brazil from Instituto Butantan were not active participants in

this session, in view of the high number of foreign specialists who had kindly

accepted to take part in this Symposium. It is obvious that with 13 foreign

participants registered, each of them having 30 minutes for presenlation and discus-

sion of his paper, there would be a 6 hours and 30 minutes long session. Thus.

there was material impossibility to register papers of our group, and we did not

do it in order to leave the time for our foreign colleagues who had a long journey

to attend this Symposium.

In 1954, Dr. Afrânio do Amaral, at that time Director of the Instituto Bu¬

tantan, gave me the attribution to direct and reorganize the Hospital Vital Brazil,

function which I carried out until March, this year. In this Service and in the

Department of Physiopathology, I and my collaborators have had the opportunity

of raising some experience in the subjects discussed in this session. During this

period, 15,709 patients bitten by poisonous animais carne to our Service to look for

medicai assistance. I, therefore, profit this opportunity to address the house as a

Moderator of one of this Symposium's sessions. Let us then start to comment the

different papers presented today.

The paper of Dr. Parrish was presented by Dr. McCollough and it gave us

the opportunity to become aware of many of the epidemiologic aspects of poisonous

snake bites in the United States. Here in the Southern hemisphere, and with

different climate, our data are, obviously, different. These aspects of accidents by

poisonous animais are very interesting and are useful, not only for the evaluation

of the problem, but for the informations which may guide to improve prevention

of these accidents as well.

Dr. McCollough showed a series of cases bitten by snakes from the genera

ürotalus and Agkistr odon from the United States. The frequency, in-

tensity, and extension of necroses are impressing. In our opinion they are typical

consequences of deficient treatment by antivenin. Deficiency of serumtherapy may

be due to three factors: unspecific antivenin, delayed treatment, and unsufficient

doses. The first and the second factor cannot be blamed in those cases, only the

deficiency of antivenins’ doses. What may have contributed to this fact is that

the authors referred to the number of vials as a criterion for dosage evaluation.

This is a mistake we all made at the beginning; it is why, at those times, we had

similar cases to those we have seen now. It is a nonsense to say that a patient

was treated by such or such number of antivenin vials. Antivenin’s potency vary

from one producing iaboratory to another and, unfortunately, there are many of

them which do not indicate on the labei the neutralizing capacity of venom in

miiigrams. Even the Instituto Butantan fell into the same error with some anti¬

venins up to a short time ago, and it still does it with arachnidic antivenins. It

is essential that all laboratories producing antivenins should indicate on the labei

the number of “units” contained for the venom. "Units”, as we propose, is the

ability of neutralizing 1 mg of venom. We use this designation and definition in

our works since some time ago, because we consider it of great practical importance,

either to the physician or to the researcher. The reason is that, once the anti-

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COMMENTS OF THE MODERATOR

venin is an agent neutralizing the venom, it is necessary to in.ject, lhe earlier the

better, such a number of “units” which will be able to neutralize all of the venom

that eventually has been inoculated by the animal. By knowing from the specialists

of each country or region the quantity of venom in mg contained in the different

species' glands, the physician will be able to evaluate the amount of "units” to be

injected in order to neutralize actually the whole venom which may have been

inoculated. It is an elemental and a simple arithmetic question without any dif-

ficulty for anyone. This criterion is as obvious as in medicine’s other fields, where

nobody would give a drug unless its quantity could be referred to in relation to

weight or unit. For instance, nobody prescribes a corticoid without indicating its

number in mg of “units”. Thus, we do not understand how one may have the

courage to indicate serumtherapy measured in mililiters or vials without knowing

how many mg of venom the antivenin is able to neutralize. We insist that this

tradition is wrong and it should be corrected. It is interesting that I have already

found, in a publication of Vital Brazil of about 1910, the statement about the

necessity of indicating antivenins’ potency in mg of venom which they are able

to neutralize; unfortunately, this was forgotten.

From this reasoning comes our opinion that the results we have seen are

only a consequence of insufficient serumtherapy and not of antivenin's ineffeclive-

ness, as it may have seemed like. Obviously, the specific treatment is useless afler

the necrosis occurrence. There is only the symptomalic, clinicai, and surgical treat¬

ment left, which, infering from Dr. McCollough’s words, has been well conducted.

About this point, we would still like to present an information: on about 1,600

cases of Bothrops bitten patients treated in the Hospital Vital Brazil in the

last 11 years, there was, practically, no need for amputations excepting those cases

which carne to the Hospital late after the bite, when dry necrosis had already

occurred.

Another treatment presented by Dr. McCollough, deserving some comments, is

the one of incisions done for elimination of venom. To us it lacks physiological

basis. These incisions intersect blood vessels and through these intersections is go-

ing lo flow the circulating blood which does not carry any venom. The venom is

in the tissue and it penetrates by lymphatic way. Circulation stops, as a conse-

quenee of the solution of continuity and the venom will stay at the site, aggravat-

ing necrosis. Besides, it will not be in contact with the antivenin coming through

the blood circulation. In envenomations with proteolytic venoms, which provoke

necrosis, incisions will enhance this effect. Something that has physiological basis

is, withdrawal of the venom from the site of lhe bite by suction of lhe site attained

by the fangs, if it is bleeding. Otherwise, to prick with a needle around the site

is an aid to the outflow of serosity. If this procedure is carried out within the

first half hour after the accident, part of the venom will be eliminaled; later it

is useless.

One more comment about the ligature which has been advised. Dr. Deoras

has already asked a rather “venomous” question during discussion, and we agree

with him. In fact, ligature is of no reason. If the venom is proteolytic, the

circulation retention keeps the venom at the site, helping to provoke necrosis. If

•the venom is neurotoxic, it penetrates with or without ligature, since it contains

an appreciable amount of hyaluronidase which helps venonVs dissemination. And

still, if the ligature is perfectly done as to really prevent the venom from penetrat-

ing the circulation, there will not be any blood circulation. Then, depending on

how long the ligature was kept, loosening it will provoke shock which may be

fatal, since the general condition is aggravated by the envenomation. Here, at

the Hospital Vital Brazil, nurses are afraid, by experience, when a patient arrives

with a good ligature; they will not open it, leaving it to the physician, because

at this very moment many patients have had a Peripherie shock. By the way, in

experiments we made with Dr. Schenberg, present at this session, we provoked

many shocks in dogs using only ligature. A well done ligature is enough to provoke

a fatal shock even in normais, there is no need for venom. As a matter of fact,

this mechanism of shock was discovered by Trueta in the II World War, during

the bombings of London.

Dr. Kornalik presented very interesting data on the problem of fibrinolysis

and blood incoagulability as provoked by snake venom. The demonstrated facts

are valuable but their interpretation may be another. In order to make it clear

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Mem. Inst. Butantan

Slmp. Internac.

33(l):241-244, 1966

GASTAO ROSENFELD

243

to tho audicnce, it would perhaps be better to explain (in our point of view) what

happens with coagulant and proteolytic venoms. In vitro, a small amount of venom

clots the blood due to its coagulant fraction, which is active even in very small

concentrations. This is not so with the proteolytic fraction. In higher amounts

the venom provokes clotting by its coagulant fraction, then the fibrin formed is

lysed by the proteolytic fraction and its high concentration permits its action bo-

fore coagulation would occur. In vivo, small amounts of venom provoke hyper-

coagulability at the first minutes, fibrinogen clotting, and then blood incoagulability,

due to a gradual and massive defibrination. In the phase of hypercoagulability,

a fugacious fibrinolytic activity appears which disappears when the blood becomes

incoagulable by defibrination. In vivo, there is no fibrinolysis or fibrinogenolysis

by the direct action of venom because such high amounts would be necessary for

provoking these effects which are, practically, impossible to obtain. Besides, they

would almost instanlly provoke death.

The presentation of Dr. Efrati’s paper was very clear and synthetic. The

clinicai picture presented is exactly the same as the one observed in accidents by

Bothrops snakes and all snakes with coagulant and proteolytic venoms. His

definition of some symptoms as being an “anaphylactoid picture” is very appropriate,

since they are due to the proteins’ decomposition by the venom and the consequent

iiberation of resulting substances in the circulation. Thus, it provokes the same

kind of shock obtained by injecting proteins or their degradation products. Dr.

Efrali pointed out that he did not observe hemolysis, in spite of the venom being

hemolytic in vitro. The reason is that through the bite, the venom is inoculated

in tissues and it penetrates slowly the circulation. It is very diluted in the blood,

never reaching a high concentration. But, if the venom is injected intravenously,

the hemolysis will appear. Dr. Efrati refered to a case in which neurological

symptoms appeared. I would like very much to discuss this with him and to find

out what kind of symptoms they were, for two reasons: first, that I did not know

that snakes from the genus Vip era had neurotoxins; second, that I would like

to know if these neurological symptoms manifest the presence of modifications

which produce what we call “neurotoxic fácies”, and which exist in all cases of

envenomation through snakes containing neurotoxins. Unfortunately, I am not able

to show a demonstrative slide. I will do it in other opportunity. Dr. Efrati’s

recommendation about the antivenin intravenous injection, the eariier the better,

is very exact and we have here the same principie. However, we do not agree

with his reference to the number of vials for the serumtherapy, for reasons we

have already mentioned. I would still like to utter my personal point of view:

I consider as very peculiar the fear of lhe physicians to inject greater amounts

of antivenin while not fearing to leave the patient exposed to the risk of death

and necrosis as a consequence of insufficient treatment.

The work of Dr. Ohsaka is extremely good and of an edifying experimentai

perfection. The way of evaluating in vivo the hemorrhagic action of venoms, giv-

ing way lo compare different venoms, is very nice and based on a well imagined

experiment technic. Dr. Ohsaka presented an important fact which is, the separa-

tion of two hemorrhagic fractions which are not bound to the proteolytic factor.

This is a new fact to us, since the general idea is that the proteolytic factor is

responsible for hemorrhage by causing the rupture of capillary walls. During the

discussion of Dr. Ohsaka's paper, Dr. Puranananda asked about the time the active

venom substances may stay in the circulation. Since there was not a clear answer,

I may inform about what happens with bothropic venom which is of the same

kind as the Habu. In a severe envenomation, while the blood is incoagulable, the

venom’s active substances are present in the circulation even 48 hours after the

bite. They are rapidly neutralized when antivenin is injected intravenously.

Dr. Schenone’s paper was very well presented and we already knew part of it.

He advises antihistaminies administration and we agree with that for a long time

ago. About the indication of corticoids, however, we disagree. We do not have

any proof that it is useful. It seems more likely to be some kind of a crutch in

case there is no antivenin. One has to do something, so one gives corticoid. It

is a rest to the doctor’s conscience to give the patient some medicai treatment.

What should be done in the eountries facing the problem of loxoscelism is to

produce a Loxosceles antivenin which really neutralizes the spider venom. as

it was done by the Instituto Butantan, here in Brazil. We have experience with

this antivenin which presented some interesting peculiarities for the physician. At

SciELO

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COMMENTS OF THE MODERATOR

the beginning, even when serumtherapy was given in time, some cases showed,

after the treatment, a hemolytic syndrome. At that time, 2 to 5 vials were

injected. Later, in other patients we started to inject 10 antivenin vials and, after

that, no more cases of hemoiysis were observed after the treatment. We do not

mention “units” but vials because we did not succeed when we suggested that this

antivenin should aiso indicate the quantity of mg or gama which it is able to

neutralize. In the discussion of Dr. Schenone's paper he was asked about the

cardiotoxic activity of Latrodectus venom which is apparent by the arterial

hypertension he has observed in these envenomations. We wouid also like to com-

ment this topic. We have, with much frequency, accidents by the spider Phoneu-

tria fera which, like the Latrodectus, has a neurotoxic venom (different from

the ophidic one, since it acts on the Peripherie nervous system). In these cases

the symptoms are identical to those refered to by Dr. Schenone, including the

arterial hypertension. However, we do not consider the hypertension as due to a

direct or indirect cardiotoxic activity on the neuro-vegetative nervous system, be¬

cause as long as the pain is suppressed by means of an hypnotic or an anesthetie,

the arterial pressure gets normal, and the hypertension reappears when the pain

returns. We consider it, by this clinicai evidence. as a secondary symptoms to the

pain and not a direct venom activity.

The paper of Dr. Chapmann is extremely interesting to us because we have

very few literature and data on ophidic accidents occurring in África. It has been

a great lesson the way he presented the well tabulated data, including symptom-

atology. Dr. Chapmann is against incision as therapeuties, since he thinks, like

we do, that necrosis provoked by the venom is already sufficient damage. Dr.

Chapmann is also against ligature for the same reasons we already discussed be-

fore. He referred to the use of ligatures as a psychological effect but it seems to me

that even this should not be tolerated. In the medicai and physiopathological point

of view, it may only be unfavourable to the patient.

Dr. Lieske brought an interesting contribution to the problem of snake bites,

showing that even in Germany accidents of this kind may occur, provoked by

snakes imported with merchandise. It is a hard problem and it can only be solved,

as sai d Dr. Deoras, providing these counlries with antivenins suitabie for the snakes

of the countries with which they have trading. We had the opportunity to know

one case in quite a peculiar way. Once, when I was in Valparaiso, Chile, talking

about poisonous animais, a physician from the Chilean navy said that this was no

problem to them because there were no poisonous snakes in his country. Some

days after being back in Brazil, a radio appeal carne to Butantan, asking urgently

for Elapidic antivenin. A dock worker in Valparaiso had been bitten by a poisonous

coral snake while unloading some banana bunches arrived from Equador. The

antivenin was sent in a few hours and, fortunately, the patient was saved. Dr.

Lieske advised the use of corticoid in the treatment of snake bites. We disagree

of this point of view based on an experiment made in collaboration with Dr. Lan-

glada, published in Memórias do Instituto Butantan, 1964. Neither Dexamethasone

nor ACTH in small, médium, and high doses showed any usefulness. On the con-

trary, they increased mortality with some venoms in experimented animais. All

the same, we use corticoid in ophidic envenomation, but only for treatment of

shock when it occurs. Dr. Lieske related some fatal cases, in spite of serum¬

therapy given in time. But, as admitted by himself, the antivenin doses were not

sufficient. So we will not discuss this point.

Thanks for all collaborators to this Symposium.

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IMUNOLOGIA

IMMUNOLOGY

Mem. Inst. Butantan

Simp. Internac.

33(1): 245-250, 1966

P. A. CHK1STENSEN

245

31. THE PREPARAT10N AND PURIFICATION OF ANTIVENOMS

P. A. CHRISTENSEN

The South African Institute for Medicai Research, Johannesburg, South África

This short talk on lhe preparation and purification of antivenoms must of

necessity he influenced by the attitude adopled at lhe South African Institute for

Medicai Research, but knowing that there is room for improvements, and having

noled lhe titles of thc many papers to he presented today, this attitude may vvell

liave changed hefore lhe end of this Symposium.

My experience with the preparation of scorpion and spider antivenoms is

rather limited, our main concern being the production of snake antivenoms, but

lhe basic: problems are the same, namely, 1, which animal to choose as serum

I)roducer, 2, which venoms to use as antigens, 3, how to use the venoms as anli-

gens and 4, how lo treat the resulling serum to make it suitable for use.

The use of sera from animais other than the horse may carry less risk of

serum reactions in some persons, but the horse is lhe natural choice in climates

where it thrives and can be obtained at a reasonable price. It is easy to handle,

yields a large volume of serum, and methods of purification of horse antitoxins

liave been thoroughly studied and are technically more advanced than is the case

with antitoxins derived from other animais.

With regard to which type of horse to choose, there can be no strict rule,

and one’s views are based on general impressions rather than controlled experi-

mentation. It is natural to prefer big horses because they yield more serum, but

young horses do nol respond better than old, rather the opposite may be true,

and leniently immunized, the life expectancy of anlivenom producing horses is

very long. though not always as long as that of a horse dying earlier this year

at the age of 29 after 181,4 years’ continuous anlivenom production.

Most laboratories making antivenoms prepare other antitoxins as well, and it

is common practice to screen new horses for naturally induced antibodies to diph-

theria and Clostridium pcrjringcns toxin and lo immunize them prophylactically

against tetanus. Sueli horses can be allocated to lhe production of diphtheria,

tetanus or gas gangrene antitoxin according to immediate requirement, and can be

transferred from the production of one kind of antitoxin to that of anolher with-

out much delay. The response of a horse to one antigen does not necessarily

indicate its potenlial response to another, and horses previously immunized with

bacterial toxins, but no longer required or discarded because of falling titre, are

as good antivenom producers as any other horse. ll can be argued that the

presence of significant amounts of other antitoxins in the raw serum will reduce

the ratio between antivenom potency and protein content of the final produet be¬

cause purification methods do not discriminate between antitoxins, but this is a

rninor objection.

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THE PREPARATION AND PURIFICATION OF ANTIVENOMS

Which venoms to use as antigens must obviously 1 >«■ delcrmined hy lhe

frequency of biles hy different snakes, the severity of tbe effects, and tbe avail-

ability of venom.

Availability of venom is essential for continued large scale serum production

but goes usLially hand in hand with tbe frequency of bites by different species

and to some exlerit with the severity of lhe effects, hecause larger snakes deliver-

ing much venom tend to do most damage.

In lhe majority of snakebites lhe cidprit is not seen or is not identified and

this is one of the reasons why polyvalent sera are preferable lo monovalent,

except in areas where dangerous bites are almost exclusively due to a single

species. The choice of venoms for lhe production of polyvalent serum must of

eourse he influenced hy the cross-neutralizing properlies of monovalent sera prepared

with the different venoms, but loo much reliance should not he placed on para-

specific action. The immunological overlapping of proteclive antibodies, in which

lhe earliest workers placed much faith, is very limited, as we know from the work

of Dr. Vital Brazil and others who followed liirn. Not only is lhe paraspecifie

titre of a serum lower than the specific hut a serum’s therapeutic value must

depend not oídy on titre hut also on lhe firmness of the union between aritigen

and antibody, and paraspecifie venom-antivenom complexes tend to dissoeiate.

Some laboratories prepare polyvalent sera hy blending several monovalent sera,

others, including ourselves, prefer lo immunize the horses with all the antigens.

The ohvious argument against blending monovalent sera is that eaeh serum is he-

ing diluted hy the addition of the others. This must happen in the case of anti¬

bodies with strict specificity hut not with antibodies to common antigens, just as

antibodies to related antigens could show some additive effect. The dilution of

antibodies could he eounlerhalaneed if lhe horses reeeiving a single venom reached

much higher titres than lhose immunized with several venoms. Horses given only

one venom could respond better hecause larger doses eotdd he injected or hecause

a ‘crowding’ effect would suppress lhe response lo important loxins in lhe horses

reeeiving several venoms. However, horses given only one venom soon reach a

state of immunity which does not improve unless lhe dose of venom is inereased

quite out of proportion lo the rise in antibody titre. Furthermore, such mono¬

valent horses usually maintain lheir titre to this particular venom after they have

been transferred lo the production of polyvalent serum.

Once immunized, a horse will tolerate large single venom doses, hut new

horses are easily killed. A horse immunized many years ago for the preparation

of monovalent Naja nívea antivenom received single doses of up to 2 g of venom

without symptoms, yet 15 mg of the same venom given in error killed a diphtheria

antitoxin produeing horse hefore the mistake was discovercd and the outeome

prevented with antivenom. To give new horses their first, basal, immunity with

unmodified venom is a tedious proeess, and the initiation of hasie immunity is

therefore the first part of the next problem, how to use venoms as antigens.

To use venom-antivenom mixtures for this purpose is also tedious hecause

such mixtures are dangerous when they are under-neulralized, and fully neulralized

they are poor antigens. Many ways of rendering venoms atoxic without destroy-

ing their antigenicity have been suggested over lhe years, hut only detoxification

with formalin seems to have been used on any scale. But lhe loss in venom

toxicity due to formalin is accompanied hy a large loss in antigenicity, and the

use of formol-toxoided venoms, or anavenoms, is wasteful of venom and can he

the cause of much suffering of lhe horses. Actually no detoxification is neces-

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Mem. Inst. Butantan

P. A. CIIRISTENSEN

247

Simp. Internac.

33(1): 245-250, 19GG

sary if lhe venom is adsorhed on some inert carrier; the harmlessness of adsorhed

venoms is presumahly due lo a decreased rale of absorplion from lhe sile of

injection and the good response is probahly due lo prolonged stimulation hy

anligens held in subcutis, apart from any possihle adjuvant effect of the adsorhant.

In principio this method originaled willi Calmelle who in 1894 recorded llial

a sniall piece of chalk impregnated with venom and coaled with eollodion and

inserled under the skin of a rahhit would serve as a continued stimulation from

an artificial gland, a suggestion he credited lo Dr. Houx. in whose laboratory

lie was working.

Criley (1956) had good resulta with venoms adsorhed on aluminium hydroxide,

a method we found unsuccessful many years ago, possihly hecause our aluminium

hydroxide gels were unsatisfactory. and I turned to the use of venoms adsorhed

on hentonite, with good resulls during the last fifteen years.

The first hasic immunity of lhe horses is aehieved with a few injections of

from 25 to 100 mg of venom adsorhed on a 2% suspension of hentonite in distilled

water. The venom Solutions are sterilized hy filtration, the hentonite suspension

hy steam under pressure. This method saves time, venom and lahour, bul it would

appear llial not all preparations of hentonite are suilahle. As soou as traces of

eirculating anlihody are deteetahle hy mouse proteetion lests, the immunizalion

is continued with venom Solutions sterilized hy filtration and preserved with 0.25%

eresol. hut without lhe addition of hentonite. The injection of a suspension of

hentonite does in some cases cause the formalion of a small sterile abscess whieh

requires ineision. hut the lesion heals in few days and does not upset the horses,

and allhough some antigenie material must he evaeuated ihrough lhe ineision,

enough remains lo slimulate antihody formalion.

The greatest advantage of the use of plain Solutions of unmodified venoms

for lhe continued immunizalion is the laek of untoward reactions in hasieally

immune horses. A healthy horse will not only live longer hut will presumahly

in the long run respond belter lo antigenie stimulation than a horse in poor

condition due to repeated injury, sueh as that caused by some adjuvants. The

use of Freund’s complete adjuvant gives amazing resulls in horses immunized

with diphtheria or tetanus toxin (Mason, 1963), hut tends to cause rather severe

reactions. l iipuhlished experimental work hy J. H. Mason has shown that the

severily of the reactions is considerahly reduced if antigen and Freund’s ad¬

juvant are ineorporated in a multiple (water-in-oil-in-water) emulsion of lhe type

described hy Herhert (1965). As far as venoms are eoncerned, we have failed

lo ohserve any difference in lhe response of comparable groups of horses im¬

munized with or without lhe addition of Freund’s adjuvant in lhe forni of simple

or multiple emulsions.

Soulh Afriea’s needs for seorpion and spider antivenoms are mel liy one or

two immunized horses, too few to allow one to forni any opinion on the value

of different methods of immunizalion, hut the methods in currenl use are hriefly

as follows.

Seorpions of the genus Parabuthus, regardless of speeies, are kept alive

and the eolleeted venom is dried under vacuum in a desiceator and dissolved in

saline as required. Filtration through Seilz pads removes about 75% of lhe

toxin. and the solution is therefore sterilized by the addition of phosphate huffer

and heta-propiolaetone lo a eoneentration of 0.2%. Having stood overnight at

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248

THE PREPARATION AND PURIFICATION OF ANTIVENOMS

aboul 8 o , the solulion is mixed with procaine immediately before injection in

order to make it painless. Sterilization liy means oí beta-propiolactone is very

convenient eyen if it does reduce lhe toxicity hy aboul 30^.

Latrodectus iridistirictus antivenom is prepared with exlraels of dried cephalo-

thoraces. The tedious lask oí isolating lhe chelicerae with the venom glands for

extraction and use as antigen was discontinued hecause exlraels oí lhe remaining

eephalothoraces were toxic and sera prepared vvilli eilher chelieera extract or

cephalothorax exlrael gave complete cross-neulralization. This was more than

likely due to incomplete removal of lhe venom glands but the practical implication

was to save the effort of collecting chelicerae. One rnighl add that a serum

prepared against lhe. exlremely loxic extract of spider abdomens is ineffective

against the true venom, just as the ordinary antivenom fails lo neutralize lhe

toxic material obtained from lhe abdomens. The extracts have hitherto been

sterilized hy filtration ihrough Seitz pads or with beta-propiolectone but we intend

lo resort to membrane-filtration in order to minimize lhe loss of toxin which is

quite considerable.

Irrespective or which kind of serum lhey produce, snake, scorpion or spider

antivenom, the horses rest for about five weeks between courses of immunization

lasting about two weeks. The crude serum, or rather plasma, obtained at the

end of each course of immunization is improved for therapeutic use hy purifica-

tion, which removes inactive material, and by concentration, which reduces the

volume lo he injected.

Purification hy means of sall fractionation carne inlo general use after Dr.

Vital Brazil had shovvn that the dislribution of antibodies in antivenoms was

similar to that in bacterial antiloxins, but, although lhey are still used, such

earlier methods have been superseded by others involving treatmenl with proteo-

lytic enzymes, pepsin in particular.

The intercst in the use of pepsin for this purpose hegan in 1902 and was

smouldering until Parfentjev (1936). Pope (1939a, 19391»J and Hansen (1941)

published methods suitahle for large-scale purification of bacterial antiloxins. In

all the three methods, the serum is treated with pepsin al a conlrolled pH, but

the methods of Parfentjev and Hansen rely on adsorption for the removal of

inert material, whereas lhe unwanted protein is removed by heat-coagulation in

Popes method which was further developed by Harms (1918). Latrodectus

mactans antivenom was suecessfully purified already in 1942 hy Pirosky and eo-

workers in Argentina using Pope’s technique, and this is the method used al

Instituto Butantan (Hoxter and Deeoussau, 1949) and at the South African

lnstitute for Medicai Hesearch (Grasset and Christensen, 1917).

The advantages of pepsin-treated sera are too well known to warrant lenglhy

discussion. Such sera are slable when stored al reasonable temperatures and.

of more importance, the incidenee of serum reactions is reduced to a low levei.

It may be of interest to note that the incidenee of serum sickness in children

treated with pepsin-refined diphtheria antitoxin has been found significantly lower

in Baniu patients than in Whites (Mason & Christensen, unpublished), which is

gratifying hecause lhe Bantu is more likely to need treatmenl with antivenom,

which vvi 11 hehave as other equine antiloxins with regard to serum reactions.

lhe draw-back lo lhe production of pepsin-refined antivenom is the eost.

A contrihuting factor is a considerable loss of active material during the process.

The well-known rlifficulties in assessing antivenom potency rnakes it difficult to

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Mem. Inst. Butantan

Simp. Internac.

33(1): 245-250, 1966

P. A. CHRISTENSEN

249

estimate the amoimt lost, but it is probably about 55%, which is not surprising

as the antibodies are distributed in both the so-called pseudo- and eu-globulins,

and even the waler insoluble globulins of viper antivenom may contain some

anlibody (Christensen, 1955).

In relrospect, there bave been no greal forward strides in antivenom pro-

duction apart from the introduction of hetter purification methods, and further

developments in this direction will probably bave to await improvements of bacterial

antitoxins, which are easier lo evaluate. The scope for improvement lies in the

broadening of the specificity of polyvalent antivenoms and lhe use of purer

antigens.

For reasons already stated, it is inadvisable to rely on paraspecific protection

if specific sera can be produced, but taking the African region as an example,

it might be necessary lo immunize wilh ten or more veuoms in order to cover

lhe more important snakes. Each venom is likely to contain a dozen or more

different antigens of which the mosl toxic is often the poorest antigen, and the

solution to the problem may lie in lhe isolalion of lhe important toxins from

these venoms in quantities large enough for use as antigens, possibly after bind-

ing to a suitable carrier in order to enhance tbeir antigenicity. Steps in this

direction bave already been taken by workers in Israel and France (Kochwa et

«/., 1959; Moroz et aí., 1963), a lead we hope lo follovv in South África.

Referentes

1. Calmette, A. — Contribution à 1’étude du venin des serpents. Immunisation des

animaux et traitement de 1'envenimation. Ann. Inst. Pasteur, 8:275, 1894.

2. Christensen, P. A. — South African Snake Venoms and Antivenoms. The

South African Institute for Medicai Research. Johannesburg, 1955.

3. Criley, B. R. — Development of a multivalent antivenin for the family CRO-

TALIDAE. In: Venoms, Washington, D.C., Buckley, E. E. & Porges, N.,

Editors, A.A.A. Sei., 1956, p. 373.

4. Grasset, E. & Christensen, P. A. — Enzyme purification of polyvalent anti-

venine against Southern and Equatorial African colubrine and viperine venoms.

Trans. R. Soc. trop. Med. Hyg., 41:207, 1947.

5. Hansen, A. — Studier over Isolering af det antitoxinbaerende Protein fru

andre Serumbestanddele. Ejnar Munksgaard, Copenhagen, 1941.

6 Harms, A. J. — The purification of antitoxic plasmas by enzyme treatment

and heat denaturation. Bioch. J., 42:390, 1948.

7. Herbert, W. J. — Multiple emulsions. Lancet, 2:771, 1965.

8. Hôxter, G. & Decoussau, D. — Concentração, purificação e controle físico-quí¬

mico dos sôros antitóxicos e antipeçonhentos. Mem. Inst. Butantan, 21:187,

1949.

9. Kochwa, S., Izard, Y., Boquet, P. & Gitter, S. — Sur la préparation d'un im-

munsérum équin anti-venimeux au moyen des fractions neurotoxiques isolées

du venin de Vipera xanthina palestinae. Ann. Inst. Pasteur, 97:370, 1959.

Mason, J. H. — The South African Institute for Medicai Research. Annual

Report, 137 & 149, 1963.

SciELO

10 .

250

THE PREPARATION AND PURTFICATION OF ANTIVENOMS

11 . Monoz, Ch., Goldblum, N. & de Vries, A. — Preparation of Vipera palestinue

antineurotoxin using carboxymethyl-cellulose-bound neurotoxin as antigen. Na-

ture, 200:697, 1963.

12. Parfentjev, /. '— U.S. Patents 2065196 & 2123198, 1936.

13. Pirosky, I., Sampayo , R. & Franceschi, C. — Obtención y purificación de suero

anti La t rodee tua. Revista de la Sociedad Argentina de Biologia, 18:169,

1942.

14. Pope, C. G. — The action of proteolytic enzymes on the antitoxins and pro-

teins of immune sera. I. True digestion of lhe proteins. Brit. J. Exper. Path.,

20:132, 1939a.

II. Heat denaturation afler partial enzyme action. Brit. J. Exper. Path., 20:

201, 1939b.

Discussion

A. Shulov: “Whether you tried in your vast experience direct bites of snakes

and direct stings of scorpions in order to increase the title of the antisera. In

our iaboratory we received good results in donkey and camels, but not in sheep,

goats and rabbits.”

P. A. Christensen: “No.”

P. Cohen: “Have you observed any differences in the antibody titers of horses

immunized with bentonite-absorbed venom compared to horses which have received

unmodified venom.”

P. A. Christensen: “Unmodified venom is not used for new horses. The basal

immunity is achieved with venom adsorbed on bentonite, but continued with un¬

modified venom. No comparison has therefore been possible.”

P. J. Deoras: “Does the speaker have any observation to make to the fact

that venom when used with débris gives reactions?”

P. A. Christensen: “No.”

C. Puranananda: “When the speaker mentioned young horse, I want to know

at what age?”

P. A. Christensen: “Difícil precisar a idade. Penso que é velho um que mor¬

reu aos 29 anos e moço um cavalo de 6 anos.”

2 3

5 6

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):251-280, 1966

PETER KRAG and M. WEIS BENTZON

251

32. ANTIVENIN TESTING AT DIFFERENT VENOM LEVELS

PETER KRAG and M. WEIS BENTZON

Internai. Lab. Biol. Standards, Copenhagen, Denmark

During thc past fevv years t!ii“ World Health Organizations has heen interested

in the slandardization of antivenins. An International eooperation hetween la-

horalories in Europe, África and Asia has lead to a study of lhe Naja anti¬

venins and the establishment of an International Standard for Naja Antivenin.

The usefulness of this new standard is for the time being under trial. and

all producers of this antivenin have got samples of it vvith a request for a

specialized study, to examine vvhether it is useful as a standard in comparison

with Naja antivenins in test against Naja venoms of different geographical

origin and from different species of snakes helonging lo this groui).

The evaluation of the Naja . Antivenin Assay (WHO/BS/604 & 708) dis-

elosed that although lhe relative potencies based on the values for the international

standard vvere agreeing for Naja nivea and for several Naja naja venoms, some

unexplained discrepaneies remained for results obtained with the venom Naja

haje; it was also noted that the quantities of venom neutralized per ml serurn

were not agreeing.

During this study a eloser co-operation was estahlished between lhe laboratories

in Bangkok, Bombay, Johanneshurg, Paris and Copenhagen. In this co-operation

it was underlined from especially Dr. P. A. Christensen, that lhe composition

of certain Naja venoms with two or more venom components may be an ex-

planalion for the discrepaneies found hetween results from different laboratories,

as it may have happened that some laboratories have heen testing under conditions

where a component number 1 was predominant, while other laboratories have

had their testing levei lying corresponding to lhe effect of venom component

number 2.

blications and reports, a

Based on a renewed study of Dr. Christensen’s

model for the titration curves was constructed.

The co-operation hetween lhese laboratories continued from the Naja anti¬

venins to antivenins helonging to the Echis/Bit is group. It was agreed that

a series of studies of lhe E chis/ Bit i s sera were needed, especially tests al

several venom leveis. Such studies were performed hefore June 1965. The studies

have covered monovalent sera tested with the homologous venom at one levei as

well as polyvalent sera tested with several venoms at different LD-50 leveis; lhe

testing of same sera with venoms of different origin were also included.

The report I am giving you today will enclose facts from this co-operation

where lhe major part of the testing work has heen done by Dr. Christensen and

Dr. P. Boquet, while the evaluation was made in Copenhagen hy Mr. Weis

Bcntzon and me.

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10 11 12 13 14 15

252

ANTIVENIN TESTING AT DIFFERENT VENOM LEVELS

The evaluatio.n

The logarithmie values for LD-50 and for ED-50 were estimated together

with the slope at lhe 50 per eent point of lhe dose/response eurves hy means

of a logit analysis (Finney, 1952)*. The results have been compiled in Tablea I

lo IV. where lhe slopes and the log ED-50 values for each combination of serum

and venom have been noted.

The survey on the results showed a wide variation of lhe slope of lhe

dose/response curve. Those for lhe venom titrations (b v ) varied from 4 lo 59,

while those for the serum titrations (b„) varied from 4 to 185.

The log ED-50 values show a elear increase with the testing levei, but for

the b s values no simple relation to the leveis could be detected.

The variation in b s could be explained from the model constructed on the

following assumptions.

A venom contains three eomponents. rhe neutralization of the single coni-

ponent with corresponding antivenins in a serum takes place independently and

in each case following the multiple proportions. 1 he free quantity of venom

could he expressed for any situation as lhe total dose minus the part of venom

neutralized hy the serum activity, and this expression could he applied for each

of several venom eomponents.

The quantity of serum needed to reduce a venom component (i) lo its

ED-50 levei could thereafter he expressed as y 50 = r/i X (v — ai) where y is

the serum quantity needed, iji the amoinit of serum required to neutralize the

component i in one /.ig o! venom, v lhe quantity of venom used, and ai the

LD-50 of venom i expressed in terms of the whole venom.

Further we know that the ED-50 for the whole venom is larger than the

serum quantities needed for this above partial neutralization of any component.

The verbal expression for the above conditions is that lhe serum quantity

should he large enough to neutralize one of the eomponents (i„) al lhe same

time as there is surplus of serum against the two other eomponents.

From the study of diagrams showing the venom dose and the ED-50 values

it has been possihle to obtain graphie estimates of the LD-50 values for two,

maybe three eomponents of several of the venoms tested. Based upon this ex-

perienee a ealculation has been performed using the abovementioned expressions

to estimate for a elose series of venom doses the effect of each component and

lhe summarized effect of all three eomponents.

For a long series of venom titrations it has been found that lhe venom

dose/response curve could be described by a logistic curve.

A neutralization curve is constructed under the assumption that 1) the log

venom dose/response curve is logistic with the same slopes for all three venom

eomponents, and 2 ) the neutralization of each component follows lhe straight

lines in Fig. 1 . The resulting curve is shown in Fig. 1 as ... If the third

component is absent. the neutralization curve will be as the curve indicated

by 000 .

• Finney, D. J. — Statistiàal Method in Biological Assay, Lontlon. Charles Griffin &

Company Limited, 1952, p. 524.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 251-280, 1966

PETER KR AG and M. WEIS BENTZON

253

For the calculation was chosen the following values for three components,

ai values 20, 50 and 80 and values 0.2, 0.5 and 1.0. The calculation was

based ou an estimate of the probability of death for each of the three venom

components.

The relation between logits to the per cent of death and lhe quantity of non-

neutralized venom will he linear with /? v as the slope. Through a differentiation

of the formula for the logit curve it was found, that the slope of the tangent to

lhe usual dose/response curve would be-^ — . As the logit curve is approximately

4 £ v

linear between 30 and 70 per cent death the "— could be estimated as the

increase in the probability of death per unit increase in the log dose (see Fig. 2).

Combining this expression with lhe formula for y r , 0 we will arrive to

b s = b v X ( — 1), that is the slope of the plain dose/response curve is

equal lo lhe product of the slope for the venom tilration and the number of

LD-50 neutralized for that particular component.

The dependency between the slope and the venom dose is illustrated in Fig. 3.

An increase according to lhe formula is found for venom doses for which oídy

the first component is the important one. The slope shows a decrease when

another component '"takes over" followed by an increase.

From the above example it is obvious that the relation between the venom

dose and the results of the serum titrations, ED-50 and b s , is rather complicated.

It has been possible to estimate ai and q■, for two or three components,

when sufficient numher of observations have been available. In some cases the

observations are fitting very well lo the calculated values. An example of such

a fitting is given below.

Bitis lachesis

Venom

Dose

ED-50

íil b.

Component LD-50

a, = LD-50 = 9.7 uê

Serum quantity

neutralizing 1 /tg

venom

component yi

Venom slope

calculated

b v

100

3.5

a„ 76 iig

0.145

200

18.5

> 62

> 38

400

47.0

a .i

0.265

600

100.0

223 Mg

a 3

0.275

800

155.0

In other cases the calculation gave for different venom doses, assumed lo

belong to the same linear part of lhe curve, clearly disagreeing values. This

may indicate that more components are existing, hui that we did not have suf-

SciELO

10 11 12 13 14 15

254

ANTIVENIN TESTING AT DIFFERENT VENOM LEVELS

ficient observations for disclosing th is fact or il may indicate thal other factors

are influencing the outcome of lhe neutralizalion experiment.

A sludy of lhe relalive potencies showed lhat in many cases a serum gave

practically same relative potency for Iwo or more lesling leveis and in some

cases the relative potency was found the same for different venoms. It was

realized that the random variation of h s and ED-50 should he described ihoronghly.

The STAST1CAL ANALYSIS

Through the logit analysis has heen estimated the Standard error of the

logarithmic ED-50 value and of the slope of lhe dose/response curves (b B for

serum titrations and h v for the venom titration). In some cases it has also heen

possihle to eslimale the common slope for all sera belonging to a group tested

under similar conditions; these values are marked b ac , in other cases are used

lhe geometrical mean value for a group of sera b a (Tahle VIII).

The ahove standard errors are only covering lhe part of variation correspond-

ing lo the variations in the sensilivity of the animais against the non-neutralized

venom fractions.

The pari of variation due to the dilution and pipetting errors will mostly

influence the 50 per cent values, ED-50 as well as LD-50. Variance estimates

covering also these error components were oblained by means of analysis of

variances for log b s and also for log ED-50 in all cases where at least two sera

have been tested against the same venom in different doses (leveis) (see Tahle IV).

For lhe slopes (b s ) il has heen found thal lhe variance estimates obtained

are fitting very well lo the expectations from the logit analysis.

A comparison of the slope b„ for the dose/response curve of lhe titration

of antivenins at different lesting leveis and the standard error (SE|>) has shown

that lhe standard error, estimated for each slope, is in close relation to the

magnitude of lhe slope. For each venom and testing laboratory corresponding

values of SE b and b s have heen plotted against each other. In all cases per

laboratory and venom these values are following a distinct slope varying from

SE 2 (1»)

0.3 lo 0.4. The variance of log h, 0.1886 X “ tinis varies between 0.0171

and 0.0304. The residual variances s g are in accordance with the value 0.0304

except for two cases (0.12+ and 0.18 + + + ).

If we accepl 0.0304 as a general eslimate of the variance of log b s the

standard error for average values of log b a is for lhe three sera 0.10, for two

sera 0.12. These values will correspond lo the following limit factors for b s : three

sera 1.60 and 0.63, Iwo sera 1.76 and 0.58.

A difference between two log b B average values will have SE values 0.14

and 0.17 for n = 3 and n = 2 respectively. Tliis indicates thal the ratio between

two average b a values has limits 1.90 and 0.53 or 2.10 and 0.48 respectively.

In practice the ratio of average b a values may vary with a 2-factor.

+ , ++, and + + +, indicate values passing the limits of significance at the 5, 1 and 0.1

per cent levei respectively.

í, | SciELO

Mem. Inst. Butantan

Simp. Internac.

33(1):251-280, 1966

PETER KRAG and M. WEIS BENTZON

255

From Table VII it is seen that lhe slopes are not depending of lhe serum

lested, Imt in some cases a dependance of the levei is found.

The standard errors obtained by lhe logit analysis of log ED-50 values vary

within an elevenfold range 139 X IO - ' 1 lo 1556 X 10 -6 . The residual variances

(Sr ) in lhe analyses of variances are generally higher as will he apparent from

Table VI. The ralio hetween the sj values and the average variances obtained

by lhe logil analysis (SE 2 ) show lhe following distribution:

iboul

3.0,2 being significant at the 5 per cent levei

all significant at the 1 per cent levei

With lhe exception of the five highly significant ratios the variations of the

residuais seem to he random. Therefore it seems reasonahle to accept that the

experimental variance is obtained hy multiplying lhe value of SE 2 by an average

ralio for lhese cases, calculated to he 1.55.

The introduction of this increased variance leaves only five significant devia-

tions: all three values from Bilis nasicornis (ratio 8.2 now reduced to 4.8) and

two values from Echis carinatus (ratio about 41 reduced to 24).

The two Echis carinatus values correspond both to venom from Paris but

tested in two different laboratories. The three Bilis nasicornis values were re-

ported from one laboratory using two venoms of different origins.

The ED-50 showed usually significant variations hetween sera as well as

hetween leveis.

An interaction was found for Bilis nasicornis and Echis carinatus showing

that the ranking of ED-50 values was different when same serum was tested by

different venoms even from same species.

A study of the relative potencies for the sera mentioned above has shown

that the inerease of the ED-50 with lhe testing levei is regular but differs in the

rate for different sera in each of the groups with lhe extremely high variance.

Therefore it is concluded that a higher variance factor lhan the above 1.55 is

not indicated as lhe increased variance is due to real differences in the composilion

of the sera. The 13 cases with lhe lower variance are fully covered hy the use

of a variance factor 1.55 as no systemalic changes in lhe relative potency is

noted here.

During the preliminary survey was plotted the relation ED-50 to the number

of ED-50 used per testing levei. Mostly the plotting diagrams showed curves

with one or more bends, where the use of a higher testing dose gave an un-

expected inerease in lhe ED-50 value. It hecame clear that the form of these

curves could only he defined when many testing leveis were examined and each

“linear” part of the curve described by three observations. Some well described

combinations of sera and venoms gave curves which had a gradual relative

inerease of ED-50 and no major linear parts at all.

It was in the following examined whether the ohserved curves fitted a linear

relation hetween log ED-50 and log ju.g neutralized venom.

SciELO

10 11 12 13 14 15

256

ANTIVENIN TESTING AT DIFFERENT VENOM LEVELS

The relation is illuslralec) in Figures 4, 5, 6 & 7 using average logarithmie

values of the ED-50 and thc logarithm lo the /i.g venom neutralized as given in

Table IX.

This linear relation can he expressed hy the following formula

Iog ED-50 — K, + K 2 X log (v — LD-50)

where v is the venom dose in /xg.

These diagrams are showing that for eacli venom these curves will lie linear,

having slopes, whieli with few exceptions, are eharacteristic for that particular

venom.

Regression analyses performed on the results for eaeh single serum showed

that the variance due lo variations around the line was only somevvhat higher

than the variance corresponding to the ED-50 variation previously descrihed.

Pari of this additional variation is due to errors in the determinalions of the

LD-50 values and in the dilutions of serum.

The linear relation hetween log ED-50 and the log jx g neutralized venom

thus gives a reasonable fit.

Tests performed in different laboratories using different venoms are fitling

in this system. The slopes of these curves are increasing from Bilis nasicornis

1.07, Echis carinatus 1.30 and Bilis gabonica 1.35 to Bilis lachesis 1.59. The

Echis coloratus curve is different from this rule. The serum collection d showed

in some cases higher slopes for Bilis gabonica and Bilis nasicornis.

On separate diagrams have heen studied the overall trend for each particular

venom and the individual curves for the comhination of venoms and sera where

significant differences hetween ED-50 values or hetween the slopes of the first

mentioned curves have heen observed.

This description of the neutralization curve seems rather different from the

previous 3 component or 2 component curve. However hy plotting the theoretical

curve (Eig. 8) constructed as mentioned above in the same way, il was found

that a straight line would fit the part where lhe second or the third component

were the important ones. Since the ahove description is more simple (contains

only Iwo unknown values against six for the three component analysis) we prefei¬

to use it for further description. The 3-component straight line is also given in

Il is seen that lhe overall fitling to the theoretical curve is good.

Table X shows for lhe relative potencies lhe influence of lhe use of venom

of the same narne but of different origin on the same sera. In nine cases were

only seen random variations and in two ( Bilis lachesis serum 504 and Echis

carinatus serum 509) a major difference was found hetween venom from Johan-

nesburg and from Paris. A similar difference was nol seen for the other sera

tested under the same conditions.

Table X gives also in some cases lhe variation in relative potency when the

lesting is performed with different venoms:

Serum 24, seven observations, only random variation; serum 12 and serum

17, Bilis gabonica and lachesis, are giving identical results, while Echis carinatus

is giving a lower relative potency.

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):251-280, 1966

PETER KRAG and M. WEIS BENTZON

257

Sera 504 and 509 are different in their composition. In nine cases ont of

18 combinations are seen relative potencies aliont —0.30. Higher potency valnes

(about zero) are seen for sernm 504 (Echis carinatus and coloratus) and for

serum 509 (Bitis nasicornis). Low relative ])otency values, about —1.00, were

observed in serum 509 (Echis carinatus and coloratus). This corresponds lo lhe

differenl program for the immunization of llie two groups of horses, serum 509

not having included horses immunized with any Echis venom.

The additional immunization of horses for serum 504 willi Echis coloratus

venom has not given any remarkable difference in potency from serum 503.

The ED-50 values corresponding to a chosen common testing value 200 jag

venom are given in Table XI (the ED-50 values were when needed ohtained hy

interpolation) .

When same serum was tested against two venoms of different origin hut

from same species, the difference in ED-50 exceeded only in 3 out of 16 cases

a factor 1.3.

For venoms used in this study it seems more appropriate to use jug venom

neutralized rather than number of ED-50 for such comparison.

It was noted that lhe neutralizing effect of lhe polyvalenl sera tested varied

from venom lo venom examined. and that this variation not followed one common

pattern.

Discussion

S. Schengerg: "Whenever making a venom standard, regional variations in

venom composition were considered? This is an important point to be considered

as accident results from the bite of a single snake.”

P. Krag:

Paris.”

“Venoms in my paper are mixtures as used in Johannesburg and

A. do Amaral: “1. To present compliments for his having followed a modern,

scientific and more reliable process in his work on venom titration and antivenin

standardization. both of which are quite serious and very complex questions.”

“2. To ask what channel is used in the injection of venom and antivenin. for

inoculating the animais (white mice).”

P. Krag: “Intravenous in white mice.”

SciELO

10 11 12 13 14 15

258

ANTIVENIN TESTING AT DIFFERENT VENOM LEVELS

TABLE I — RESULTS FROM JOIIANNESBURG IN TESTING 18 ANTIVENINS.

NINE AGAINST ONE VENOM, NINE AGAINST ANOTHER VENOM

Challenge Venoms

Name:

LD-50:

Slope b v :

Dose:

Number of LD-50:

Bitis gabonica

14.2 ng

400 jig

Bitis lachesis

7.2 mS

400 MS

Serum

Codes

log ED-50

bs z

Serum

Codes

log ED-50

181

2.24

301 y

a = 1

1.96

91*

301

1 = a

1.86

72*

425

1.73

31*

327

2.04

426>

c = n

1.83

426J

n = c

2.04

> 101*

463

1.93

576

2.04

498

1.95

655

2.10

555

1.80

816

1.90

32*

638

1.82

822

2.04

686

2.00

826

2.04

718

1.91

Range for nine

results

Common slope b c

0.38

23 to > 101

0.27

12 to 91

* Slope estlmated through a Iíárter evaluation.

■ v Sera a & c are identical to sera I & n respectively.

z The slope is expressed as the increase in mortality percent, when serum dose is (at

50% levei) reduced with 10%.

slope

for

the

dose/response curve

(mortality/serum

dose)

common

slope

for

the

dose/response turve

(mortality/vcnom

dose)

2 3

6 SClELO 1Q ii 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1):251-280, 1966

PETER KRAG and M. WEIS BENTZON

259

TABLE II — RESULTS FROM PARIS. CRUDE MONOVALENT SERA

SEVEN ANTI-B/77S SERA WITII ONE VENOM

SEVEN ANTl-ECHIS SERA WITII ANOTHER VENOM

Name:

LD-50:

Slope b v :

Dose:

Number of .LD-50:

Challenge venoms and testing leveis

Bitis gabonica 0

31.0 mU

155 310 465 MU

5 10 15

Echis carinatus

28.0 MU

140 280 Mg

5 10

Serum 140 bs

Serum 161 b s

log ED-50

1.85

2.31

log ED-50

2.09

2.52

Dev. of log ED-50

-0.46

Dev. of log ED-50

-0.43

Serum 11/6 b«

106

Serum 332 b*

log ED-50

1.93

2.46

log ED-50

1.84

2.29

Dev. of log ED-50

-0.53

Dev. of log ED-50

- 0.45

Serum 387 bs

Serum 1061 bs

log ED-50

1.67

2.18

log ED-50

1.84

2.33

Dev. of log ED-50

-0.51

Dev. of log ED-50

-0.49

Serum 9//5 bs

Serum 1063 bs

65*

log ED-50

1.87

2.31

log ED-50

1.83

2.20

Dev. of log ED-50

-0.44

Dev. of log ED-50

-0.37

Serum 384 bs

Serum 1066 v bs

34*

185

log ED-50

1.95

2.49

log ED-50

1.91

2.38

Dev. of log ED-50

-0.54

Dev. of log ED-50

-0.47

Serum 616 b s

Serum 816 b s

log ED-50

1.51

2.07

log ED-50

1.94

2.40

Dev. of log ED-50

-0.56

Dev. of log ED-50

-0.46

Serum 617* bs

Serum 781* bs

log ED-50

1.85

2.33

log ED-50

1.77

2.20

Dev. of log ED-50

0.48

Dev. of log ED-50

-0.43

Common slope b<-

17.5

26.3

Common slope br

29.2

70.7

except 617 & 781

except 781

Notes to Table II

Slope estimated through a Karber evaluation.

Samples of serum 1066 also tested alter a period ot freezing (bs reduced to 1/3, ED-50

unchanged; see document on the elfect of freezlng/thawlng on sera).

No estimate of slope (dose/response 0/6; 0/6; 2/6).

Sera 617 & 781, strain of mice: Paris (all other sera HLA-mice).

Additional information for Nitis gabonica venom:

Serum 7H1'- showed at testing levei 6 LD. log ED-50 = 3.00 (ED-50 = 1000).

SciELO

10 11 12 13 14 15

260

ANTIVENIN TESTING AT DIFFERENT VENOM LEVELS

TABLE III — JOHANNESBURG AND BOMBAY

ECHIS/BITIS TESTING RESULTS OCTOBER 1964 TO JUNE 1965

Venoms and

LD-50

Sera

Testing leveis (Number of LD-50)

112

B. gab.

14.2 ng

b v = 16

k — o, bsc

q-t* log ED-50

1.86

. 2.24

B. ariet.

a-e, b.c

1.73

. 2.00

g-j* log ED-50

7.2 ug

bv = 23

VG' r b.

>34*

log ED-50

1.39

2.34

Dev. of log ED-50

0.95

Tropv bs

22*

log ED-50

1.78

2.14

Dev. of log ED-50

0.36

353 v bs

45*

log ED-50

1.20

1.63

2.13

Dev. of log ED-50

0.43

0.93

Testing leveis (Number of LD-50)

2.5

B. nas.

840 bs

23.6 ug

log ED-50

1.56

2.14

bv = 15

Dev. of log ED-50

-0.87

-0.29

2.43

504 b„

log ED-50

1.53

1.89

2.20

Dev. of log ED-50

-0.67

-0.31

E. car.

753 b.

49*

21.9 Hg

log ED-50

0.97

1.38

1.89

2.23

bv = 12

Dev. of log ED-50

-0.41

0.51

0.85

E. car.

Bombay

44.0 Hg

b v = 59

log ED-50

1.51

See also detailed list for these sera Table I.

Test results for venom N. nivea, see table in report on Naja antlvenin studies.

Samples of sera VG, Trop & 353 vvere also tested after a period of lreezing; b» & log

ED-50 showed no systematic ehanges, see document on the effect of freezing/thawing

on sera.

Slope estlmated through a Karber evaluatlon.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1) :251-280, 1966

PETER KRAG and M. WEIS BENTZON

261

TABUE IV — RESULTS FROM PARIS. FOUR AKTl-BiriS/BCHIS SERA* 12, 17, 23 & 24

ALL PURIFIED, AGAINST FOUR VENOMS. PARIS MICE

Biti8 gabonica

LD-50:

17.8 iig

28.2

Slope b v :

Dose:

340 ng

141

282

423

564 Hê

Number of LD-50:

Serum 12 b»

log ED-50

1.86

2.31

Dev. of log ED-50

-0.45

Serum 17 b s

log ED-50

2.11

2.38

2.49

Dev. of log ED-50

0.27

0.38

Serum 2S b.

log ED-50

1.65

2.12

1.96

2.38

Dev. of log ED-50

0.47

0.42

Serum 21/ b*

40*

51*

log ED-50

1.69

2.08

1.90

2.32

Dev. of log ED-50

0.39

0.42

Name:

Bi tis lachesis

LD-50:

9.3 Hg

12.6

Slope b v :

Dose:

186

372 gg

126

189

252 Hg

Number of LD-50:

Serum 12 bs

log ED-50

2.11

2.35

Dev. of log ED-50

0.24

Serum 17 bs

log ED-50

1.81

2.28

Dev. of log ED-50

0.47

Serum 28 b s

log ED-50

1.41

1.97

2.40

1.75

2.22

Dev. of log ED-50

0.56

0.99

0.47

Serum 24 b»

log ED-50

1.45

1.92

2.40

1.70

2.23

Dev. of log ED-50

0.47

0.95

0.53

SciELO

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262

ANTIVENIN TESTING AT DTFFERENT VENOM LEVELS

TABLE IV (continued)

Origin:

Name:

Echis carinatus

LD-50:

30.9 Mg

24.0

Slope bv:

Dose:

309

464

618 /ig

240

360

480 Mg

Number of LD-50:

Serum 12 bs

log ED-50

1.89

2.37

Dev. of log ED-50

0.48

Serum 1 7 bs

21*

106

log ED-50

2.34

2.52

1.87

2.20

Dev. of log ED-50

2.09

0.25

0.43

0.33

Serum 23 b«

log ED-50

2.12

2.32

Dev. of log ED-50

0.20

Serum 21/ b»

log ED-50

2.07

2.33

Dev. of log ED-50

0.26

Notes to Table IV

Bitis naslcornis

Johannesburg Paris

5 LD-50

Dose:

151 Mg

lio Mg

Serum 23 b s

log ED-50

2.22

1.64

Serum 2b ba

log ED-50

2.23

1.54

The sera were tested with lhe venoms commonly used in Paris, LD-50 ‘21.9; b v 17, and

with a sample received from Johannesburg, LD-50 30.2; bv 4. The slope of the dose/

response curve was exceptionally low also vvhen compared with results from Johan¬

nesburg, Table V.

Samples of these sera were also tested after a period of freezing. No systematic

changes in b* or in log ED-50 were noted, see the doeument on the freezing/thawing

effect on sera.

Slope estimated through a Karber evaluation.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1) :251-280, 1966

PFTER KR AC and M. WEIS BENTZON

263

TABLE V — JOIIANNESBURG. BITIS/ECHIS SEPTEMBER 1965. THREE SERA TESTED

WITH FIVE VENOMS (FOUR OF WHICH OF TWO DIFFERENT ORIGINS)

Name and Origin:

Bitis gabonica — Jòhannesburg

LD-50:

11.5 Hg

Slope b v :

Dose:

80 hZ

200 HZ

400 hZ

600 Hg

800 Hg

Number oi LD-50:

17.5

Serum 503 b»

38*

> 52*

52*

iog ED-50

0.40

0.95

1.32

1.57

1.87

Deviation'*

-0.55

0.37

0.62

0.92

Serum 504 bs

log ED-50

0.61

1.23

1.58

1.87

2.13

Devlation

-0.62

0.35

0.64

0.90

Serum 509 b«

30*

log ED-50

0.70

1.29

1.70

1.91

2.16

Devlation

-0.59

0.41

0.62

0.87

Name and Origin:

Bitis

gabonica —

Paris

LD-50:

25.8 hZ

Slope b v :

Dose:

200 HZ

400 hZ

600 HZ

Number of LD-50:

Serum 503 b»

30*

log ED-50

0.89

1.52

1.82

Deviation

-0.63

0.30

Serum 504 b s

log ED-50

1.22

1.82

2.17

Devlation

-0.60

0.35

Serum 509 b»

log ED-50

1.07

1.79

2.02

Devlation

-0.72

0.23

Name and Origin:

Bitis lachesis — Jòhannesburg

LD-50:

9.7 HZ

Slope b,:

Dose:

100 HZ

200 Hg

400 Hg

600 Mg

800 HZ

Number of LD-50:

Serum 503 b«

> 62*

log ED-50

0.55

1.25

1.67

2.00

2.19

Deviation

-0.70

0.42

0.75

0.94

Serum 504 b»

71*

log ED-50

0.95

1.54

2.03

2.28

2.50

Deviation

- 0.59

0.49

0.74

0.96

Serum 509 b*

40*

log ED-50

0.84

1.40

1.87

2.16

2.44

Deviation

-0.56

0.47

0.76

1.04

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10 11 12 13 14 15

264

ANTIVENIN TESTING AT DIFFERENT VENOM LEVELS

TABLE V ( continued )

Name and Origin:

intis

lachesis —

Paris

LD-50:

10.7 Ag

Slope b v :

Dose:

200 A g

600 Ag

Number of LD-50:

Serum 503 b»

log ED-50

1.22

1.91

Deviation

0.69

Serum 504 b»

< 42

log ED-50

1.78

2.52

Deviation

0.74

Serum 509 b«

log ED-50

1.51

2.28

Deviation

0.77

Name and Origin:

Bitis nasicornis — Johannesburg

LD-50:

25.2 Ag

Slope b v :

Dose:

100 IJLg

200 Ag

400 Ag

800 Ag

Number of LD-50:

Serum 503 b.

53*

> 52*

57»

> 52'

log ED-50

1.23

1.65

2.02

2.35

Deviation

-0.79

-0.37

0.33

Serum 504 bs

No test

log ED-50

1.47

2.02

2.46

Deviation

-0.99

-0.44

Serum 509 b»

> 52'

> 63*

log ED-50

1.29

1.65

2.04

2.42

Deviation

-0.75

-0.39

0.38

Name and Origin:

Bitis

nasicornis —

Paris

LD-50:

22.8 Ag

Slope b r :

(14)

Dose:

100 Ag

400 /íg

Number of LD-50:

4.4

17.5

Serum 503 b«

log ED-50

1.09

2.00*

Deviation

-0.91

Serum 504 b s

log ED-50

Deviation

Serum 509 b„

log ED-50

Deviation

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Slmp. Internac.

33(1): 251-280, 1966

PETER KRAG and M. WEIS BENTZON

265

TABLE V ( continued )

Name and Origin:

Echis carinatus — Johannesburg

LD-50:

22.1 Hg

Slope bv:

69*

Dose:

50 Hg

100 Hg

200 Hg

400 Hg

800 Hg

Number of LD-50:

2.25

4.5

Serum 503 b s

> 52*

log ED-50

0.46

0.97

1.51

1.93

2.25*

Deviation

-1.47

-0.96

-0.42

0.32

Serum 504 bs

53*

34*

log ED-50

0.42

0.95

1.54

1.99

2.31*

Deviation

-1.57

-1.04

-0.45

0.32

105 Hg

134 Hg

4.75

6.06

Serum 509 b»

log ED-50

2.00

2.30

Deviation

Name and Origin:

Echis carinatus — Paris

LD-50:

31.8 Hg

Slope bv - .

55*

Dose:

100 Hg

200 Hg

300 Hg

400 Hg

Number of LD-50:

3.1

6.3

9.4

12.6

Serum 503 b»

53*

> 52*

57*

log ED-50

1.55

1.95

2.11

2.36*

Deviation

-0.81

-0.41

-0.25

Serum 504 b 5

102*

53*

log ED-50

1.40

1.99

2.37

2.55*

Deviation

-1.15

-0.56

-0.18

Serum 509 b.

log ED-50

2.30

Deviation

Name and Origin:

Echis coloratus

LD-50:

16.0 /ig

Slope b,:

Dose:

100 Hg

200 Hg

400 Hg

Number of LD-50:

6.3

12.5

Serum 503 b s

log ED-50

1.18

1.37

2.00

Deviation

-0.19

0.63

Serum 504 b s

log ED-50

1.23

1.56

2.21

Deviation

-0.33

0.65

SciELO

10 11 12 13 14 15

SciELO

10 11 12 13 14 15

Hem. Inst. Butantan

Slmp. Internac.

33(1) :251-280, 1966

PETER KRAG and M. WEIS BENTZON

267

Notes to Table VI

(Serum collection code a-i also used in Tables VII to IX)

The collection of sera 4 used for studles on ED-50 and testing levei.

a J I 181, 301, 327, 426, 576, 655, 816, 822, 826

ft J I 301, 425, 426, 463, 498, 555, 638, 686, 718

Sera

140, 146, 387, 945, 384, 616

23, 24

17, 23, 23F* *\ 24

503, 504, 509

840, 504

161*, 332, 1061, 1063, 1066, 1006F**, 816, 781*

12, 17

23, 23F X , 24

503, 504

■ v Table II, serum 781, not included in the estimate oi common b«.

x Table II, serum 161, only used with Its ED-50 vaiue (b s levei 10, no estimate pos-

sible).

* Number of b s values are e.xceeding those of ED-50 values by one.

* Table I, serum collections a and /?, tested at one levei only, not used for study on

ED-50 and testing leveis.

" The sera marked F vvere tested among sera described in Tables II & IV, they are

all tested after a period in frozen State.

X, XX and XXX indicate values passing the limits of signiflcance at the 5, 1 and 0.1

per cent levei respectlvely.

(X ) corresponds to cases vvhere the degree of significance is reduced when the increased

SE is used.

SciELO

10 11 12 13 14 15

TABLE VI — ANALYSIS OF VARIANCES FOR RESULTS FROM TABLES II TO

268

ANTIVENIN TESTING AT DIFFERENT VENOM LEVELS

1, | SciELO

TABLE VHIa

SURVEY OF SLOPES bsc, bs OR bs AT DIFFERENT TESTING LEVELS

Venom

Origin & bv

li. gabonica

Sera

Slope

bsc

0,0*

18.

"bs

22.

b.c

bsc

Trop

353

bsc

b s

"b«

bsc

40 ft

26.,

33 15

B. Jachesis

J 23

12.

22 2

12 2

23 s

22.

42,

21,

> 37 2

31.

51 2

19,

The index at bsc and b s indicates the number of bs values ií different from 3.

J & P indicate origin of venom.

Iligh variance, see Table VI s = 0.1765.

TABLE VlIIb

SURVEY OF SLOPES bsc, bs OR b s AT DIFFERENT TESTING LEVELS

Venom

Origin & b v

Sera Slope

2i/ 2

e b«c

15.

22 2

2fi,

d bsc

27 55

54 30.

d b»c

(14)

f bs.-

29,

71„

753 b»

27,

21 1

15,

Bomb. bs

12,

17 Fs

18, 46

< 21 í

g + h b»

34. 41, 55»

i bsc

16 2

13.

56,

24 2

i bsc

32.

38.

64, 55

i bsc

30,

■

E. carinatus

69-

E. coloratus

13..

Slope estimated through a Kárber evaluation.

The letters n, fi, and a . .. i indicate the different collections of sera used.

J, P & B indicate origin of venom.

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1) :251-280, 1966

PETER KRAG and M. WEIS BENTZON

277

TABLE XI — ED-50 ESTIMATED AT A COMMON LEVEL 200 Hg NEUTRALIZED.

SURVEY OF LD-50 VALUES FOR THE VENOMS USED

Sera

Labor¬

atory

N a

me of Venom

B. gabonica

J P

B. lachesis

J P

B. nasicornis

J P

E. carinatus

(color. )*

J By P

<*, fi.

a & f

J & P

30-71 30-182

19-35

120-251

753

Trop

353

840

251

504

148

Bombay

32y

148

257

148

110

129

123

100

126

107

503

52 43

38 (28)

100

504

126 v 93

41 (35)

132

509

52 48v

LD-50:

24 23

22 (16)

44y

K„

1.35

1.59

1.07

1.30

1.83u

1.40v

K 2 : The

slope of the curve log

ED-50/log Hg

venom

neutralized (see

Figs. 4, 5, 6

& 7);

no K 2 value for E. coloratus (curve not linear).

* E. coloratus results noted under B in ( ).

y Test in Bombay with a Bombay venom.

a, 11: ED-50 values obtained by extrapolation using the slope K, 1.35 & 1.59 resp.

u Sera 503, 504 & 509 Paris venom in laboratory J.

v Serum 504 venom J and serum 509 venom P showed high slope values (K 2 = 1.40).

J & P indieates origin of venom as well as testing laboratories.

SciELO

10 11 12 13 14 15

278

ANTIVENIN TESTING AT DIFFERENT VENOM LEVELS

Fig. 1 — Relation between ED-50 and the

venom levei (dose in microgram) cons-

tructed example vvlth 3 venom components

(...); with the third component absent

use ... until venom dose 70 and .. for

higher venom doses. The 3 straight lines

represent the ED-50 values expected for

each component separately. The full dravvn

parts of these lines represents for each

levei the component requlring maximum

of ED-50.

Flg. 2a — Dose/response curve for a

venom titration.

v = venom dose

bv = slope of dose/response curve

Fig. 2b — Dose/response curve for a serum

titration.

y = serum dose

b» = slope of the dose/response curve

testing levei 2.5XLD-50

VENOM OOSE

Fig. 3 — Relation between the slope (b s ) of serum titration

curves and the venom levei (dose ln microgram). Constructed

example with 3 venom components (.—.—.). The 3 straight

lines represent values expected, if each component is considered

separately (— — —-).

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(1)1251-280, 1966

PETER KRAG and M. WEIS BENTZON

279

LOG CDjo CDjq

NEUTRAl i

?00 400 800

LOG *iq nCUT RA L (2£ 0

Fig. 4 — Bitis (jabonica. Average values

of iog ED-50 plotted against log micro-

gram venom neutralized. 4 diffcrent group

of sera (a, b, c, d); one (d) tested against

2 venoms of different origin.

NEU T BAUZr D

200 400 800

LOG M8 NEUT84LI7EC

Fig. 5 — Bitis lachesis. Average values

of log ED-50 plotted against log mlero-

gram venom neutralized. 3 different groups

of sera (b, e, d); one (d) tested against

2 venoms of different origin.

LOGEDjo ED»o

:r ;

7T7

80 '00

LOG yq HCUTR

Fig. 6 — Bitis nasicornis. Average values

of log ED-50 plotted against log micro-

gram venom neutralized. 2 different groups

of sera (d, e); one (d) tested against 2

venoms of different origin.

SciELO

10 11 12 13 14 15

280

ANTXVENIN TESTING AT DIFFERENT VENOM LEVELS

LOG EDjo tOso

LOG /iq NEUTBAU2ED

Fig. 7 -— Echis carinatus & Echis coto-

ratus. Average values of log ED-50 plot-

ted against log mlcrogram venom neutral-

ized. 4 different groups of sera (f, g, h,

i); one ot these (1) tested against 2 Echis

carinatus venoms ot different origin and

against one Echis coloratus venom.

Flg. 8 — Observed ED-50 values for serum

503 — — — calculated ED-50 from the

expression log ED-50 = K, + K, log

(v — LD-50). K, a constant correspond-

ing to potency of serum. K, the slope of

the curve. log. ED-50/log mlcrogram neu-

trallzed venom. (Biíis Zocliesis-serum 503 —

- K, =1.71).

SciELO

10 11 12 13 14 15 16

Mem. Inst. Butiintun

Simp. Internac.

33(1): 281-291, 1966

O. ZWISLER

281

33. THE ROLE OF ENZYMES IN THE PROCESSES RESPONSIBLE FOR

THE TOXICITY OF SNAKE VENOMS (AN IMMUNOLOGICAL STUDY)

O. ZWISLER

Behringwerke AG., Marburg-Lahn, Germany

I want yon to know some immunological experiments which we recently <li<l

in the Behringwerke AG, Germany, experiments by which we intended to elncidate

As yon know, snake venoms are a mixture of different j)harmacologicaIIy

active components. There are a numher of papers in which lhese principies have

been classified in accordanee to lheir different mode of action (1-6), but I want

to make a simple differentiation according to our knowledge of their substrates

or reeeptors tliey act upon.

For an investigation whether the enzymes in snake venoms play an important

role during the course of intoxication we have chosen different ways of experi¬

ments. In one series of experiment we compared the neutralization of lethal

action and defined enzyme activity by polyspecific antisera from horses, in an

other one we added an enzyme to venoms, which were either devoid of lliis enzyme

or in which this enzyme was strengthened by lhe addition and tested the toxicity

of this mixtures. In further investigations we produced monospecific antisera

against some enzymes and some toxie components and determined lhe protective

action of these antisera against the whole unfraclioned venoms.

Titrating commercial antisera from horses with the venom, which has been

used for immimization, the dose/response curve does not follow the law of multiple

proportions and soon parallels the serum axis, showing a low conlent of antihodies

against the toxins (7).

On the other hand, in case of elevated venom doses were there was in vivo

no more protection by any volume of antiserum, the enzymes in this venom-dose

were completely inhibited. In Fig. 1 a typical result is shown (8). There is

only one enzyme. we have determined, the 1-amino acid oxidase, which was not

at all inhibited, but the explanation for this special case is given later.

We asked ourselves, whether these findings resulted from lhe choice of lhe

horse as antibody-donor and from lhe method of immunization used for production

of antitoxic snake sera or whether we were ohserving a general feature of anti-

venomous sera. In attempt to answer this question we used other animais, i.e.

rabbits, for immunization using various adjuvants. The venom we used was

Bilis gabonica venom, which we know to be very good antigenic and against

which generally animais produce potent antisera in a relatively short time. After

two subsequent courses of immunization the sera were tested in vivo for their

protection against the whole venom and in vilro for their enzyme-inhibiting

2 3

5 6

10 11 12 13 14 15

282 T1IE KOLE of enzymes in tiik rrocesses RESPONSIBLE FOR THE

TOXICITY OF SNAKE VENOMS (AN IMMUNOLOGICAL STUDY)

properties. In all cases lhe anti-enzyme lilers were higher lhan lhat againsl loxic

components (Fig. 2). Allhough lhese experimental arrangements can give in-

sighl into lhe conlrihulion of enzymes to the killing power of venom. further

refinements were needed for detailed analysis.

]ii order to examine the contribution of individual enzymes to the loxic ef-

ficiency of a snake venom, Kaiser and Michl have proposed the utilization of

monospecific antisera, which are directed againsl only one of the many enzymes

in the venom (9). Tliis approach allows lhe selective inhihition of a single enzyme

under physiological conditions. In ihis case a decrease in toxicity may he cx-

pected, provided lhe enzyme makes an essential contribution to the poison-

ing process. On the other hand, an antiserum against loxic components, hui

without any anti-enzyme aetion should give protection against 1 the whole venom.

if participation of enzymes in the process of intoxication can be neglected.

In order lo obtain materiais for immunization venom components were

separated hy electrophoresis in polyacrylamide gel (10). Figure 3 shows the

electropherograms of some VIPERIDAE venoms. The various activities were

localized in the gel slah whether hy in situ mettiods or

pieces, elution of lhe componenl contained herein and lesting their aetivity in viro

and in vitro (Fig. 4). For immunization, the gel strip, containing the respeclive

aetivity, was homogenized in an appropriate amount of saline and was injected

subculaneously into rabbits. The polyacrylamide aets as an adjuvant. By this

method we have prepared monospecific antisera against 1-aminoacid oxidase, phos-

phodièsterase and a fibrinolytic agent from the venom of Vipcra ammodytcs.

We have similarly prepared monospecific antibodies lo phospholipase A from

venom of Echis carinatus and to acetylcholinesterase from Naja haje venom.

The immunoeleetrophoresis of these antisera, compared with the precipitation

pattern of the respective polyspecific antisera from horses are shown in Figure 5.

Anti-toxic-sera were prepared against a loxic fraction from Echis carinatus

venom. Work with this venom is convenient since its toxic components are not

separated by electrophoresis (11), so that one small section of the gel contains

practieally the whole toxicity of the unfractionated venom. Further on we isolated

crotactin from the venom of Crolalus terrificus (12) and prepared the respective

antiserum. The immunoeleetrophoresis of these antisera are shown in Figures

6 and 7. The protective potency of all these antisera was examined againsl

whole venom.

As it is known, the phospholipase A can he completely inhiblted hy anti¬

serum, but in vivo the monospecific antiserum even in a surplus necessary to

neutralize the phospholipase A-activity in a small venom-dose is not capahle lo

j>rotect against the whole venom. So we cannot confirm the sentence of Kellaway,

written 25 years ago (2) : “It is hy no ineans certain that pari or all of the

neurotoxic effect of some venoms may not he ultimately he found to be attrihutahle

to the phosphatidases”, but our findings are in accordanee with some recent zoo-

toxie investigations made with purified phospholipase A. showing the low toxicity

of this enzyme (13,14).

Using phosphodiesterase-antiserum, there is also no inhihition ol the toxicity

of the whole venom, though this antiserum shows a sufficient inhihilory eapacily

for the respective enzyme, though the purified enzyme shows a certain toxicity (15).

The fibrinolysin-antiserum inhihits the fibrinolytic component of lhe Vipcra

ammodytes-xe nom in vitro. This can he shown hy the inhihition of the lysis

of thrombin induced fihrinclots and otherwise hy comparing the clotting times

■tho(

hy '-utling the gel inlo

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O. ZWISLER

283

after injections of lhe fihrinolytic component or mixlures of the antiserurn with

whole venom into guinea-pigs. Intravenous injections of the fihrinolytic principie

delay the clotting of guinea-pig lilood up to ahout 600 seconds (determined in

whole venom into gu;

delay the clotting of

nol treated animais to he ahout 300 seconds), while injections of a mixture of

antiserurn and whole venom accelerate the eoagulation. In this case clol formation

can be seen within 60 to 90 seconds, thus showing once again the dualistic action

of snake venom containing a pro- and an anticoagulant principie upon eoagulation.

Although the fibrinolysin-antiserum did not neutralize the lelhal action of un-

fractionated venom.

Two very interesting examples of an enzyme-antienzyme-system we found in

the cases of the acetylcholinesterase and the 1-aminoacid oxidase.

In immunoelectrophoresis and applying other gehliffusion techniques, we

found that a precipitation linc retains acetylcholinesterase activily even after pro-

longed washing with buffers or saline, and which can he made visihle by specific

staining with indoxylacetate or acetylthiocholinchlorid (16) (Fig. 8). Inhibition

of acetylcholinesterase by monospecific antiserurn and also by two antisera from

liorses against Naja haja-venom could be determined quantitatively by in vitro

assay, the inhibition was incomplete as il was lo be expected from the aetivity

in the precipitation lines. Even after addition of large volumes of concentrated

antiserurn lo a constant amount of Naja haje-ve nom and varying tlie incubation

lime, temperalure and huffer syslems, lhe degree of inhibition was always 51%.

We never obtained precipitates with acetylcholinesterase-activity, so thal the aetivity

could only be measured in the supernatants (Fig. 9). After geldiffusion of snake

venom against the corresponding antiserurn in most cases on precipitation line can

be seen, which has 1-aminoacid oxidase aetivity. This phenomenon, indicating

incomplete inhibition, was examined with monospecific antiserurn, rcacting with

the venom of Vipera ammodytes. By estimating lhe residual aetivity in lhe well-

washed and finely resuspended antigen-antibody-precipitates and its corresponding

supernatants it was shown that the enzyme was completely precipitated by lhe

antiserurn and that 36% of the enzyme aetivity was inhibited. The antiserurn

crossreacted with the enzyme of other venoms of viperids but nol with venoms

of crotalides and elapides giving nltimately the same degree of inhibition (Fig.

10). However, more antibody was required in lhe heterologous tlian in lhe

homologous system to lead to the given degree of inhibition (17). It may be

that by this means the relationship of different species can he sludied. We never

got in vitro precipitates with antisera from horses, so that the lack of inhibition

of the 1-aminoacid oxidase in the forementioned case lilration of the antienzyme

aetivity of Vipera russelii- antiserurn can be explained in this way.

Tested in vivo, neither the acetylcholinesterase- nor 1-aminoacid oxidase-anti-

serum afforded protection against the lelhal action of lhe corresponding venoms.

Although the inhibition of these two enzymes in vitro by lheir respective antisera

is incomplete, it can nol be deduced, that this may be the reason for the lack

of efficiency against unfractionated venom. Remember lhal antisera against

urease, though lhere is only an inhibition of 20% of the enzyme aetivity, is

capablc to protect animais completely against the deleterious action of ihis en¬

zyme (18). It seems lo be sufficient for protection that the active agent rcacts

with the antibody. The complex which forms seems lo be eliminated from the

organism at a higher rate. These residis are resumed in the next picturc (Fig. 11).

Furthermorc we wcre interested in lhe importance of lhe hyaluronidase dur-

ing the eourse of intoxication. We never succecded in isolating this enzyme, so

2 3

5 6

10 11 12 13 14 15

284 THE ROLE OF ENZYMES IN THE PROCESSES RESPONSIBLE FOR THE

TOXICITY OF SNAKE VENOMS (AN IMMUNOLOGICAL STUDY)

that we were not able lo produce any antiserum. Since lliere is no proof that

the hyaluronidase in snake venoms is eompletely different from that derived from

testes, we added this latter enzyme to two different snake venoms and determined

its influence on toxicity and death rate. The venoms we used were from Naja

nigricollis, wliich was eompletely devoid of hyaluronidase and showed, injected

subcutaneously, no haemorrhagic action, and from Vipera ammodytes, which

showed considerable hyaluronate lyase activily and produced severe hemorrhages.

The addition of various amounts of enzyme from testes did not increase lhe loxicily

of botli venoms applied s.c. or intramuscularly nor could we see a ehange of time

between lhe moment of injection and death (Fig. 12). The only ehange which

was to be deteeted was an increase of the local action of the Vipera ammodytes-

venom, while the venoin of Naja nigricollis though in mixture with hyaluronidase,

remained without any local efficieney. This experimental arrangement allows to

exclude the participation of hyaluronidase during intoxication by snake venoms.

Now let us regard the protection, which is aehieved by toxin-antisera. Both

were eompletely devoid of enzyme-antibody. Nevertheless these sera afforded pro¬

tection against toxicity, that is they protect animais against the intoxication with

whole venom, containing lhe not inhibited spectrum of all enzymes (Fig. 13).

Suming up these findings demonstrated here, give further support lo the view

that the toxins are the chief noxious principies in snake venoms, residis which

were shown by other immunological means by Kochwa and col., 1920. In order

to obtain snake venom antitoxins of which the dose response curve is straightened,

there must be a second immunization with enriched toxins or toxic components

which are bound on high molecular carriers thus showing a better immuno-

gcnicity (21).

May be all these results are valuable for the production of potent antisera,

for which, as you have seen, is advisable to use selected toxins with an especially

high content of toxins.

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O. ZWISLER

285

Keferences

1 .

2 .

6 .

8 .

10 .

11 .

12 .

13.

14.

15.

16.

17.

18.

19.

20 .

21 .

Houssay, B. A. — C. R. Hebd. Soc. Biol., 105:308, 1930.

Kellaway, C. H. — Ann. Rev. Biochem., 8:541, 1939.

Essex, H. E. — Phys. Rev., 25:148, 1945.

Porges, N. — Science, 117:47, 1953.

Slotta, K. H. —• Experientia, 9:81, 1953.

Boquet, P. — Toxicon, 2:5, 1964.

Christensen, P. A. — South African Snuke Venoms and Antivenoms, Johan-

nesburg, 1955.

Dickgiesser, F. & Zwisler, O. — Behringwerk- Mitt., 43:279, 1964.

Kaiser, E. & Michl, H. — Die Biochemie der tierischen Gifte. Franz Deuticke

Verlag, Wien, 1958.

Zwisler, O. — Zschr. Immunforsch., 129:444, 1965.

Master, R. W. P. & Rao, S. S. — Biochem. Biophys. Acta, 71:416, 1963.

Neumann, W. P. & Habermann, E. — Biochem. Zschr., 327:170, 1955.

Klibansky, C., Condrea, E. & de Vries, A. — Am. J. Physiol., 203:114, 1962.

Brewster, H. B. & Gennaro jr, J. F. — Toxicon, 1:123, 1963.

Russell, F. E„ Buess, F. W. & Woo, M. Y. — Toxicon, 1:99, 1963.

Uriel, J. — Immunoelectrophoretic Analysis. Amsterdam, ed. Grabar, P. &

Burtin, P., p. 30.

Zwisler, O. — Experientia (in press).

Summer, J. B. — Erg. der Enzymforsch., 6:201, 1937.

Kochwa, S., Gitter, S., Strauss, A., Moroz-Perlmutter, C. & de Vries, A. —

Fifth Int. Meeting of Biol. Standardization, Weizmann Science Press, 1959,

p. 483.

Kochwa, S., Izard, Y., Boquet, P. & Gitter, S. — Ann. Inst. Pasteur, 97:370,

1959.

Moroz, C., Goldblum, N. & de Vries, A. — Nature, 200:697, 1963.

Discussio.n

A. do Amaral: “Just to compliment him for his splendid paper wherewith he

just delighted us, as specialists in this quite attractive field of investigation.”

1 .

2 .

6 .

8 .

10 .

11 .

12 .

13.

14.

15.

16.

17.

18.

19.

20 .

21 .

2 3

5 6

10 11 12 13 14 15

ml Antiserum

esterase. 7. Hyaluronidase.

ADJUVANS

dcl

ENZYME ACTIVITY OF dcl

Phospho-

Lecithinase A

Proteinase

cliesterase

Bajol/Arlacel Rabbit Nr. 1

> 24

> 18

> 24

> 18

> 12

> 9

> 12

> 9

> 8

> 6

Bentonit Rabbit Nr. 1

> 20

> 15

> is

> 16

> 12

> 16

> 12

> 9

> 12

> 9

> 8

> 6

Alton);, Rabbit Nr. 1

> 24

> 18

> 24

> 18

> 20

> 15

> 20

> 15

> 20

> 15

PAA Rabbit Nr. 1

> 24

> 18

> 24

> 18

> 20

> 15

> 4

Fig. 2 — Neutralization of toxicity and enzyme activity by antisera. Venom: Bitif,

(jabonxca. Antisera: rabbit. Neutralized by 1 ml antiserum.

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O. ZWISLER

287

Bitis nasicornis

Bit is gabonica

Cevastes vi per a

Echis carinatus

Vi pera berus

Vipera lebetina

Vipera aspis

Vipera ammodytes

Vipera russelii

Vipera xanthina

Fig. 3 — Polyacrylamide-electrophoresis of some VIPERIDAE venoms.

k i m i i

lÊm ■ MBâÊWÊÊ

Naja hoje

9 I ! t

1 1

I 4 I

Vipera ammodytes

10 | | 7 UI

5 I UI I 7 I

Echis carinatus

I W I I 4/8 I I 5 |

Fig. 4 — Localization of some venom components after PAA-gel-eleetrophoresis. 1. Acetyl-

cholinesterase. 2. Fibrinolysine. 3. 1-Aminoacidoxiclase. 4. Haemorrhagine. 5. Phospholipase A.

6. Phosphodiesterase. 7. 5’-Nucleotidase. 8. Neurotoxin. 9. Protease. 10. Esterase (Tame).

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10 11 12 13 14 15

2 3 4 5 6 SCÍELO 10 1X 12 13 14 15 16

Fig. 6 — Antiserum against Echis- toxin. A. Echis — Toxin 1%. B. Antiserum

against Echis — Toxin. C. Antiserum agains Echis carinatus from horse. D. Echis

carinatus 2%.

Fig. 7 — Antiserum against Crotactin. A. Crotaetin 1%. B. Antiserum against Crot. terr.

from horse. C. Crotalus terrificus 2%. D. Antiserum against Crotactin.

Fig. 8 — Incomplete inhibition of cholinesterase. Center well:

2% Naja haje. Outer vvells:Anti -Naja haje serum from horse.

Upper half: stained with indoxilacetate for acetylcholinesterase

activity. Lovver half: without staining.

i, | SciELO

pMo! Acetylcholin /h

Fig. 9 — Inhibition of Acetylcholinesterase by antiserum.

VENOM

Activity (Q,.j)

Preeipilation

by antiserum

(ml)

ACTIVITY IN

% Inhibition

Supernatant

(Qoj)

Precipitate

(Qo 2 )

Vipeva ammodytes

0.1 mg = 44

0.35

Vipeva uvsinii

0.12 mg = 44

0.37

Vivera lebetina

0.08 mg = 44

0.41

Bitis gabonica

0.21 mg = 44

0.45

Cevastes cevastes

0.09 mg = 44

0.85

Echis cavinatus

0.07 mg = 44

1.25

Fig. 10 — Inhibition of 1-Aminoacidoxidase by antiserum.

Serum agalnst

Species

1 ml antiserum neutralized

dcl

Enzyme activity oí dcl

Phosphodiesterase

Vipeva ammodytes

< 2

l-Aminoacidoxidase

Vipeva ammodytes

< 2

Fibrinolysin

Vipeva ammodytes

< 2

Acetylcholinesterase

Naja haje

< 2

—

Lecithinase A

Echis cavinatus

< 2

Fig. 11

Protection by antienzyme-sera.

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O. ZWISLER

291

VENOM DOSE

I.E. TUR.

HYALURONIDASE

MICE

TOTAL VOLUME Q2ml

DEAD

ALIVE

—

100

—

2°ç

30 t

—

30 t

4 °t

—

40f

40 í

50 t

—

50 r

60 i

—

6 °S

601

70 í

—

70j

70 f

» o t

—

Fig. 12

Snake venom and hyaluronidase.

Serum against

Species

Neutralized by 1 m] antiserum

cicl

Enzyme activity of dcl

Toxin

Crotalus terrificus

Echis carinatus

100

—

Fig. 13 — Protection by antitoxin-sera.

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AFRÁNIO DO AMARAI

293

34. VENOM AND ANT1VEN1N SPECIFICITY: MODEHN CONCEPT

AFRÂNIO DO AMARAL

( Brasil )

An expression of lhe changes our knowledge of hiological phenomena constant-

ly undergoes may he found in lhe gradual evolution of the notion of specificity

of venoms and antivenins since the beginning of the preseut century.

At the implanlalion slage of Serumtherapy, when Calmette (1894). liased on

tests and injections made vvith the first halehes of antivenin he had prepared to

counleract the effeels of the envenomation eaused hy “Cohra” [Naja naja), stated

lhe anti-neurotoxin was devoid of specificity, a serious generalization of lhe con-

cept tlius expressed hy a such a prominent pioneer of Science, commenced rapidly

spreading out. Fortunately, Calmette’s radical theory did not last long. As a

matter of fact, since 1897 and, more precisely, 1901, following Vital BraziFs

original demonstration in favour of lhe opposite view, a succession of experimental

studies was puhlished hy specialists from all over lhe world (McFarland, 1901;

Rogers and Lamh, 1906; Ishizaka, 1907; Noguchi, 1909; Arthus, 1911; Gomes,

1920; Houssay and Negrete, 1923; Amaral, 1921-27, besides other investigators

al a more recent period) showing specificity to be a normal phenomenon between

venoms and antivenins. The adoption and generalization of this coneeption, how-

ever, did not occur before successive tecbnical advancements were made in Bio-

Chemislry and Physico-Chemistry so as to dissipale many doubts and remove

serious obstacles researcbers had encountered in their way.

The existence of specificity in this field is no longer a matter of conlroversy.

Any restrietion still to be heard in this respect is rather a reaction to prove

lhe notion of specificity lo be broader lhan we were at first led to suppose.

Il is true we admit that this property. normally confined to the morpbological

range of any species of veneniferous animal, is aj>t eventually, but partly any-

how, to reach elosely allied fornis through the intervention of factors leading to

specialion.

Be as it may, such an extensiveness woidd in no case eross lhe boundaries

of lhe genus wherein those fornis happen lo have been placed. In thesis, such

a limitation does exist in view of the fact that the specificity principie implies

lhe solidary and uniform intervention of the entire succession of activities on the

pari of lhe numerous constituents of the molecnles forming every venom, mosl

of which have a protein nature.

The behaviour of such substances in regard lo lieat. diffusion, dialysis,

ebemical affinities and bio-immunologic reaclions is so consistent as lo have led

researcbers lo include them in lhe group of the “antitoxinogens” (Zinsser, 1923).

2 3

5 6

10 11 12 13 14 15

294

VENOM AND ANTIVENIN SPECIF1CITY: MODERN CONCEPT

hle lo connect venoni

te prescnce of certain

In lhe course of numerous investigations it was pos

nocuousness lowards lhe organisni of other animais with

toxins and especially of a large series of enzymes.

The number of enzymes found in venoms has lieen steadily inereasing as a

direel result of many improvements introdnced in lahoratory technic, which are

rendering their identificalion possihle.

The very number of loxins lhat have been recognized in a few venoms. sueb

as "crotoxin" connected with lhe South American ralllesnake (Slolla et al,, 1938)

has inereased ihrough furlher researches (Gonçalves, 1950; Neumann and Haher-

mann, 1955), “crotamin” (1) and “crotaetin” deserving special menlion as new

principies.

As a maller of fact, variation in lhe composition of lhat crotalic venom was

foreseen some forty years ago (Amaral, 1925-6) (2), when lhe presente of yellow

pigment was consistently nolicetl in lhat excrelion so as to characterize lhe ratlle-

snake population found in North-Eastern Brazil.

Furlher knowledge of lhe Chemical eonstitution of lhat venom was enhanced

hy lhe application of improved and more sensitive technical processes such as

electrophoresis (Slolla, 1958; Gonçalves, 1950), chromalography and douhle miero-

diffnsion (Schenherg, 1959-68). Under the stimulus received from such findings,

lhe charaeler ization of olher venoms was also atlcmpted with a view lo identifying

the nature of their chemical constituents, the constancy of which, connected with

genetic factors, is used as a rneans for telling apart specimens representing even

suh-racial forms.

As regards lhe presence of immunologic variants already traced in some

venoms (lhat of li. neuiviedii, for instance), their possihle connexion with racial

differences is a question under investigation (Schenherg, 1963).

Jn view of the existence of variations in the eonlent of lhe venom from

different specimens of the same morphologic speeies, even lhe composition of

mixtures or hatches (prepared either at different periods or al only one occasion)

of several samples of a definite venom even though il he secured from numerous

specimens all from lhe known speeies range rnay not always he lhe same (Schoct-

tler, 19511. This variahilily is likely lo assume a clinal or ecologic charaeler

related It) the ‘"inche” where those specimens have heen captured (Amaral, 1956).

In the light of these observations il is easy lo admit lhe existence of "hiological

races” in such speeies.

A good exarnple of lhat occurrence we poinled out (Amaral, 1956. WHO/

B5/373) while comparatively examining Iwo populations of li. jararaca, one from

Cruz Machado (Paraná), the other from Titnhó (Santa Catarina), separaled

from each other hy the Iguaçu river. Morphologically, they were undistinguishahle.

Pharmacologically, allhough lhe venom from specimens really from C. Machado

showed a toxicity (MLD) comparahle lo that from specimens really

following

lalter venom

dermically (slow,

not to advise the use, either in litration

venins, of intravenous injections, since

intra-venous inocula liou

was ahout 50%

enzymic

jrenc

ral

than

This

more deadly

effects).

of venoms

this is apt

the

from Timbó

effects), lhe

given hypo-

(rapid hlood-clolling and loxinic

former when

s one of lhe reasons for us

or in standardization of anti-

to provoke the primary in-

tervention of substances involved in lhe hlood elollinc meehanism.

(1) Acting like “apamln”, the bee-venom baslc polypeptid.

(2) Rev. Mus. Paulista, 15:91; Hull. Antivenin Inst. uf America, 192S, 8:6.

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AFRÂNIO DO AMARAL

295

Moreover. lhe neurolropic venoms (lypically toxinifcrons), when kt*|>l uiuler

orclinary, uneontrolled, conditions, are likcly lo keep lheir aclivity for a long

lime. whilsl lhe cytotropic ones (lypically enzymophorous) gradnally lose activily.

This inactivation as measured ihrough toxicologic lests may reach 60 to 80%

of the original figure.

In the lighl of lhese facts lhe conception of homogeneity and stahility of

the general composition of venoms (at leasl those imder scrutiny) is no longer

tenahle.

Specificity reflects a eombinalion al definite proportions of a series of prin¬

cipies in any venom; its derangement vvill follow any handling or Ireatment llial

may he partly or totally destruetive to sueh hiochemieal principies hy enhancing

splitting or cleavage of lheir hasic constituenls and hy altering their halanee

and normal ratio.

Although most of sueh principies are known to act as “antitoxinogens” a

fevv of them (ineluding mucin, an impurily mixing witli lliem through the mueh

used and ahused process of oversqueezing the snake glands) appear to be non-

antigenic. This might explain why, in certain cases, not all of lhe active prin¬

cipies are nculralized even hy an otherwise potent uni-specifie anlivenin. More¬

over, even uni-specific antivenins, vvhen given late in the course of ophiotoxicoses,

are likcly not lo neutralize those new noxious sidislances residting from the inter-

action of preformed, normal constituents of the venom vvith the victinTs lissue

and hlood proteins.

In their present general connotation, zootoxicoses represent chain-reactions

iniliated hy proteinases and intensified hy the intervention of other enzymie

snhstances (hesides specific toxins) successively acting on different tissues as well

as on the very products issuing from cell lysis (Amaral, 1959) (3).

Due to lhe variahle composition of the venom from specimens representing

a definite morphologie species hut proceeding from different clines or “niches”

the preparation of an uni-specific antivenin for exclusive use in the correspond-

ing area appears lo deserve consideration. However, this ideal solution, no matter

how justifiahle it may he from a scicntific standpoint, really is economically

unsound. Indeed. it would involve the necessity of multiplying heyond accepl-

ahle limits lhe collecting and preserving in a separate Container every individual

venom (this without mentioning lhe influence of possihle seasonal variations in

venom composition) to he employed in immunizing any group of animais for

serum production.

In view of the economie contra-indication lo the rontine use of sueh a

solution, one might resort lo the following expediency:

a) seeuring, preferahly through electrie stimulation, lhe venom from specimens

proceeding from the greatest numhcr of localities lying within lhe recognized

range of lhe corresponding species;

b) |ireparing antivenins for the greatest number of species (races and sub-

races) within a certain genus so as lo possibly cover all the types and

subtypes of toxic and antigenic representative principies of that group (multi-

specific hut uni-generic antivenins);

(3) Ciência e Cultura, 11:176.

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296

VENOM AND ANTIVENIN SPECIFICITY. MODERN CONCEPT

<•) lilrating, biometrically, cvery batch of antivenin against either lho venoms

employed in immunizing the animais or al least lhat in the molecule of

which lhe grealesl numher of active principies common to thal group mav

be found.

Due to the eomplexity of Iheir coniposilion, vcnoms seem lo stimulate, at

variable degrees, the production of neutralizing suhstances: anti-neuroloxic, anti-

cytotoxic or anti-enzymic. In certain venoms, especially in lhe highly enzyino-

phorous group. the rate of inert, non-antigenic, suhstances appears to be highcr

than in the toxiniferous group. This may explain why il happens quite often

for the anlivenins produced even through an advanced irnmunizalion procedure

not to be so potenl as the usual bacterio-antiloxins. In South America, the auti-

cytotoxie poleney of antivenins happens to be ponderally higher than lhe anli-

neurotoxic potency. As a matter of fact, among venoms used in immunization

lhe neurotoxic ones have a greater activity, as expressed in MLDs, than the

cytotoxic.

For litrating antivenins the best proeess — although not yet the ideal nor

equally efficient for every case — among those thus far devised and developped

implies the biomelric calculation of the results secured on wliite miee, all lioino-

zygotie young males of the same weight, serially injected subcutaneously with

decreasing dilutions of antivenin mixed with a fixed dose of the corresponding

venom (XMLDs), this antigen being lyophilized and preserved away fmm light

and humidity (Amaral and Schoettler, 1956, WHO/BS/364).

Brazilian contribution — The. Brazilian contribution lo the advancement

of this branch of Science lias a rather long and uneven evolution. At first, it

coineided with the very growth of this Instituto, which happened to become one

of lhe world centres devoted to the sludy of zootoxicoses thanks to V. BraziPs

liioneering activity. This fact notwithstanding, the present slage of our know-

ledge has not resulted either from a rapid |>rogress or from unforseen discoveries.

On the contrary, it has come about through several, successive and disconlinuous

attempts at abaling lhe surrounding gloom until a few rays of light would appear

lhat started clarifying this highly tangled field of investigation.

We are just beginning to unhide a few of the curious meehanisms. which.

by intervening with the development of lhe most complex phenomena connected

with the physiopathologic activities of venoms, have for so long a time kept

among the N atura e arcana (the 1'hyseos mysteria of Aristolelianism) the real

meaning of those toxic seeretions in lheir multiplc effects on the human and

animal tissues.

Our modesl role and meager share in those developments had incipience, firsl-

Iy while we had the Iuck to be one of V. BraziPs collaborators at Butantan (as

a matter of fact, we represent the only survivor of lhat group still to be intercsted

in zootoxicoses); secondly, as the result of a mere accident, after we were promoted

lo head lhe Butantan I.aboratory of Medicai Zoology, previous lo our being com-

missioned in this Institute’s directorship as one of V. BraziPs first sueeessors. In

lhat double capacity have we been both an instrument and a witness in the

amazing, gradual unveiling of lhe secrets surrounding the phenomena of venoms.

Although quite long, lhe history of our “prise de contact” with prohlems

related to venom and toxin titration, is not sufficiently known. For this reason

we feel lhat, at the last quarter of our life of scientific researeher, we ought al

least lo louch upon the series of the most impressive experiences we had while

dealing with such questions. And so, il mighl be pertinent for us to do it al

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thc very moment so large a group of exponential figures in the fiold of zoo-

toxicoses is altending lliis International Symposium. This will also give us an

opportunity to explain to all of you, particularly lhose who have shown interesl

in learning lhe scientific evolution of Butantan particularly for lhe last 40 years

or so (which were complicated by the worlds greatesl economic depression and

political instabilily leading lo lhe recenl international conflict), lhe changes lhat

have taken place here, lo wil:

By lhe middle of 1919, when V. Brazil decided lo retire from lhe position

hc so much elevated in exeellence at this Institute, we were engaged in an

investigation, on the mosquitoes of São Paulo, at lhe Parasilological Laboratory

under J. Florêncio Gomes. Much to our misfortune, Gomes was then caught hy

the epidemic influenza called “espanhola” and died from il. We were thus

compelled, all of a sudden, to assume the heavy duties of chief of Medicai

Zoology, this being Butantan s main Section.

1 — As though Ibis responsability were no! suffie.ient lo challenge our youlh s

energy, one more, out of the six laboratories existing at Butantan al that

remote period, that devoted to Tetanus Serumtherapy had also he assigned

lo us, as one more contribution to avoid Butantan’s work from collapsing.

There, while using the process of Anderson and Kosenau for lesling

tetanus loxin on guinea-pigs reared al lhe Instilule Breeding Slalion, we

came aeross an extraordinary case of individual variation as disclosed by

their ca])acity to reacl against the established MLD of that antigen.

2 — In lhe 1919-21 period we found thc H. jararaca venom, as extracted from

several specimens, purified hy centrifugation and intravenously injected (ac-

cording lo the technic used here) into adult pigeons (350 g) (of the race

Columba livia domestica) not te show consistent toxieily, its MLD varying

sometimes between 0.016 and 0.025 mg.

3 — In that period we also verified that the venom of H. insularis, a Crotalid

we described from Queimada Grande Island, where il lives on trees and

feeds on small hirds, was much more toxic lo the pigeon than the venom

of the homologous “jararaca” of the mainland, living on the ground and

feeding generally on rodents(4).

4 — In 1921, we showed that the experimental intoxicalion caused hy the Texas

rattler (C. atrox) venom, although yielding much more markedly to the

injection of the speeific antivenin (that we had jusl prepared by using a

batcli of venom sent by our good collaboralor, B. Ditmars, of the Bronx

Zoo), also favourably reacted to the injection of lhe antivenin speeific for

the S. A. rattler still called terrificus. The cross-tests as performed intra¬

venously into pigeons showed lhe following striking differences in toxieily:

A) Venoms: atrox MLD .... 0.200 mg (in weight)

terrificus MLD . 0.001 mg (in weight)

B) Sera: anti -atrox titre ... 15 MLD (3,000 mg in weight) of atrox venom

23 MLD (0.023 mg in weight) of terrificus venom

anü-lerrificus titre 1.000 MLD (1,000 mg in weight) of terrificus venom

6 MLD (1,200 mg in weight) of atrox venom (5)

(4) Cot. Trab. Inst. Butantan, 1920, 2:53; Anex. Mem. Inst. Butantan, Ofiologia, 1921, I:

44, 88.

(5) Cot. T.ab, Inst. Butantan, 1921, 2:171.

2 3

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VENOM AND ANTIVENIN SPECIFICITY: MODKRN CONCEPT

These observations, which date- from ahout 45 years ago, were all liornc in

our mind and, if nol published, registered in our note-books for further invesliga-

tions whenever we could find the means for correctly performing them and suh-

jecting their results to scientific analysis. No matler how faithfully wc had ac-

complished our studenEs duties at Collegc and the Medicai School, we could not

satisfactorily explain the amazing facts we have just tried briefly (not lo tire

Ihis audience) to point out, in the lighl of the knowledge of hiological phenomena

prevailing al least in our midsl at lhat really remote period of our aelivity.

Faced wilh the impossibility of finding the reason for such differences and

variations, and led hy lhe desire to penetrate lhe secrets underlying lhose in-

Iricacies, we decided to lake advantage of the traveling príze we had received

from the Medicai School in order lhat we could lake post-graduate eourses at

lhe outslanding University centres and research lahoratories hoth in North America

and Europe, our longer stay ahroad heing facilitated hy a special fellowship we

received from the International Health Board. We had thus a chance carefully

lo study different subjects eonneeled with th is Institutes work line and especially

lo enlighlen our mind concerning hio-tests and litrations.

Through the personal contact we sueceeded in eslahlishing with a few of

lhe great scientists of lhat time, nol only (and partieularly) at Harvard l niversity

hui at other American and European inslilutions, wc Iearncd lhe ways and lechnic

of investigation as followed hy such prominent men as Th. Barbour (Comp.

Zoology) — wilh whom we enjoyed the privilege of collaborating for several

years — and C. Parker (Comp. Physiology) — in Camhridge; W. Cannon, C.

Drinker, W. Porter and A. Redfield (Exp. Physiology), W. T. Ilichards and

E. Bovie (Physico-Chemistry), E. J. Cohn (Physiol. Chemistry), H. E. Hendcr-

?on (Blood Phys.-Chemistry), E. W. Wilson (Bio-Mathematics) , R. Pearl (Bio-

Statistics), W. Castle (Gcnetics), H. Zinsser (Immunology), M. Rosenau (Sero-

logy), E. E. Tyzzer (Comp. Palhology), R. Strong (Trop. Medicine) — all in

Boston; J. Macleod and F. Banling (Bio-Titralion:insulin) in Toronto; E. V.

McColIum (Nulritional Chemistry I — in Baltimore; Wm. Park (Serumlherapy)

in New York; J. Kolmer (Lah. Testing) — Philadelphia and E. C. Kendall

(Hormonal Biochemistry) in Rochester, our experience in Europe having

covered the Eister Instilute (London), lnst. Pasteur (Paris), St. Serum Inst.

(Copenhagen), Inst. f. Sehiffs-u. Tropenhygiene (Hamhurg) and Institulo Siero-

lerapico (Milano). During our second sojourn ahroad and hefore returning

liornc we talked with some of lhose teachers and investigators over the main

prohlems hoth of technic and personnel we had lo face al Butantan. In

Europe, we also secured advise as lo lhe selection of a group of specialists we

wanted to invile lo come lo São Paulo as collahoralors in our plan of expanding

ihis Institule so as to transform it into a centre of Experimental Medicine especial¬

izei! in Human Palhology. This seemed. indeed, to he the logical, lhe necessary

step for us to take, in the development of the ideal that led V. Brazil to found

this institution. We thus atlemptcd lo catch up with the never so mohile trend

scientific investigation was already revealing al lhat lime.

That plan as presented lo and approved hy our Government following their

invitation for us to return and modernize Butantan, called for lhe organization,

lo starl hy 1951, of whole new Departments to deal, respectively, wilh Experi¬

mental Gcnetics (and Cyto-Emhryology) , Bio-Chcmistry (and Pharmacology) ,

Physico-Chemistry, Immunology (and Serum-Therapy), Vinis (and Virus-The-

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rapy I, Physio-Palhology (vvilh Physiology, Endocrinology and Histo-Palhology),

Parasitology (with Enlomology) and Medicai Botany (with Pharmacognosia), most

of which were then a novelty in our “milieu”.

Needless lo say lha! thc dynamics of lhat transformation implied the real

integration of research ihrough a close cooperation of the numerous scientists

involved therein, willi lhe hope lliat it rnighl set an example in an environmenl

sucli as onrs, so vvell knovvn for ils individualism. A sketch of lhat plan may

he found in Mem. Iiist. Butantan, VI. 1931 et X. 1935 in their “Noticiário"

section.

Concerniug venoms and antivenins: we immediately decided lo synchronize

the (iction o) the rietv group o) scientijic collaborators and lo tuke advantage oj

the modern laboratory equipment we were installing at Butantan. in order to

tackle lhe fundamental prohlem of properly analysing the chemical eomposition

of venoms with a view lo purifying them in such a way as to make it possihle

for the exact properties of their active constituents lo he pharmacologically de-

termined. Moreover: the dream we slarted lo cherish at Harvard as early as

1921 calhai for the eventual synthetic produetion of pure principies to he applied

as antitoxinogens instead of whole, crude, venoms, so lhat lhe preparalion of

really specific antivenins couhl leave lhe empiric stage in which it lay for so

long a lime, and follow a really scientific direction, thus also opening the way

for the establishment of a rational procedure in venom titration and antivenin

slandardization.

Besides several promising colleagues such as Lemos Monteiro, J. Travassos

and Vallejo-Freire (Vinis), Flavio da Fonseca and Paulo Artigas (Parasitology

and Enlomology), J. K. Valle and B. F. Mello (Physiology and Endocrinology)

and olliers, lhe following specialists carne lo work here: G. v. Ubisch (Prof.

Heidelherg Univ.), in Genetics; K. H. Slolta (Prof. Breslau Univ.) and his as-

sistants H. F.-Conrat and G. Szyska, hesides Kl. Neisser (Berlin Univ., assistant

to Nohel Prize Prof. A. Windaus), in Bio-Chemistry; D. v. Klobusitzky (Frank¬

furt Univ. Prof. W. Paulfs pupil) and P. Koenig (Wien Univ.), in Physico-

Chemistry; Prof. Thales Martins (Insl. Oswaldo Cruz, Rio) and M. F. Amorim

(São Paulo Med. Facully), in Physio-Pathology; lhe greal Prof. Pirajá da Silva

(Bahia Med. Facully, retired), in Medicai Botany, hesides the famous Prof.

Ludwig Fraenkel (Breslau Univ.), who joined us as a volunteer at the Endo-

crinological Laboratory; the dynamic Werner Schoettler (who as a student at

Berlin Univ. had already heen one of our collaborators through the Antivenin

Instituto of America); and, at a later date. the cautious Saul Schenberg, who.

like. W. Schoettler, hecame engaged in pharmacological experimentation.

Resui.TS Among lhe many faels lhat were brought to light at Butantan

at lhat period. besides many olliers but unrelated to lhe ohject of lhis Symposium.

the following seem to deserve special menlion at Ihis time:

a) At the Genetie Dept., v. Ubisch showed the guinea-pigs reared at our Breed-

ing Stalion not to represent a homozygotic colony but to descend from an

extensive and long-standing hybridizalion between Cavia /xtrcellus and C.

rufescens , hence lheir variable response to toxin and venom.

bl Our local pigeon is aboul twice as susceplible to lí. jararaca slabilized venom

as ils IN. American homologous, in the light of comparativo experiments we

2 3

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VENOM AND ANTIVENIN SPECIFICITY: MODERN CONCEPT

macle at Butantan Ophiologie Dept., at Harvard Univ. while teaching lliere

and at the Antivenin Institute of America in its organization period.

c) Al the Physico-Chemica) Dept., “bothropotoxin” as a Idood coagulant was

prepared from II. jararaca venom by v. Klobusitsky and Koenig.

d) Al the Bio-Chemical Dept., Slotta and eolls., started the isolalion of the neuro-

toxie principie from terríficas crotalie venom, lhe substance still bound lo

phospholipase A having been called “crotoxin” and “crotaclin” (N. et H.),

when separated from il. This pioneering |>iece of work lias enhanced further

investigalors in lirazil and ahroad (M. Gonçalves, Ribeirão Prêto Med.

School, 1950; A. Barrio, Inst. Malbran, 1954; S. Schenberg, 1959, O. V.

Brazil et al.) lo develop the analysis of lhal substance and recognize “cro-

tamin” also as an active principie in that venom.

e) Al lhe Ophiological Dept., an extensive study of the Brazilian and the Neo-

tropie serpents was made, Iwo general Cheek-Lists having appeared pre-

paralory lo the puhlieation of our “Iconographic Catalog of the Serpents

of Brazil” willi coloured plates and Portuguese and English texts (in jiress).

In the 1929-1937 period, our official journal “Memórias do Instituto Butan¬

tan” was issued quite regularly, numerous studies having been published in

its volumes IV lo XI (8) as original contributions from some Departments

of this Institution. Vol. IV of our “Memórias” eovered 2,764 pages, all

laken up by the afore-mentioned Cheek-Lists besides many olher articles

prepared by the Director of this Institute and Chief of its main Ophiological

Department. Previous lo lhal period, a preliminary study as based on lhe

examination we earried oul from 1920 to 1924 of lhe differential characters

of over 6.000 specimens (mostly living ones) of the main species of Neotropie

pit-vipers of the genus Hothrops, whieh had been misidentified by the

great G. A. Boulenger (in Cal. Sn. Bril. Mus. IV. 1896), was published as

Xo. 2 Contribution from the Harvard Institute for Tropical Biology and

Medicine, 1925. That revision pointed oul many misleading differenees par-

ticularly connected with lhe ontogenetic evolution and frequent individual

varialions in lhe chromatic characters of those pit-vipers. And as a comple-

ment to sueh a work we published an artiele (in Amer. .1. Trop. Med., IV.

5 1921) on lhe differentiation of II. atrox. />. jararaca and II. jararacassu

venoms by lheir M. L. I)., coagulability l>y heal, ])roteolylie, hemolylie and

hemoeoagulant aelivitics. venom-anlivenin cross-neutralization and serum-pre-

cipitin lests.

f) Al the Ophiological and Physio-Palhological Depts., lhe method of treating

human ophioloxicoses was sernlinized and. following improvement, lias ol

late been inlroduced into the routine work at the Butantan Infirmary.

In this connexion we may say that our firsl personal contribution lo-

wards rationalizing specifie therapeuties of envenomation consisted in the

establishment of the following fundamental principie that had thus far been over-

looked: the dose of any antivenin lo be given lo a patient (eilher human or

animal) must be inversely proporlional lo bis hody weight, sinee the lighler the

victim proporlionately the greater the eoneentration of the venom in his tissues

(Amaral in Hall. Antivenin Inst. America. 1:77, 80, 1927, et Mem. Inst.

Butantan, 5:223, 1930).

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AFRÂNIO DO AMARAL

301

g) Through the joint work of the Ophiological and Pharmacological Laboratories,

an analysis of the most rational process for testing venoms and titrating anti-

venins lias been under way for many years although not with the desirable

continuity.

As explained in a special report we prepared in collaboration with W. Schoet-

tler for publication through the WHO (BS/364/1956) and now brought up to

date, in lhat work a few preliminary precaulions must he taken so as to eliminate

many causes of error and reduce variants lo an acceptable minimum, to wit:

A) Concerning the snake:

a) Speciméns must proceed from or be secured in definitely identified

places;

h) When taken into the lahoratory, every specimen must he kept undis-

turbed and under constant environmental conditions, in a separate cage

(with number and fnll data on tag), to be properly fed and cared for

(WHO/BS/373/1956).

I>l Regarding the venom:

a I Forceful extraction must be avoided to preveni mixing lhe proper ex-

cretion with other gland constituents and mucus;

I>) For tilration and standardization purposes, it is advisable to secure the

venom hy means of the electrically induced hite through a special ruh-

her membrane so as to avoid injuring the snakes teeth and exceeding

lhe norma] limits of pressure set hy the natural contraction of the

muscles involved in lhe hite mechanism;

e) The venom, ejected into a lahoratory glass, must he immediately and

successfully centrifuged and dehydrated, either from the liquid or from

the frozen State, under vacuum at either room or lower temperature,

or through Stokes’ lyophilizer at 0.1 mm llg;

d) As to slorage, apparently keeping in neutral-glass tubes filled with N

at athmospheric pressure and kept in dark at — 10"C is a safe way to

warrant preserving every active principie in a venom. For obvious

reasons, the desirable establishment of any “reference preparation” must

he hased on a rationally extracted and properly preserved venom.

C) Respecling venom testing:

Among the technics llitis far devised for tliis purpose there seems to deserve

preference, for reasons hoth economic and biologic, the probit method through

hypodermic injeclions into standard homozygotic mice, every precaution he-

ing taken to warrant a correct statistical computation of results and all

tangible causes of error heing avoided, inclusive sucli shortcomings as:

age/weight varialions; sex differences (pregnanl female mice usually heing

more resistant tlian lhe inale) ; greater or lesser local trauma, with loss of

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VENOM AND ANTIVENIN SPECIFICITY: MODERN CONCEPT

plasma as licld up in lhe oedema area (resulting partieularly írom injection

of enzymophorous venoms), responsible for lhe dchydration followed hy

collapse. of some of the lesl animais; onvironmental physieal farlors prevail-

ing at lhe hreeding quarters, etc.

Dl As lo antivenin tilralion:

Preliminary, the following facts musl he horne in rnind; a) The MLD of a

venom lype per unit of weight varies from species (perhaps also from raee

lo raee) of animal; the relalive poteneies of antivenins, for reasons imlil

unknown and ealling for further investigation, may show variations when

assayed on different species of lahoratory animal; different animais shov\

difíerent suseeptihility lo <lifferent venom types. h) The ahsolute and re-

lative rcsistance of rnan lo venoms is unknown. e) The inlimate mechanism

of dealh of man and even lahoratory animais in every lype of ophiotoxieosis

is slill a matter of speenlation and so is ils possible and complete inhihition

hy antivenin.

Hiímahks In view of so many fundamental and unknown faetors heing

involved in lhe mechanism of dealh eaused hy the numerous lypes of venom. it

seems neeessary to extern! our comparative serutiny to larger animais such as

dogs and monkeys as an approach to our learning the ways man reacts to venoms

and antivenins. We feel that, at the present stage of our knowledge, even the

raethod (whieh we eonsider reasonahle) hased on the delermination of the relalive

poleney of an antivenin hy assaying various amounts of serum against a fixed

dose of venom would he generally aeeepted only when ils effieieney should pass

a lesl performed on larger animais. In this connexion we might mention that,

while organizing the Antivenin Instituto in lhe U.S.A. some 10 years ago. we

decided to resort to Rliesus monkeys on whieh to lesl the aetivily of the firsl

hatehes of lhe Nearelie antivenin we had prepared. in order lo give satisfaetion

lo the offieers of the Hygienic Lahoratory. in Washington, as to lhe iherapeutie

effieaey of that produel. Coneerning dogs and monkeys as testing animais, one

important aspeet that seems to have been overlooked is that, in order for the

rnosl tangihle varianls to he removed, il would he neeessary for lhose animais

also lo he reared at Hreeding Stalions so as to warranl the formation and main-

tenance of homozygotie stoeks.

Coxci.UDiNG itiNTs — The reorganization of Butantan always as a State

inslilution. having eommeneed in 1931, was near eompletion hy 1937. having

thus passed a rnosl diffieidl period following lhe 1930 Revolution and opening

lhe era of sueeessive politieal crises, social inirest and eeonomie distress Brazil

starled lo experienee. Mueh to our regrei and shame, at lhe very moment we

were heginning lo get the fruit of our reorganizational plan, lhere eame the

well-knovvn "eoup d Élat” of 1937, responsihle for lhe improvised statization of

this eountry. As happened in Europe all through traditional eenlers oí eidlure

such as Germany, Italy and other eountries, in Brazil the new regime rnade havoe

and preferred negalive seleelion as the ride for filling posilions of responsahilily.

Instituto Butantan as a State organizalion was profoundly affeeted: many of

lhe mernhers of ils seienlifie staff were displaeed. Whilst some of lhem luekly

were engaged hy local Biologieal Laboratories and olhers were made teaehers al

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AFRÁNIO DO AMARAL

303

Medicâl Schools, a few had to leave Brazil Lo continue serving Science. Amongst

these we may mention G. v. Ubisch who went lo work in Genetics at Leyden

University; K. H. Slotta, who, as the head of the Biochemical Dept. of lhe

Miami University Medicai School, immediately look up chemical analysis of the

ative constituents of hlood involved in coagulation; and H. F.-Conrat, who joined,

at lhe University of Califórnia, the group under Prof. W. M. Stanley, wilh vvhom

he soon succeeded in separating and re-synthetizing the active molecule of a

virus of vegetahle mosaic; not to mention Prof. L. Conrat, who left for Uruguay

where he continued cooperating towards lhe progress of Medicai Science.

thology

and

more rccent years, scientific research was taken up again on Physio-pa-

hy G. Rosenfeld and his assistants, on Bio-chemistry hy S. B. Henriques

his group, as well as on Cyto-genetics, first under the leadership of Prof. G.

Schreiher with collaboration from H. Belluomini and others, and lately hy W.

Beçak and assistants, on Arachnidology hy W. Bücherl and on Ophiology hy

A. K. Hoge.

Fifteen years had not yet elapsed when, in view of the Brazilian recons-

titutionalization, we made a new attempt at modernizing Butantan (cf. “Noticiá¬

rio” in Mem.. Inst. Butantan, XXVI, 1954). In order that it might he freed

from strange influences used lo causing periodic slowing crises at Butantan, we

tried to have il changed inlo an autarehie organization. I nfortunatelv, Brazil,

slill as an

opinion apt

lo he ready

lo keep Ihis

rendered to

heing held.

underdevelopped country in want of an organized, watchful public

to uphold any gensible plan of scientific investigalion. proved not

yet to take Science seriously or cven to comprehend our programme

institution true lo the sprit that dictated its foundation, as an homage

its first director, in whose honour ihis International Symposium is

Anyhow, Butantan scientific structure has suffered so dee|)ly, that,

our decision to retire from official duties hcre, after having honestly

following

and faithfully tried to serve Brazil for 50 years, and confine our attention to

the international organizations I International Commission on Zoological Nomen-

clature, in London, and World Health Organization, in Geneva) wherewith we

have long hecn connected, our successor, Dr. A. Vallejo-Freire, decided to devote

his energies to lhe extreme ("heróico” as it is called in Portuguese) plan of

estahlishing here a foundation whereby research might recover vitalily. This is

our earnest hopc, which we feel is shared hy you all. who know the progress of

hurnan knowledge lo lie on investigalion.

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83(1) :3ü5-32(j, 1906

P. A. CIIRISTENSEN

305

35. VENOM AND ANTIVENOM POTENCY ESTIMATION

P. A. CHRISTENSEN

The South African Institute for Medicai Research, Johannesburg, South África

lt is common knowledge lliat venoms contain a wealth of enzymes and otlier

biologically active substances, and that their potency in terms of such substances

may be ascertained by measuring llieir effect on suitable substrates. Mos!, if nol

all, the active venom components are antigenic and an antivenom’s potency could

refer lo its ability to inhibit the effect of any such substance, lnit in íbis discus-

sion of potency measurement of venoms and antivenoms, I intend to confine my-

self to the measurement of lhe lethal effect of venoms and the life-saving properties

of antivenoms as assayed in animais. Furthermore, having very limited experience

with venoms of scorpions and spiders which constitute a minor problem in Southern

África I will be basing my arguments on snake venoms and their antivenoms and

draw my examples from work with African venoms.

For the determination of venom potency we are restricted lo observing either

the proportion of animais dying from suitably chosen doses,

of animais, given larger graded doses of venom.

or lhe time to death

for a venom would

rabbit, guinea-pig,

If one used lhe quantal res|>onse method, i.e. observed the percenlage death

caused by graded doses of venom. the magnitude of the median lethal dose (LD50)

course depend on which animal one cmployed for lhe test,

mouse or pigeon, lo mention some of lhe more commonly

also the order of toxieily of different venoms could not be

same irrespective of which animal was used because lhe sus-

different venoms may vary from one kind of animal to another.

the order of toxieily for a series of different venoms lested in one

usei

animais, and

expected to be the

ceptibility to

Furthermore, tne orcter 01 toxicny ior a series 01 ctinerent venoms

kind of animal may depend on the roulc of injection. As an example one could

mention that different li o t h r o p s venoms examined by Scbottler here at Institute

Butantan could be placed in one order of toxieity when lhe mice were injected

intravenously and in a different order when the test was carried out subcutane-

ously. This, as he pointed out. would indicate that lhe main lethal faetor differed

for the two routes of injection (Schottler, 1955/6, 1958).

In this respect, African venoms beliave in a more regular fashion. Table 1

shows the intravenous and subeutaneous LD50 delermined in mice weighing from

16 to 18 g for lhe venoms of the important African snakes. The values for some

Naja species from the Asian mainland and lhe Philippines which show a cerlain

relationship to African Naja venom have been included for comparison.

These particular values refer lo venom samples set aside as laboralory re-

ference preparations and other samples may give slightly different values but the

order of potency for different venoms is quite typical. Il is as lhe table 1

shows elosely the same for the two routes of injection which gives no reason to

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306

VENOM AND ANTIVENOM POTENCY ESTIM ATION

ihink that different eomponenls are active in the two ways of testing. The ver)

slighl tlifferenee hetween the intravenous and subculaneous LD50 for lhe elapid

venoms is presumably explained hy a small rnolecular size and rapid ahsorption

frorn the suhcutis whereas lhe larger loxins in viper venoms are surprisingly in-

effeetive subcutaneously.

Il is always interesting lo have an idea ahont lhe order of loxicily of venoms

from different snakes for differenl kinds of animais, hui nohody would allempt

lo compare lhe potency of two different venoms and expect lo gain more than

an impression of Ifieir relative loxicily. Venoms from different speeies are totally

dissimilar and one cannot ohtain a potency estimate which is valid in lhe accepted

sense of this lerm. Even lhe comparison of lhe loxieities of samples of venom

from lhe samc kind of snake may not lie a simple matler.

If two preparations eontaining one and the samc toxin in different concen-

Irations were compared one would expect to gel the same estimate of their relative

potency irrespective of the kind of animal used and the route of injection, as

long as the animal is susceplihle to the toxin and hoth preparations are tested

hy the same route, but this cannot be taken for granted in the case of venoms

which contain more than a single lethal toxin.

If a venom contains Iwo loxins, il could happen that one is lhe dominanl

toxin in one experimental animal, the other in another, and one toxin may be

particularly active intravenously, the other subcutaneously. The relative contenls

of the Iwo loxins may not be lhe same in two samples of the same venom in

which case the apparent relative loxicily will depend on the test animal and the

route of injection. Il is well known that such variations in venom eomposition

do occur; the most striking and extreme example is the crotamine-secreting and

non-secreling South American rattlesnakes demonstrated by Brazilian workers

(Gonçalves, 1956; Schenberg, 1959).

In order to increase the likelihood of comparable results it is lhereforc neees-

sary to define the exact technical details of the tests with regard to test animal,

and route of injection. Factors such as lhe injeeted volume, the speed of intra¬

venous injection, and lhe temperature at which the injeeted animais are kept

must be kept in mind as possible sources of variation. Working with mice and

African venoms, I have never observed signs of significant differences in the

siisceptibility of males and females, but Dossena (1950) in Switzerland elaimed

that female mice, rats and guinea-pigs were more resistanl than males to Naja

nivea venom and according to Schottler (1952) there is an indication that this

applies lo Crolalus and fí othrops venoms also, yet Henriques and her co-

workers (1959) noted later a higher LD50 for male mice. but slressed, as did

Schottler, lhat the results were inconclusive, aml it is probably fair lo conelude

that lhe sex of the mice used in venom work is of no great imporlance.

lhe results of loxicily tests carried out in mice nnder similar conditions in

different laboratories on identieal venom samples have occasionally given widely

discrepanl LD50 values indicating differences in the susceptibilily hetween slrains

of mice. This, in itself. need not mean that Iwo laboratories cannot arrive at

lhe same relative potency estimate for Iwo venom preparations, but il is jnsl

possible that the susceptibilily of mice lo lhe different loxins in a venom could

vary independently, which could lead lo complications, particularly in antivenom

potency assays but also in loxicily tests. if in some mouse slrains a minor toxin

took on lhe role of lhe dominant toxin, even if this may be unlikely.

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Whichever llii' lest animal and whatever lhe route of injeclion il is common

practice to compare tlie toxieilies of different venom preparalions in parallel Jine

assays hy converling lhe observed mortalities into probits which are linearly related

lo lhe logarithm of the venom dose, and I should like lo call atlention lo an

interesting suggestion made hy Sehdttler (1958) to overcome the difficidty lhal

showing partial survival separates

arises when only a single group of animal;

groups showing 0% and 100% mortality.

fail

per

and

for instance 10 mice are used

lo kill any mice, d :i kill some,

Schôlller suggests lhal lhe response

,5% because an expected mortality

when only 10 mice are tested, and

dose and the two lower doses, d 4 and d 2 ,

lhe higher doses d 4 and d-, kill all the 10 mice,

lo d 2 could be recorded not as 0% but as 2

of from 0% to 5% would residi in no deaths

similarly lhe response to d, could be recorded as 97.5%, the mean of lhe expected

mortalities 95 and 100%, which would residi in no survivors.

The magnitude of the LD50 depends on lhe effect of lhe dominant toxin

or on ils combined action with other components il they happen to have some

forrn of joinl action, but il gives no information about lhe toxicity of minor

loxins which cannot be determined except after their isolation although il some-

limes rnay be possible to gel a erude estimate of lhe order of toxicity in lerms

of whole venom from lhe residis of neutralization lesls with antivenoms and some

examples of th is will be presented later.

The higher the toxin dose the shorter lhe time lo dealh, and everybody

routinely examining lhe lethal effect of loxins will have some idea about the

amount injected from lhe lime il takes lo kill the animal. There is however,

no simple relationship between dose of venom and death-time. Jf the time to

dealh (T) is plotted in a graph against lhe dose (D) one gels a hyperbolic curve

wilh asymptotes determined hy a dose (d) which kills in unlimited time and a

death-lime (t) for an infinilely large dose. Many workers early in Ihis century

sought a general formula to express th is relationship and the English immunologisl

A. T. Glenny suggested in 1914 that lhe different Solutions were modifications

of a formula.

(D - d)" (T

= K,

here alpha and K are

conslanls characteristic for each toxin.

of lhe relationship between dose and

In order to rnako use of the relationship between dose and death-time for

lhe estimation of the relative potency of different preparations of a toxin, it

would be desirable to transform lhe doses or death-times, or both of these. in

such a way that they hecome linearly related.

Working with hacterial loxins. Ipsen 11941) found that lhe equation

(D/d - 1)“ (T/t - 1) = K

fitted his data betler lhan Glenny’s formula. The parameters in this equation,

d. t, «, and K, of which lhe three last are constants, may be determined graphically

(Ipsen, 1941; Christensen and Finney, 1953), and a function of the death-time.

Ipsen s logarithmic dose-time equwalent, j (T), is defined as

/(T) - log D - log d = log (Kl/(T - t)-J- + 1).

This logarithmie dose-time

>m dose over a fairly wide

equivalenl is linearly related

range of doses and has been

to the logarithm of

used successfully in

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VENOM AND ANTIVENOM POTENCY ESTIMATION

potency assays of venorns from África and Formosa (Christensen and Finney,

1953; Christensen, 1955; Lin, 1959).

Also a simpler function of lhe death-time, namely the logarillim of lhe death-

time, is for some venorns linearly related lo the logarillim of the dose over a

range of doses wide enough lo make it equally suitable for use in such assays

(Christensen and Finney, 1953; Christensen, 1955).

This graded response method is very satisfaclory in thal residis with fairly

narrow limils of error are obtained with very few mice in two or ihree hours,

hut also this method has ils limilations in work with whole venom. The dose (d)

which kills in infinite lime is presnmahly determined hy the dominant loxin, hut

as one moves to larger and larger venom doses, more and more other toxins will

come into play and the dose-time relationship must he determined hy lhe eomhined

actions of several toxins, with the result thal two preparations of venom from the

same species which have not exactly the same toxie pattern could give graded

response lines which are not truly parallel, just as one cannot expect that a

purified toxie fraction will yield a curve which only differs with regard to posilion

from lhe curve ohtained with the starting material, crude venom. An example

of this was recently presented hy Boquet and his eo-workers (1966) in experiments

with N. nigricollis venom and a purified letlial component of this venom called

alpha-toxin.

Hoj res of using this method to determine the amount of unhonnd toxin in

nnder-neutralized venom-antivenom mixtures, which apart fiotn its theoretical in-

terest might offer a means of obtaining a quick, even if rough, estimate of anti-

venom potency, are therefore not likely to he fulfilled, nor is the method likely

lo he of any great value as a screening test dnring the purification of venom

toxins, hut it may he of value as an additional means of selecting venorns with

similar properties and of checking stored preparations such as those set aside for

the assay of antivenom potency.

The preparation of antivenom for lhe first lime just beforc the lurn of the

century was a natural sequence lo the discovery of bacterial antitoxins, and the

preparation, purification, and potency assay of anlivenoms have ever since closely

followed the lines adopted for bacterial antitoxins, though not with the same

measure of success.

As far as potency assay is concerned, this has not been for lack of interest,

because a large proportion of venom literature has dealt with lhe specific and

paraspecific potency of anlivenoms, hut it is regrettahle that much of the earlicr

work must he considered wasted because the particular behaviour of venorns and

antivenorns was either ignored or misinterpreted.

Whenevor it is possihle it is alvvays desirahle lo replace methods which de-

mand lhe use of animais with tests carried out in glassware and similarly tests

carried out in for instance the skin of animais are preferable to tests which neces-

sitate tlieir death as long as these ways of testing measure lhe same thing. But

lhe lethal venom toxins have not, as yet, been shown lo have any specific enzyme

activity or other effect which can he measured in such simpler tests. As far as

African antivenoms are concerned, tlieir ahility lo inhibit various effects of ve-

noms, such as tlieir haemolytic, proteolylie, coagulant or anti-coagulant, and hae-

morrhagic actions, will not even serve as a guide for their protective titre. Some

venorns are however more likely to lead lo permanenl damage due lo tissue

destruction than lo loss of human life and one cannot exclude that for instance

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lhe anli-haemorrhagic potency of a serurn woiild be a hetter indicator of its

lherapeutic value than its ability lo save lhe life of mice, bul standardization

iii Ibis respect is nol as simple as il mighl seem because llu- venom haemorrhagin

is not a single factor; those interested in this aspect should consult the recent

study of Kondo and bis eo-workers (1965b).

The precipitalioM of lhe flocculation lype occurring in mixtures of venoms

and antivenoms bas been studied by numerous workers and ibere is no doubt

lhat the lethal loxins are capable of flocculating with their antitoxins as exemplified

by tbe early sludies by Calmelle and Massol (1909) on lhe liberation of toxin

frorn such floccules, and by Bier's work witb purified C rolai us toxin at Ins-

tituto Bulantan (Bier, 1917), but lhe reaction is of little praetical imporlance

in work wilb erude venom because of lhe mullilude of individual antigen-antihody

flocculations which take place, making interpretation in terms of titre impossible.

The potency of antivenoms has therefore to be assessed in animais and, as

for bacterial antitoxins, the assay usiially involves tbe preparation of a set of

mixtures eontaining eilber a fixed amount of serurn and increasing amounts of

venom or a constant amount of venom and varying amounts of serurn, which

arrangement one adopts is rather immaterial for the result.

The mixtures can be injeeted at once, but are often lefl for some lime al

a certain lemperature in order lo allow anligeit and antibody to combine before

the mixtures are tested for toxicity. Tbis is probably of little importance when

one is dealing with a venom and its specific antivenom, particularly if ihis is a

refined serurn frorn hyperimrnune borses, bul il nray, as will be sbown, some-

times influence the result. Tbe outcome nray also depend orr the route by which

lhe venom-anlivenotn mixtures are injeeted, and some workers favour the sub-

cutaneous route, because the snake injects its venom under lhe skitt rather than

into tire blood strearn. Some go further, and condemn the injection of ready-

rnade mixtures as art artificial procedure which should be abandoned in preference

for a melhod by which lhe animais are tested for protection by injecting venom

aml serurn separately, Urus irnmitating the sequence in naturally occurring strake-

bite. There nray be something in favour of such arguments, but it is by far the

simples! to mix the reagents before injection, a principie adopled in work witb

olheis toxins and antitoxins and practiscd in rnost venom laboralories, and it is

reasonahle lo assume lhat a preparation found lo be truly hetter hy this melhod

would also be more effective in the treatment of snakebite.

Most laboratories express the potency of a serurn as the amount of venom

neulralized by a unit volume of serum using a particular venom preparation set

aside for lliis purpose. Whether the amount of venom or the amount of serum

is kept constant is, as already stated, largely immaterial for tbe result, but in

order to have a meaning, lhe estimate of potency should be independent of lhe

conditions of the test, and if a certain amount of venom is found to be neulralized

by a given amount of serum, then any multiple of this amount of venom should

require the same multiple of serum for its neutralization, as originally stated by

Ehrlieh. It vvas soon seen that tbis rule did not apply to venoms and antivenoms.

Among the firsl to realize this was Vital Brazil who observed that certain anti¬

venoms neulralized relatively more venom in higher dilutions than in stronger

coneenlrations, observations which were confirmed and extended by many workers

both in tbis and otber countries.

This “law of combination in multiple proportion” implies that the “neutral

points” obtaincd I»y testing serum with venom at many concentration leveis will

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VENOM AND ANTIVENOM POTENCY ESTIMATION

fali on a straight line if recorded graphically in a system of co-ord inales in whieli

lhe axes express lhe concentrations of serum and venoni in lhe injected mixtures.

i( lakes a certain amounl of venom lo

lherefore, pass ihrough lhe origin hut

from this poinl. If lhe neutral poinls

toxie enough lo kill half lhe injected

intersect the dose axis at a point eor-

ln assays carried ont at a single dose

is linear and lhe assay car-

Kven without lhe addition of serum

kill. and lhe neutralization line cannol,

must eut lhe dose axis at some distanee

were recorded lo indieate mixtures jusl

animais, the neutralization line should

responding to the LD50 of lhe venom.

levei, ihis would he ol no conseqnenee if lhe curve

ried out at a levei high enough to ensure thal lhe injected mixtures eontain a

large numher of lelhal doses. This is the case when bacterial antitoxins are

assayed in the usual way, hut with antivenoms it may not he possihle lo employ

a very large lesl dose because lhe serum potency is too low. Under sueh eir-

eumstances account must he taken of the faet thal the neutralization curve cuts

the dose axis at some distanee from lhe point of origin, otherwise il will ohviously

lead lo results whieh seem to disohey the law of multiple proportions and give

tilres whieh are exaggerated and whieh seemingly inerease wilh deereasing numher

of lelhal doses in the injected mixtures. Although others may have realized this.

/ /

il was left for Banie and Ljuhetie (1938) lo stress that the amounl of serum

required to proteet a mouse from lhe effect of two lelhal doses is larger than

twiee lhe amounl protecting against one lelhal dose, and to suggest a method

of assay whieh made allowanee for this. Later the same year their method was

modified hy Ipsen 11938) who proposed to determine the slope of lhe neulral¬

ization curve whieh expresses the titre of a serum.

The method assumes linearity of the neutralization curves and however im-

portant they were, these studies did not solve the difficulty; tliey may in faet

have retarded progress hy goading experimentors lo assume that linearity is lhe

rule. whieh it ohviously is not.

Neutralization curves may sometimes he perfectly linear hut if examined over

a suffieiently wide dose range they will commonly show a change of slope, some¬

times ahrupl. hut often gradual, giving the impression of a gentle eurvature convex

towards lhe venom axis. Some workers realized that eurvature ralher than linear¬

ity is the rule and atlempted lo descrihe the relationship hetween serum dilution

and amount of venom neutralized in mathematical terms (Houssay and Negrele.

1921), and Kiehbaum (1917) ohserved a linear relationship hetween the logarilhm

of lhe amount of neutralized venom ly) and the logarilhm of the serum dilution

(x) effecting this neutralization according to lhe equation, log y log a + h . log x.

where a and h are constanls, a heing lhe amounl of venom neutralized hy 1 ml

of undiluted serum.

This tendeney lo aeeepl noneonformity with lhe law of eomhination in mul¬

tiple proportions as a cornmon phenomenon in antigen-anlihody reaetions may

have heen due to a eonfusion of certain concepts. This law States, as already

mentioned, that if a certain amount of toxin is neutralized hy a given amount

of serum then any multiple of this amount of toxin will require the same multiple

of serum for its neutralization. But, as Jerne wrote in his monograph dealing

with avidity, '‘this law does not irnply, as il mighl he thought and as il is some¬

times understood, that the amount of toxin neutralized hy antitoxin is proportional

to lhe quantity of antitoxin added” (Jerne, 1951). This, whieh Jerne called

“the principie of proportional neutralization”, would mean that lhe amount of

serum required exaetly to neutralize a fixed amount of venom would he exaetly

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P. A. CHUISTENSEN

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twice lhe amount whieh could neutralize half lhe venom in lhe mixture. Jerne

showed that although neither lhe "law” nor lhe “principie” are followed as a

general rule, lhe eonformity vvith holh law and principie is so good in lhe

particular case of sera from hyperimmune animais that deviations would nol he

disclosed hy ordinary measuremenls.

All antivenoms roulinely examined fali inlo lhe category of hyperimmune

sera and therc is no reason lo expeel gross deviations from eotnhinalion in mui

>unl rol inn ro l| ,,

whieh can he tesled

n any case, the type

tiple proportions especially nol as lhe coneenlralion range

is very restricted even wilh lhe hesl antivenoms availahle.

of eurvalure seen wilh venoms and antivenoms is eonvex towards lhe venom axis.

nol as for non-avid sera eonvex towards lhe serum axis. Furlhermore, why shonld

one gel perfectly linear curves wilh some antivenoms but nol wilh others, and

why shonld the anti-coagulant property of a serum lested wilh a coagulant venom

foliou lhe law of multiple proportions hui lhe protective aetivity fail to do so.

as reeorded hy Eiehhaum in experiments wilh R o t. h r o p s venom (Eichhaum.

1947 ).

The explanation for lhe particular shape of anlivenom titration curves shonld

have heen clear ií more altention had heen paid lo the observation made hy

Schlossherger, Bieling and Demnitz (1936). They noted that lhe amount of

venom neutralized hy some sera would increase wilh increasing serum dose up

lo a point beyond whieh furlher serum addition was without effeet because such

sera lacked antihody lo some venom componenl. Schottler observed the sarne

happening wilh Bothrops jararaca venom and cante to the satne conclusiou, hui

appeared to accepl lack of comhiualion in multiple proportions as nol surprising

but "a rather eommon phenomenon of antigen-antibody reaetions" iSehottler,

1952).

The curve drawu in figure IA shows lhe same silualion for liilis lachcsis

anlivenom lested intravenously in mice. The amount of venom neutralized in-

creased as the serum dose was inereased to 0.2 ml, hui twice this amounl ol

serum had no furlher effeet because lhe serum, a raw serum from a single horse.

eontained no antihody to a venom componenl whieh began lo assert itself when

lhe venom dose was inereased beyond ahout 0.3 mg. Two loxins were involved,

a dominant toxin whieh determined the LD50 of the venom and whieh was

neutralized aeeording lo lhe law of multiple proportions, as indicaled hy lhe

lower lefl pari of lhe curve, and anolher toxin of whieh an 14)50 was eontained

in ahout 0.5 mg of whole venom and whieh was nol at all neutralized hy ihis

serum. The curve will nol show a sharp angle as indicaled in figure 111 because

lhe minor toxin acls hefore a dose of 0.5 mg is reached. A hypothelical dose-

morlality curve for this minor toxin has heen drawu in lhe lower right comer

of figure 111 in order to ease lhe explanation. If lhe minor toxin had heen

removed from lhe venom without affecting lhe dominant toxin. one would expect

lhat 0.15 mg of venom would require ahout 0.67 ml of serum lo he neutralized

lo such an extent that 50% of the lested mice would survive lhe effeet of lhe

mixture. Wilh lhe minor toxin present — and assuming lhat the dose-mortalily

curve for ihis toxin is correct — this dose, 0.45 mg of venom. would conlain

enotigh minor toxin lo kill 25% of lhe mice whieh survived the effeet of the

free dominant toxin. This would rnean lhat nol 50% hui only ahout 37% of

the mice would survive and thal more serum would have lo he added lo give an

"over-all" survival rate of 50%. This extra addition of serum would he a liltle

smaller for a dose slightly less than 0.15 mg, and a little larger for a dose het-

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VENOM AND ANTIVENOM POTENCY ESTIMATION

ween 0.45 mg and 0.5 mg, and llius the curve would shovv lhe genlle change «f

direction shown in Figure IA and not a sharp anglc as in Figuro 115.

The part of lho curve determined hy lhe minor loxin would not he vortioal

if lho serum containod moasurahlo amounts of antihody lo this component hut

would show somo slope. The stoopness of lho slope would dopond on lho con-

centration of specific antihody lo ihis componenl in lho serum.

This interprotation doais adoquately with all situalions oncounlored whon anli-

venoms aro lested with orudo venoms, and il was suhstanliated somo yoars ago

hy results ohtainod in oxporiments with Naja nivea vonom whioh contains ihroo

antigenically distinct loxifís, callod alpha. heta and gamma loxin in docroasing

ordor of toxic importanoe. Tested wilh specific serum lliis vonom gavo the usual

typo of neutralization curvo showing a change of slope, quito ahrupl as oflon is

lho caso wilh olapid venoms. The curve consisted of Iwo linear paris, ono duo

to the neutralization of alpha loxin, thc other duo lo the neutralization of gamma

loxin; the eoneentralion of antitoxin lo lhe heta toxin was too high in this serum

to interfero in the assay (Christensen, 1955). Further and very noat evidence

for lhe correctness of this explanation of the shapo of venom-antivenom neutral¬

ization curves was supplied hy Kochwa and his co-workers (1959), who showed

lhat a rahhit afler immunization wilh V /pera xanthina paleslinae venom yielded

a serum whicli duo to the ahsence of antihody to lhe venom’s neurotoxin gave a

neutralization curve of the type shown in Figure IA hut produced a serum whioh

conformed with the law of múltiplo proporlions after antigenie stimulation wilh

purified neurotoxin.

The position with regard lo the neutralization of venoms hy antivenoms may

lie summarized as follows:

All venoms oontain more than oru

toxins, eaoh of whioh comhines wilh ils

proporlions.

The neutralization curve will have

axis corresponding lo lho 1,1)50 of the

• and oflon several antigenically differenl

antitoxin according to the law of multiple

ils origin al a point on the venom doso

mosl dominant loxin, whioh will he in

agroement with lho LD50 of wholo vonom unless several antigenically distinct

loxins have joint action and all contributo lo the offect produced hy the 1,1)50 of

whole venom.

The neutralization curve is only truly linear il lhe serum under tost conlains

relatively leasl antihody lo the dominant toxin, and a valid eslimale of the relativo

potoncy of difforent sera will he obtained if lliey all give this typo of curve.

In cases whore a serum contains relatively less antihody lo a component of

minor toxic importanoe, the curve will show a change of direction if the poteney

of the tested serum allows the use of sufficienlly high vonom doses. The poteney

ostimate will dopond on whioh of lho components is activo in the losl hut a valid

eslimale of the rolalive poteney of differenl sera can he ohtained if correspond¬

ing parts of the neutralization curves are compared.

The point al whioh lho curvo hegins to change direction and the steepness

of lho changed slope will dopond on lhe magnitude of the 1,1)50 of the minor

component in terms of whole venom and on the concenlration of antitoxin lo this

component. Extrapolation to the vonom axis will givo an eslimale of lho amouul

of venom whioh conlains an 1,1)50 of this componenl.

Further ehanges in direction may oceur if lho serum s eoneentralion of anti¬

toxin lo ovou less toxic components is still lower.

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The ahruptness willi whicli the curve changes direction depends on tlie sleop-

ness of lhe dose-response curves for the toxins. This interaction of different toxin-

auliloxin Systems explains why the endpoint iu potency assays may be perfeclly

sharp at lower concentration leveis, liecome "vvoolly in a certain range, and re-

gain sharpness al higher leveis, or in other words, why the loxin-antitoxin response

curve may appear flalter and lhe resulls show signs of heterogeneity at some

concentration leveis.

An estimate of the relative potency of tvvo sera will he invalid if the chosen

lesl levei involves different venom components for lhe two sera.

Having determined the neutralization curve for a serum intravenously in miee

one would expect to get a curve of similar shape if the test was repealed suh-

cutaneously or even carried out in another animal if the relative toxic importance

of the different components was the same by either route and for different animais,

which could he true for some venoms hut nol for others.

1 have nol had an opportunity to test different animais in this way, hut I

have examined one. serum with different cohra venoms and another with two viper

venoms intravenously and subcutaneously in miee.

EXPLANATION OK THE TABLES

Table 1 — See text for details.

Table 2 — Shows the intravenous and subcutaneous potency of four sera relative to

that of the Naja antivenom standard, calculated from the ED50 of the sera tested with

a dose of venom fixed at five ttmes the LD50. The venoms llsted in Table 2 have been

separated into four groups. The two venoms in the first group, Naja naja venoms from

Thailand and the Philippines, are grouped together because they both gave simple linear

neutralization tines in the tests by both routes with standard serum. This in itself does

nol mean that they only possess one lethal toxin but it means either that their main

toxin by far supersedes any other toxtn or that the standard contains relativeiy least anti-

toxin to the dominant toxin in these venoms and the table shows that the ratio between

relative titres for a serum determined by the two routes is for all sera as close to the

value 1 as one would expect in toxin-antitoxin titrations. The observation that the order

of inereasing potency of the four sera against the Thai N a j a venom is 1, 2, 3, 4 but

for N. naja philippinenses venom 1, 4, 3, 2 indieates that the two venoms dominant

toxins are antigenlcally distinct.

The two venoms in the second group, those of Indian N. naja and N. melanoleuca,

are placed together because two venom components were demonstrable by both routes in

the tests with standard serum, but for both venoms the subcutaneous and intravenous

neutralization curves were almost parallel. For the Indian N a j a venom the order of

Inereasing potency of the sera is 1, 2, 3, 4, as in the case of Naja venom from Thai-

iand and the numerical value for a serum is in fair agreement for the two venoms which

were about equalty toxic, indicating that the dominant toxin in these two venoms is in

all probability identlcal. With a test dose of 30 or 40 micrograms it is likely that only

the dominant toxin was active in the tests.

Reference to Figure 8 justifies the assumption that the second N. melanoleuca com-

ponent was active in the tests which explains the different order of potency of the sera,

here 4, 1, 3, 2, but for each serum the two values showed good agreement as could be

expected if the neutralization curves for the two routes were parallel for all the sera.

The venoms in the third group, those of N. nívea and H. haemachatus, are akin

because only the dominant toxin was active subcutaneously. Higher doses of N. nívea

venom than those useri would have caused discrepant results because the second toxin

would have been active intravenously, as it is, agreement between paired results ís good

for this venom yet the order of potency for the four sera, if significant, would indicate

that the dominant toxins of N. naja and N. nívea venoms are antigenlcally different,

which in any case I think they are.

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10 11 12 13 14 15

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VENOM AND ANTIVENOM POTENCY ESTIMATION

The values for //. haemachatus venom shows signs of disagreement for results between

routes and between sera which were to be expected knowing that only the dominant

toxin is involved subcutaneously whereas either of the two toxins could be active intra-

venously, depending on the properties of the sera.

Some irregularities of the data entercd in Table 2 for the paraspeciflc venoms in

the last group were to be expected beeause the ratio of subcutaneous to intravenous titre

of sera tested with such venoms not only differs from unity but varies from one serum

to another, as discussed in relation to Figures 9 and 10.

Why is it that some venoms behave intravenously and subcutaneously in the expected

manner with such sera and others do not? The answer seems to lie in the problem of

specificity.

The standard was prepared from the sera of horses already immune to the venoms

of N. nívea and H. haemachatus and subsequently immunized with N. naja venom, and

tested with these venoms it behaved as expected. The satisfactory result obtained with

IT. naja venom from the Philippines and N. melanoleuca venom could be explained by

a close antigenic relationship between these venoms and one or more of the venoms used

as antigens in the preparation of the standard, whereas the poor result obtained with

N. nigrtcollis and N. haje venoms could be due to qualitative differences between these

two venoms and those used as antigens, resulting in a lack of firmness of antlbody

binding.

If this is the case, one would expeet sera prepared with the venoms of N. haje and

N. nigricollis to behave in a “specific” way in tests with the homologous venom, unless

the lack of firmness of antibody binding is due to peeuliarities in the structure of the

toxin molecules, and not only the fault of the serum.

In order to test this, the preparation of monovalent sera against these tw*o venoms

was begun. The results with serum prepared with N. nigricollis venom are unfortunately

not yet available, but the results obtained intravenously and subcutaneously with N. haje

antivenom, a pepsin-treated solution of globulin from one horse after two short eourses

of immunization, are shown in fig. 11.

Table 3 — In each of these experiments, a set of mixtures was prepared with a

fixed amount of venom and graded volumes of serum and these mixtures were injected

intravenously in mice in a volume of 0.5 ml under varying conditions as outlined in

Table 3. Nine mice were used per mixture for each set of conditions, a fast injection

Jasted less than 2 seconds, a slow injection between 15 and 20 seconds.

The result of the first experiment eonfirmed that these factors are unimportant

with N. naja venom and specific anti-venom. The second experiment indicated that the

mice receiving warm mixtures of IT. haje venom and standard serum fared better than

those receiving cold mixtures and, even if it is not statistically signlficant, the difference

between the ED50 values for the first two groups in the second experiment suggested

that the speed of injection might influence the result and this was eonfirmed by the

third experiment. In the last of the experiments recorded in Table 3, N. haje venom

was tested with the specific monovalent serum at the extremes of the test conditions

using two venom doses, 105 and 150 micrograms. For both doses the FD50 was smaller

when the mixtures were warm and injected slowly after having stood for an hour, but

not significantly smaller than for the cold mixtures injected rapidly immediately after

preparation.

The conclusion to be drawn from these experiments in that technical details such

as the time and temperature at which the mixtures are held before injection, and the

speed with which they are injected are at most of llttle Jmportance if a venom is tested

with specific serum, and that the titre assígned to such sera will be about the same

whether the mixtures are injected under the skin or into a veln. But sera with para-

specific action will bind the venom loosely and the result will depend on all these factors.

The paraspecific venom-antivenom eomplex is presumably dissociating when injected

intravenously whereas the mixture injected under the skin will benefit from incubation

at a high temperature and become concentrated beeause water is more rapidly absorbed

than the venom-antivenom eomplex which therefore will have less tendency to dissoclate.

The discusslon has so far only been çoncerned with variations encountered in tests

with a particular set of venoms set aside as laboratory test preparations but variations

in the composition of venom from the same speeies will probably prove the greatest dif-

fieulty to uniform antivenom potency control. Figure 12A and B and Figure 13 are

included here to exemplify this.

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10 11 12 13 14 15 16

Mem. Inst. Butantan

Simp. Internac.

33(1): 305-326, 1966

P. A. CHRISTENSEN

315

TABLE 1 — THE INTUA VENOUS AND SUBCUTANEOUS LD M IN MOCROGRAM OF

DIFFERENT SNAKE VENOMS FOR MICE WEIGHING FROM 16 g TO 18 g

VENOM

INTRAVENOUS

LD r)0 5% fiducial limits

SUBCUTANEOUS

LD 50 5% 1 ‘iducial limits

Naja naja philippinenses

3.3

3.7 —

2.9

3.6

4.1 —

3.6

Naja naja (Thailanrt)

5.9

6.6 -

5.4

6.6

7.4 —

5.7

Naja naja (índia)

6.2

6.8 —

5.9

7.7

9.0 —

6.6

Naja nivea

9.7

10.8 —

9.1

12.3

13.0 —

11.6

Naja melanoleuca

18.5 —

14.9

28.5 —

22.0

Naja haje

23.5 —

19.2

33.8 —

25.6

Naja nigricollis

28.9 —

19.9

51.4 —

43.5

Hemachatus haemachatus

35.1 -

26.1

34.4 —

26.0

Dendroaspis polylepis

8.3

8.9 —

7.7

9.3

10.7 —

8.3

Dendroaspis viridis

12.0

13.3 —

10.9

13.5

15.2 -

12.2

Dendroaspis jamesoni

18.6 —

14.4

19.3 —

15.6

Dendroasp is a n g us t ice ps

57.5 -

37.8

64.9 —

49.1

Bit is lachesis

9.7

10.6 —

8.8

107.5 —

59.4

Bitis gabonica

11.5

12.9 -

10.4

100

117.0 —

85.5

Bitis nasicornis

28.3 -

22.8

184

209.5 —

163.7

Echis carinatus

30.9 —

16.3

177

177.4 —

134.3

TABLE 2 — THE POTENCY OF FOUR SERA

RELATIVE TO THAT OF

STANDARD Naja

ANTIVENOM DETERMINED 1NTRAVENOUSLY AND SUBCUTANEOUSLY IN

MICE

WEIGHING FROM 16 g TO 18

g USING

A TEST

DOSE OF

FIVE TIMES THE LD, 0

' DIFFERENT VENOMS

VENOM

Route

Test

dose /ug

SERUM

Stand.

naja (Thailand) intraven.

0.64

0.73

0.79

0.96

subcut.

0.67

0.78

0.87

0.90

n. philippinenses intraven.

16.5

0.70

1.65

1.15

0.85

subcut.

17.5

0.78

1.57

1.13

0.91

naja (índia) intraven.

0.53

0.73

0.86

0.94

subcut.

0.72

0.84

1.01

1.00

melanoleuca intraven.

0.86

1.56

0.97

0.62

subcut.

125

0.90

1.47

1.03

0.65

nivea intraven.

0.65

0.90

0.71

0.96

subcut.

0.72

0.94

0.72

0.90

haemachatus intraven.

150

1.20

2.00

1.36

0.89

subcut.

150

0.91

1.59

1.09

0.96

haje intraven.

105

1.08

1.79

2.03

0.91

subcut.

145

0.89

1.53

1.06

0.72

nigricollis intraven.

115

1.17

1.59

1.04

0.70

subcut.

240

0.92

1.15

0.93

1.15

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VENOM AND ANTIVENOM POTENCY ESTIMATION

TABLE 3 — THE EFFECT ON THE ASSAY RESULT OF THE CONDITIONS OF THE

TEST WITH REGARD TO SPEED OF INJECTION AND THE TIME THE VENOM-SERUM

MIXTURES ARE HELD AT DIFFERENT TEMPERATURES BEFORE INTRAVENOUS

INJECTION IN MICE

Test

ED.„

fiducial

VENOM

dose

Serum

Cnnditions of test

in uR

in /d

limits

Naja

naja

standard

fast, immediate,

16°

13.7

slow, 60 mins.

37°

13.9

14.6

13,1

Naja

haje

105

standard

fast, 20-40 mins

2C°

64.0

233

• 58.6

fast, 20-40 mins

2C°

57.4

64.6

— 56.7

slow, 90 mins.

37°

50.5

55.0

41.5

Naja

haje

105

standard

fast, 60 mins.

37»

58.5

67.6

— 54.5

slow, 60 mins.

370

48.4

51.4

43.4

Naja

haje

105

monovalent

fast, immediate

16°

101.6

106.3

— 96.3

slow, 60 mins.

370

92.4

98.2

— Sd.7

Naja

haje

150

monovalent

fast, immediate

16°

166

177

— 158

slow, 60 mins.

37°

161

170

— 152

ExPLANATION OF THE FIGURES

Figure IA & B — See text for details.

Figure 2 — Shows the neutralization curve obtained with N. naja venom from índia

and a particular serum which recently was established as the International standard for

Naja anti-venom potency. The upper curve was determined intravenously, the lower

curve subcutaneously, and the slopes of corresponding parts of the two curves agree quite

well, which means that the titre of the serum expressed in mg venom neutralized per ml

would be closely the same whichever way the mixtures were injected, but, being different

for the two venom components, the value would depend on the levei of the test.

Figure S — The curves for a sample of N. naja venom obtained from Thaiiand are

shown in Figure 3. Only one venom component is active throughout the tested range

and the titre in mg per ml would not only agree quite closely irrespective of the route

of injection but would also be independent of the concentration levei.

Figure j — Shows the same to apply to resulls obtained wdth a sample of N. naja

venom obtained from the Philippines. That the subcutaneously determined curve happens

to be placed above the other is probably due to experimental varlation as the two tests

were not carried out at the same time, and not because this venom is more toxic sub¬

cutaneously than intravenously.

Figure 5 — The curves for N. nivea venom are shown in Figure 5. It appears that

two venom components operate intravenously but only the dominant toxin is active sub¬

cutaneously. Maybe the effect of the second component would have shown up if even

more concentrated Solutions had been injected, but this component is obviously of little

or no importance subcutaneously in mice. Expressed as mg neutralized per ml, the titre

of the serum would be about the same for the two routes at low dose leveis, but dis-

agreement would be found at higher leveis where two different properties of the serum

would be compared.

Exactly the same position applied to the results obtained with Hemachatus haema-

chatus venom (Figure 6) and with two viper venoms, those of Bitis gabonica and B. la-

rhesis, tested agalnst a polyvalent Bitis-Echis antivenom (Figure 7).

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10 11 12 13 14 15 16

Mem. Inst. Butantan

P. A. CIIRISTENSEN

317

Simp. Internuc.

33 ( 1 ): 305 - 32 ( 5 , 1966

Figures Cd 7 — II could reasonably be suggested lhat the potency of sera against

venoms such as those giving the results shown ln Figures 5, 6 and 7, ought to be tested

subcutaneously in mice if the second venom eomponent is equally non-toxie injected under

the skin ot human beings, as happens in snakebite.

Figure 8 — The results for N. melanoleuca venom, shown in Figure 8 are very similar

to those obtained vvith N. naja venom (Figure 1) but the parts of lhe curves due to the

second eomponent Show signs of dívergence resulting in a slightly higher estimated titre

in the subcutaneous test.

Figure ,9 — Even more marked differences in slope for the parts of the curves de-

termined by the second eomponent was seen when N. haje venom vvas tested by the tvvo

routes (Figure 9), and the roule of Injection would clearly have to be specified if the

titre in terms of weight of reference venom neutralized per ml of serum was to have

any meaning. Even if the titre were lower, it could be argued in cases like this that

lhe sera should be tested intravenously because the neutralization in persons treated for

snakebite probably takes place in the blood-stream rather than at the site of the lesion.

If the ratio of subcutaneous to intravenous titre remained constant from one serum

to another, this situation could aiso be quoted in support of the use of a reference

preparation of serum rather than venom; but this is not necessarily so.

Figure 10 — As an example, Figure 10 shows the subcutaneous and intravenous N.

nigricalis antivenom potency of tvvo sera of which the one was the Naja anti-

venom standard, the other a similar preparation of unmodified serum 1'rom one horse.

The coinciding intravenous curves for the tvvo sera, shown farthest to the left in Figure

10 , indicate that the two sera were equally potent against both the toxic venom compo-

nents. As far as the dominant toxin is concerned, this was confirmed by the subcutaneous

test, but tested by this route the potency of the raw serum appeared to be far superior

against the venom’s second toxin. If two different laboratories were to test these two

sera vvith the same preparation of venom, one laboratory intravenously the other sub¬

cutaneously, the expected result would be in disagreement about the absolute amount

o2 dominant toxin neutralized by the two sera but agreement with regard to their relative

potency against this eomponent, a situation which would favour the expression of potency

in terms relative to the potency of a standard serum. As far as the second venom com-

ponent is concerned, the tvvo laboratories would report conflicting results vvhether the

titre was expressed in relative terms or as the amount of venom neutralized per ml, and

nothing would be gained by the use of a serum standard.

From the results discussed so far, it appears that whether or not subcutaneous and

intravenous tests yield the same result depends on the venoms and sera under test and

the next step was to attempt to find the reasons vvhy.

Figura 11 — The two curves are in very good agreement with each other and with

the contention that concordance between lhe intravenous and subcutaneous titres is the

rule for specific sera, but not for paraspecific sera.

In all the tests so far recorded no particular attention was paid to the time for

which the venom-serum mixtures were left before they were injected, or to the temperature

at which they were held, because past experlence had shown this to be of little importance,

but during these tests the impression was gained that such factors might influence the

result with some venoms, N. haje venom in particular, and Table 3 summarises some

experiments carried out to clarify this point.

Figures 12 A d II — Figure 12 A shows the neutralization of the Johannesburg B. ga-

bonica reference venom by three specific sera and Figure 12 B, the neutralization of a

sample of B. gabonica venom obtained from Institut Pasteur in Garches. One of the

three sera is obviously the better in tests with both venoms but the order of potency

though almost the same, is reversed for the other tvvo sera indicating basic differences

and not just quantitativo variations in the composition of the tvvo venom samples.

Figure 1.1 — Two of these sera were specific against Echis carinatus venom aiso,

and tests with E c h i s venom from the same tvvo sources gave the result shown in

Figure 13. The relative potency of the two sera mny happcn to be roughly the same

against the second eomponent, but these two venoms must be fundamentally different.

These examples have all been dravvn from experiments with African snake venoms

but the results are probably applicable to venoms in general, and confirm that true

2 3

5 6

10 11 12 13 14 15

318

VENOM AND ANTIVENOM POTENCY ESTIMATION

standardlzation ls Impossible as long as we have to test the sera with crucie venom

preparation. Some form of potency control Is, however, urgentiy needed in orcier to

protect the public against worthless sera, and minimum potency requirements vvill have

to be baseei on referenee preparations of either venoms or sera.

That baeterlal antitoxins are standardized on the basis of serum standards is In it-

self no reason why the use of a venom referenee preparation shouid be rejected. The

toxieity and antibody-binding power of dried venoms seem to be extremely stable and

the main objectlons to the use of a referenee preparation of venom appear to be firstly,

that it may not ensure the correct evaluation of sera in areas where snakes with a dif-

ferent venom composition predominate, and, secondly, that once nearing exhaustion, it

would be impossible to replace it by another preparation with identical qualities, as

pointeri out by Sehottler (1958). To these objections one coulcl add the financial dlf-

ficulty. An International referenee preparation would have to be freely available, and

considering the amount of venom required of dlfferent species to serve the needs of many

laboratories for a number of years, the capital outlay would be substantial. A US dollar

would buy about 1,000 intravenous lethal mouse doses of an average priced Afriean

venom, just about enough to compare the potency of two sera at two concentration

ievels. A referenee preparation of antivenom is a great deal cheaper to produce, it might

be capable of dealing with ‘abnormal’ venoms, and its eventual replacement, though dif-

ficult indeed, would not be impossible. The present Naja antivenom standard, the only

antivenom standard establlshed so far, is a polyvalent preparation of whieh 2.69 mg

represents the unit of potency against toxic component of Naja venom in general.

Its replacement would undoubtedly require the redefinition of the unit not only for

eaeh Naja species but also for each of the toxic components contained in such venoms,

and this would be an extremely diffleult undertaking. While on the subject of this

particular preparation, and in view of the resuits just presented, I think that it will

prove unsuitable as a standard in tests with paraspecific venoms such as those of N. haje

and N. nigricollia.

The use of antivenom referenee preparations will not for the time being do away

with the need for referenee venoms if independent laboratories are to get concordant

resuits in potency assays and minimum potency requirements are to be defined but, even

so, one shouid welcome the estabiishment of international referenee preparations of anti-

venoms, and the definition of potency in terms of units because it lends itself to concise

Information between laboratories, and the time will come when the dlfferent venom

components have been isolated in quantity and valid potency estimates can be obtained

in assays of antivenoms with such purlfled toxins as already done by Kondo and his

co-workers for Habu venom (Kondo et al. 1965a).

But potency control is not an international problem; it is a matter for local iicensing

authorlties to decide in consultation with antivenom producers.

It would of course be Impossible to suggest a single method of potency control which

would be acceptable to everybody and fair to all sera but, in concluslon, I shouid like

to add some remarks with regarei to a reasonably practical approach in places where

some form of potency control ls not already establlshed, and where anti-snakebite sera

are accepted on the wording of the labei they carry.

There are usually very few producers of antivenom in any country and it shouid be

possible for them to agree to contribute — maybe according to their own requirements — to

a common pool of referenee venoms to be controlled by the iicensing authority. At the same

time it would be advisable tò put aside suitable freeze-dried sera, which would serve as

a means of checking the constancy of the venoms and of settling disputes between pro¬

ducers and authority, and such sera would be of value when new venom preparations

had to replace the old; 1 but whether the potency requirements are expressed in terms of

absolute or relative potency would be immaterial.

Antivenom potency tests are simple to carry out, require little equipment and space,

and the iicensing authorlties in countries relying entirely on imported antivenoms shouid

aequire a small stock of appropriate referenee preparations, lay down their own minimum

potency requirements, and ciemand samples for test before importation is permitted.

The optimai eondittons of the potency test, for example with regard to test animal,

route of injectlon, and other technlcalities, need not be the same for all venoms but

shouid be defined in detail. The alm would be potency control, not true stanclardlzation,

and the eventual replacement of the referenee venoms by other preparations with closely

similar toxlcities, gradeei response curves, and antibody-binding power, would be good

enough to guarantee that about the same levei of quality was maintained.

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10 11 12 13 14 15 16

serum

P. A. CIIRISTENSEN

Mem. Inst. Butuntan

Slmp. Internac.

83(1):305-326, 1966

319

mg Bitis lachesis VENOM

Fig. 1 — A: Neutralization curve obtained in mlce tested intravenously with Bitis lachest■

venom and unmodified serum from a horse. B: See text for detaíls.

Fig. 12 — Neutralization curves for Indian Naja naja venom and standard Naja anti-

venom determined in mice.

O Intravenous test

® Subcutaneous test

SciELO

10 11 12 13 14 15

per cent mortality

serum

Fig. 3 — Neutralization curves for Naja naja venom from Thailand and standard Naja

antivenom determined in mice.

O Intravenous test

o Subcutaneous test

002

004

006

0-08

mg N. naja phillipinenses VENOM

Fig. 4 — Neutralization curves for Naja naja philippinenses venom and standard N a j a

antivenom determined in mice.

O Intravenous test

® Subcutaneous test

2 3

SciELO

10 11 12 13 14 15 16

mi serum „ m i serum

Mem. Inst. Butantan

Simp. Internac.

33(1):305-326, 1966

P. A. CHRISTENSEN

321

-ig. 5 -

Neutralization curves for Naja nívea venom and standard Naja antivenom

determined in mice.

O Intravenous test

mg H. haemachatus VENOM

Fig. 6 — Neutralization curves for Hemachatus haemachatus venom and standard Naja

antivenom determined in mice.

O Intravenous test

® Subcutaneous test

SciELO

10 11 12 13 14 15

ml ser um

322

VENOM AND ANTIVENOM POTENCY ESTIMATION

Fig. 7 — Neutralization curves tor a polyvalent refined B i t i s - E chi s antivenom

tested with Bitis lachesis venom (A) and Bitis gabonica venom (B) in mice.

O Intravenous test

• Subcutaneous test

Fig. 8 — Neutralization curves for Naja melanoleuca venom and standard Naja anti¬

venom determined in mice.

O Intravenous test

• Subcutaneous test

SciELO

10 11 12 13 14 15

ml serum

0-1 0-2 0-3

mg N. haje VENOM

Fig. 9 — Neutralization curves for Naja haje venom and standard Naja antivenom

determined in mice.

O Intravenous test

o Subcutaneous test

mg N. nigricoUis VENOM

Fig. 10 — Neutralization curves for Naja nigricoUis venom and tvvo sera determined

in mice.

O Standard Naja antivenom tested intravenously, and

o subcutaneously

+ Unmodified antivenom tested intravenously, and

subcutaneously

■ Coincident point

SciELO

10 11 12 13 14 15

ml ser um

0 - 1 -

0-1

mg N. haje VENOM

Fig. 11 — Neutralization curves for Naja haje venom and a refined monovalent N. haje

antivenom determined in mice.

O Intravenous test

• Subcutaneous test

Fig. 12 — Neutralization curves determined intravenously in

mice for three polyvalent refined antivenoms tested with Bitis

fjabonica venom from Johannesburg (A) and Paris (B).

SciELO

10 11 12 13 14 15

Fig. 13 — Neutraüzation curves for two polyvalent refined

antivenoms determined intravenously in mice with Echis

carinatus venom from Paris and Johannesburg.

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munological study of Vipers xunthina palestinae venom and ,,reparation of

potent antivenin in rabbits. Immunol.. 82:107, 1959.

18. Kondo, H., Kondo, S.. Sadahiro, 8., Yamauclú, K., Ohsaka, A. & Murata, R.

- Standardization of antivenine. I. A method for determination of anti-

lethai potency of Habu antivenine. Japa) i. J. Med. Sei. Biol., 18:73, 1965a.

19 Kondo, H., Kondo, S., Sadahiro, S., Yamauchi, K., Ohsaka, A. & Murata, R.

- Standardization of antivenine. II. A method for determination of anti-

hemorrhagic potency of Habu antivenine in the presence of two hemorrhagic

principies and their antibodies. Japan. J. Med. Sei. Biol.. 18:127, 1965b.

20. Lin, C.-U. — The relationship between the dose of Taiwan cobra (Naja atra)

and Taiwan habu ( Trimeresurus mucrosquamatus) venom and time of death

in mice. J. Immunol., 77:87, 1956.

21. Schlossberger, H., Bieling, R. & Demnitz, A. — Untersuchungen über Anti-

toxins gegen Schlangengifte und die Herstellung eines Heilserums gegen die

Gifte der europáischen und mediterranen Ottern. Behringwerk-Mitteilungen,

7:111, 1936.

22. Sehottler, W. II. A. — Probiems of antivenin standardization. Buli. World

Hlth. Org., 5:293, 1952.

23. Sehottler, W. H. A. -- Miscellaneous observations on snake venoms and anti¬

venins. Memórias do Instituto Butantan, 27:85, 1955/6.

24. Sehottler, W. II. A. — Reference toxins for antivenin standardization. Buli.

World Hlth. Org., 19:341, 1958.

25. Schenberg, S. Geographical pattern of crotamine distribution in the same

rattlesnake subspecies. Science, 129:1361, 1959.

2 3

5 6

11 12 13 14 15

Mom. Inst. Hutantun

Slmp. Internac.

33(1):327-330, 1966

CHALOEM PUHANANANDA, RRASIT

LAUIIATIRANANDA and SOMSRI GANTIIAVORN

327

36. CKOSS IMMUNOI.OCICAL RKACTIONS IN SNAKIvVKNOMS

CHALOEM PURANANANDA, PRASIT LAUHATIRANANDA,

SOMSRI GANTHAVORN

Queen Suombka Memorial Institute, Bungkok, Thailand

The production of antivenine seriiin has started since 1913 at the Queen

Saovabha Memorial Instilule, started with antivenine sera against Cobra ( Naja

naja) and Kiissell s viper ( Viper russcllii). The sera are prcparcd in monovalenl

spccific form mi«l divalent by mixing the Ivvo sera logether. Later on sera against

Malayan pit viper ( Agkislrodon rhodostoma) , Banded Krait {Húngaras jascialus)

and King cobra {Naja liannah ) were prepared. The polyvalent serum vvas eon-

sidered to he unpractical because il is known that the regional distribution of

deadly poisonous snakes in Thailand is definite. It is lherefore advisahle to

j>repare monospecific serum. By doing Ihis, the patient will get a correet treat-

ment without any surplus of undesirable serum which in turn will expose the

patient to serum sickness.

Il is lherefore important lo prove that one serum is officicnt for only one

kind of venom., This can he proved hy cross neutralization in experimental animal

and supported hy immunoelectrophoresis (Scheidegger, 1955).

Materials \\i> methods

The experiments were earried oul both in vitro and in vivo.

In vitro tests The vcnoms used as antigen were of Thailand origin. The

neurotoxic group eonsisted of Naja naja siamensis (Thai cobra), N. hannah (King

cobra) and Húngaras jascialus (Banded Krait). The hemotoxie group eonsisted of

Vipera russcllii (Hussell s viper), Agkistrodon rhodostoma (Malayan pit viper) and

Trimeresurus popeorum (Creen pit viper). These venorns were dried in the des-

sieator under reduced pressure immediately after milking, exeept N. n. siamensis

whieh was lyophilized. Horse antivenine sera specific for each of five venorns

were used as antibodies (antivenine against T. popeorum has not been produced).

These sera were lyophilized and reeonstituled in distilled water before use in lhe

experiments.

The microimmunoelectrophoresis method described by Scheidegger (1955)

was used in lhe experiments by putting venorns in both Wells (0.2 cm in dia-

meter). Two different venorns were eleetrophoresed at constant current of 0.6

MA/cm.for 5 hours. Then. the antivenine serum specific foi the venom in the

upper well (for eontrol) was put in the middle through (0.2 X 6.7 cm). The

eleetrophoresed venorns were allowed lo reaet with lhe antivenine serum for about

18 hours and lhe excess proteins were washed oul by physiologieal saline and

SciELO

10 11 12 13 14 15

328

CROSS IMMUNOLOGICAL REACTIONS IN SNAKE-VENOMS

distilled waler sueecssively. The .«lides llicti were dricd and stained hy buffalo

blue lilaek dye.

In vivo tests — Cross neulralization vvas delermined liy animal protection

lests in vvhitc mice. The toxicity of each venom was conducted hy injecting

varying amounts of venom dissolvei! in physiotogieal salino inlo groups of white

mice, 3 mice in lhe preliminary lesls and 10 mice in lho final losl. willi a eonstant

volume of 0.5 ml of venom solulion for oaoh mouse weighing 16-18 granis. The

residis were judged hy lhe numher of dealhs in each animal group 24 hours

after injection and lhe I.D.,,, was ealeulaled hy lhe melhod of Kiirhar (The Slaled

Serum Inslitute, Copenhagen).

In lhe neulralization lesls. each of lliroe mice was injeeled inlravenously wilh

varying amounts of venom dissolvei! in 0.25 ml physiologieal salino mixed wilh

0.25 ml anlivcnin solulion. The venom-alivenin mixtures were allowed Io stand

at 37"C for 30 minutes hefore injecting inlo rniee. Survival of tesl animais 24

hours after injeelion was lhe crilerion for judgement of neulralization, and lhe

potency of serum for proleetion was expressed as mg of venom nculralized hy

10 ml of serum.

Illisi I.TS in vitro ti:sts

In lhe reaetion hetween anli-Cohra serum and Cobra and King eohra venom.

and of Cohra and Banded Krail venom. there are al least ten preeipitalion lines.

formed hetween lhe homologous reaetants and a lino is formed helween lhe sera

and King cohra venom. whereas al leasl iwo lines oecur hetween lhat and Banded

Krait venom.

In lhe reaetion hetween anti-King eohra serum and King eohra and Cohra

venom, and lhat against King eohra and Banded Krail venom rospeetively, there

arc al least five lines hetween lhe homologous reaelants an three lines hetween

sera and Cohra venom. while two lines are formed hetween thal and Banded

Krait venom.

In lhe reaetion hetween anli-Banded Krail serum and Banded Krail and Cohra

venom, and lhat of Banded Krait and King eohra venom rospeetively, Iwo intenso

preeipitin hands and al leasl two fainled hands oecur helween lhe homologous

reactanls. There are ahoul six lines hetween lhe sera and Cohra venom while

two fainled lines are formed hetween lhat and King eohra venom.

In lhe reaetion hetween anti-BusseH's viper serum against Russells viper and

Malayan pit viper, and lhat of lhe rarne homologous venom and Crecn pit viper

venom rospeetively, at least eight preeipitin hands oecur hetween the homologous

reaetants. A fainled Iine is formed hetween lhe sera and Malayan pit viper.

whereas ahoul five fainled lines oecur hetween sera and C.reen pit viper venom.

In lhe reaetion hetween anli-Malayan pit viper serum, and Malayan pit viper

and Russells viper venom, and of lhe same homologous venom and Crecn pit

viper venom rospeetively, there are al least eight preeipitin hands formed helween

the homologous antigen. Two fainled lines oecur helween the sera and Russell s

viper venom, while about four lines oecur hetween lhat and Crecn pit viper

venom.

lhe cross immunological reactions helween anli-Cohra sera and Russell s viper

venom and vice versa exists. wilh no precipitation Iine hetween the anli-Cohra

serum and RusselTs viper venom. There are ahoul six fainled lines helween anti-

RusselTs viper sera and Cobra venom. This may lie due lo lhe fact thal lhe

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 327-330, 1966

CIIALOEM PURANANANDA, PRASIT 99Q

LAUHATIRANANDA and SOMSRI GANTHAVORN

In vivo l(‘sl:

TABLE I — CROSS NEUTRALIZATION OF NEUROTOXIC VENOMS AND THEIR

ANTIVENINS

ANTIVENINE

VENOMS

Cobra

King cobra

Banded Krait

Cobra .

1 2

4.0 (20)

4.4 ( > 4)

2.0 (2)

King cobra .

1.0 (5)

4.0 (4)

1.6 ( < 2)

Banded Krait .

< 0.4 ( < 2)

1.6 ( < 2)

7.0 (7)

TABLE II — CROSS NEUTRALIZATION OF IIEMOTOXIC VENOMS AND THEIR

ANTIVENINS

VENOMS

Russeü's viper

A. rhodostoma

Green pit viper

RusselTs viper .

5.0 (48)

A. rhodostoma .

14.0 (6)

TABLE UI — CROSS NEUTRALIZATION BETWEEN

NEUROTOXIC AND IIEMOTOXIC VENOMS AND

THEIR ANTIVENINS

ANTIVENINS

VENOMS

Cobra

Russell\s viper

Cobra

4.0

1.0 (8)

RusselTs viper

0.6 (3)

5.0

1 — Quantities of venoms in mg neutralized by

10 ml of sorum. (Judged by survival at 24

hours of all 16-18 grams vvhite mice given

intravenous injection of varying amount of

venoms in saline plus 0.25 ml antivenim. Ve-

nom Solutions were mixed and allowed to

stand for 30 minutes at 3?°C before injectlng.)

2 — Amount in LD,„ of venom.

2 3

SciELO

10 11 12 13 14 15

CROSS IMMUNOLOGICAL REACTIONS IN SNAKE-VENOMS

antiserum possesses antibodies lo minor componenls of anligcn. Thus lho bands

are formed hy lhe intermingling of lhose oompononls of antigen and antihodics.

linl in lhe first case, lhe antihodics to idonlical componenls llial constilule in

lhe Cobra vcnoin may not be high enongh lo forni precipitation line with lhe

minor fractions of Russells viper venom.

I )lSCl'SSIOM

S. Minton: "1. We find very poor correlation between immunodiífusion lines

and neutralization of venom in vivo. For example, we find fairly good neulraliza-

tion of several Naja venoms and Sea Snake Antivenin (Commonwealth Serum

Labs) although this serum gives very little in the way of precipitin lines.”

“2. The finding of common precipitin antigens between serum of snake and

venom of same snake is contrary to our experience.”

“3. I am surprised at the number of common antigens between Cobra and

RusselFs Viper venoms. This is contrary to our experience."

S. Minton: "1. What do you consider a satisfactory neutralization titer in

terms of mouse LD/50’s neutralized by 1 c.c. ?”

“2. How frequently are Bungarus bites seen in Thailand?”

C. Puranananda: “1. It was answered by Dr. Krag. Method used in Bangkok

is Ipsen’s.”

“2. Very seldom, because Bungarus fasciatus is a docile snake.”

A. Shulov: “3. What is the reason of using ponies instead of horses. Did you

try donkeys? According to lhe advice of Dr. Bouquet, Paris we tried with satis¬

factory results.”

“2. Is the percentage of good producers higher among ponies than among

horses?”

C. Puranananda: “1. Sim, estou de acordo. Em meu trabalho mencionei po-

neys, burros e mulas em quantidade”.

“2. Sim. Somente não no caso de venenos hematóxicos, onde há melhores

resultados com cavalos da China e mulas.”

F. Kornalik: “I would like to corroborate Dr. Purananandas’ findings about the

presence of toxin-like antigens in the serum of King-cobra, which would react with

a specific anli-toxin serum. This has been described up lill now three times for

the serum of Viper a ammodytes.”

N. McCollough: “We have not been able to correlate the gravity of serum

sickness and the amount of antivenin given. 70-80% of lhose who receive anti¬

venin become victims of serum sickness and its degree seems to be an individual

response. Would you commenl.”

C. Puranananda: “The cases of snakebites after treatod with antivenine serum

resulted in serum sickness in our country, become important to our notice and we

iiave to be careful about cases of repeated bite. In some cases of bite amongst the

snake charmers, who carne in with second and third treatment, some of them

carne with serious symptoms and intravenous injection is indicated. II is therefore

very important lo reduce the amount of serum used possible.”

A. do Amaral: “1. How do you determine the vencm toxicily?”

“2. What process of antivenin titration do you use at Bangkok?”

C. Puranananda: “1. Replicd by Dr. Krag. 2. Ipsen’s method.”

2 3

5 6

11 12 13 14 15

Mem. Inst. Butantun

Simp. Internac.

33(1):331-337, 19(56

AKIRA OIISAKA, HISASHI KONDO, SATORU KONDO,

MASAMI KUROKAWA and RYOSUKE MURATA

331

37. PROBLEMS IN DETERMINAT10N OF ANTIHEMORHHAGJC POTENCY

OF HABl (TRIMERESURUS FLAVOV1R1DÍS) ANTIVENINE IN THE

PRESENCE OF MULT1PLE HEMORRHAGIC PRINCIPLES AND THE1R

ANTIBODIES

AKIRA OHSAKA, HISASHI KONDO, SATORU KONDO, MASAMI KUROKAWA

and RYOSUKE MURATA

National Imtitute of Health, Shinagawa-ku, Tokyo, Japan

Intkoduction

We demonstrated lhe presence of Iwo hemorrhagic principies, HR1 and

HB2 (1), in the vcnorn of Trimeresurus jlavoviridis, which is called "Ilahu” in

Japanese. The Iwo principies are not distinguishable hy hemorrhagic action on

lhe rahhit skin (2-4) hut are distinguishahle immunologically from each olher(5).

Consequenlly, preparations of Hahu antivenine contain lhe corresponding Iwo anli-

hodies, anti-HRl and anli-HR2, in varying proportions (5).

Should the potency of such an antivenine he titrateei against the test toxin

containing lhe Iwo hemorrhagic principies, wc must ask what lhe result of lhe

tilration indicatcs, lhe potency of exclnsivcly anti-HRl or anti-HR2, or some other

implicalion (3, 5).

Recently we proposed a model for the mechanism involved in tilration of

lhe antihemorrhagic potency of an antivenine containing Iwo anlihodies wilh a

test toxin containing Iwo corresponding hemorrhagic principies (5).

The pnrpose of this presentation is lo discuss the general implieations of

onr model in slandardizing anlivenincs and also hactcrial antitoxins.

DeKINITION OF THE TERMS

The minimum hemorrhagic dose (MHD) of venom is defincd as lhe leasl

quanlily of venom in gg causing a hemorrhagic spot of 10 mm in diamclcr 2 1 hr

aflcr intraeiitaneous injection into lhe rahhits(2).

Effectivc dose (ED) is defined as a qnantity of an antivenine in ml which.

whcn mixed wilh a given dose of a test toxin, makes a mixture produeing a

hemorrhagic spot of 10 mm in diamclcr whcn 0.2 ml is injected intracutancòüsly

into the rahhit (5).

SciELO

10 11 12 13 14 15

332

PROBLEMS IN DETERMINATION OF ANTIHEMORRHAGIC POTENCY OF IIABU

(TRIMERESURUS ELAVOVIRIDIS) ANTIVENINE IN THE PRESENCE OF

MULTIPLE HEMORRHAGIC PRINCIPLES AND TIIEIR ANTIBODIES

One unit of antihemorrhagic activity is defined as tbe amount of antivenine

containing one ED at the levei of 100 MHD (5). The antihemorrhagic potency

of an antivenine is expressed as units per ml.

The design jor lhe delermination of ejjeclivc dose. (ED) oj an antivenine by

the rabbit skin test

Table 1 shows an example of the design for delermination of effective doses

(EDs) of an antivenine by lhe rabbit skin test (5).

TABLE 1 — AN EXAMPLE OF THE DESIGN FOR DETERMINATION ON EFFECTIVE

DOSES (EDs) OF AN ANTIVENINE BY THE RABBIT SKIN TEST

Test toxin : Crude venom ( batch No 48)

Antivenine preparction : Antivenine No. 23

Hemorrhagic acti-

vity of test toxin

in MHD*

Rabbit

No.

Cross-diameters of hemorrhagic spot in mm

against varying onount cf cr.tivcnine

0.00063

0.00050

0.00040

0.00032

0.00025

0.00020 m *

3 0

1556

12x12

12x13

14x13

14x15

16x15

1 557

12x13

13x13

13x15

15x16

18x16

1 559

15x13

16x16

15x17

17x16

0.00200

0.00160

0.00125

0.00100

0.00080

0.00063 fn|*

1 0 0

1 556

13x15

15x15

16x16

16x18

18x18

1557

4 ;

4 :

1 1 x 1 1

13x14

16x16

18x19

1 559

4 ;

1 1 X 11

15x14

17x18

1 9 x 20

20x21

0.00630

0.00500

0.00400

0.00320

0.00250

0.00200^

3 00

1556

13x15

15x15

15x16

17x19

18x19

1557

1 1 X II

I4xlõ

17x18

19x21

1559

1 1 x 1 1

15x15

17x17

19x19

20x23

ft Per injeeted dose

Test toxin Solutions containing 30, 100 or 300 MHD per 0.1 ml wcre prepared.

An aliquot from each of lhese Solutions was mixed with the equal volume of

each of serial dilutions of lhe antivenine graded with 1.25-fold intervals. The

mixtures were kopt standing for 1 br at room lemperature. Each was injeeted

into the depilated back skin of three rabbits at a dose of 0.2 ml and hemorrhage

developed was measured after 21 br from the visceral side of the removed skin

as described in the literature.

l he ED of the antivenine against each levei of test toxin was expressed in

lhe volume of the antivenine reducing lhe size of the hemorrhagic spot to 10 mm

in diameler, when 0.2 ml of the venom-antivenine mixture was injeeted into the

rabbit. In practice, the amount of antivenine in mixture causing a hemorrhagic

spot of 10 mm in diameler was determined by interpolation.

SciELO

10 11 12 13 14 15

Mem. fnst. Butuntun AKIRA OIISAKA, HISASHI KONDO, SATORU KONDO, 333

f3(l) •3 I 313 r 37 aC Í966 MASAMI KUROKAWA and RYOSUKE MURATA

A 'eutralization curves oj antivenines with the crude venom or the partially purijied

hemorrhagic principies

Effective doses (EDs) of several antivenines were determined with a crude

venom (batch No. d8), HR1 and HR2 as test loxins(5). The residts were plot-

ted lo give the neutralization curves shown in Eig. 1 where the ordinate is ED

of llie antivenines and lhe abscissa hemorrhagic activity of the test loxins in MHD,

holli in logarillunic scale.

Statistical analyses proved that all the neutralization curves obtained with

lhe crude venom as well as lhe venom fractions (HR1 and HR2) are linear and

parallel to each other. This was shown also with a number of other antivenines.

The common slope (b) of the neutralization curves was 1.12. (5)

Eig. 1 shows that: 1) the amount of each antivenine required to neutralize

a given MllD dose of HR2 was larger than that needed to neutralize an equal

dose of HR1; 2) the ED of the three antivenines varied depending on the test

toxin used. The ED of antivenine No. 23 was lhe largest against HR2 and lhe

smallest against HR1 among the three antivenines tested. This situation was re-

versed with antivenine No. 7. The ED of antivenine No. 9 was between tliose

Hemorrhagic activity of test toxin In MHD( scale in log.)

Fig. 1 - - Neutralization of antivenine against three test toxins

as determined by skin test. Test toxin: Crude venom (batch

No. 48) and venom fractions (HR1 and HR2).

SciELO

PROBLEMS IN DETERMINATION OF ANTIHEMOHRHAGIC POTENCY OF 11ABU

CritlPrUiRESUlWS FLAVOVJRIDIS) ANTIVENINE IN THE PRESENCE OF

MULTIPLE IIEMORRIIAGIC PRINCtPLES AND TIIEIR ANTIBODIKS

of the other two anlivenines against either HKI or HR2; 3) lhe Kl)s of lhe

ihrec antivenines against HR2 and againsl lhe crude venom were in lhe same

order.

From these rcsulls we (5) poslulale: I) lhat Ilh’1 and lll!2 are nol <lis-

tinguishable in hemorrhagic action on the rahbit skin but are dislinct immuno-

logically from each other, since lhe amounls of an anlivenine required lo nenlralize

lhe two principies of an eqnal MHD dose were different; 2) lhat each anli¬

venine contains Iwo distinct antihodies corresponding lo each of lhe two hemor-

rhagic principies; d) lhat lhe ratio of lhe quanlily of lhe Iwo antihodies differed

from one anlivenine to another.

Shonld the potency of sueh an anlivenine be titrated against lhe test toxin

conlaining lhe two hemorrhagic principies, lhe question arises as to what the

results of such litration indicate, lhe potency of only anti-HRl or anti-HR2, or

some other implication.

This question can nol be answered unless the meebanism involved in tilration

of the anlivenine potency in the presence of two hemorrhagic principies and the

corresponding antihodies is elucidated.

Mechanism involved in litration of antivenine potency in lhe presence of two

hemorrhagic principies and their antihodies

For elucidating lhe mechanism a model shown in Table 2 was proposed (5).

In the table a white cirele represents a eertain quantity of toxin (HHR1) moleeules

immunologically eqnivalenl lo that of lhe corresponding antihody (anti-HRl)

moleculos as represented liy a double cirele. A filled circle for HR2 and a circle

with a dark spol for anti-HR2 represent quantities immunologically eqnivalenl

to each other.

Assume that the ratio of HR1 to HR2 in a lest toxin is 4:2 and that of

anti-HRl lo anti-HR2 in an antivenine 4:3 (see antivenine A in Table 2). When

HR1 is neutralized equivalcntly by anti-HRl, HR2 lias already been neulralized

by the excess amount of anti-HR2. In this case the end point of litration in

the rahbit skin is entirely dependent on neutralizalion of HR1 by anti-HRl and

only the potency of anti-HRl can be determined. On the other hand, wilh an¬

other antivenine having the ratio of anti-HRl lo anti-HR2 of 6:2 (see antivenine

B), when HR2 is neutralized equivalently by anti-HR2, HR1 has already been

neutralized by lhe excess amount of anti-HRl. In this case, only lhe potency of

anti-HR2 can be determined.

In other words, according lo this model either of lhe two anli-hemorrhagie

activilies will be determined depending on lhe ratio of HR1 lo HR2 in a lest

toxin and on lhat of anti-HRl to anti-HR2 in a test anlivenine.

This model is valid. however, only if lhe following fonr assunqitions liold

good: 1) that the hemorrhagic activilies of HR1 and HR2 are additive but

neither synergistic nor inhibitory; 2) that each of the Iwo neutralization re-

action systems. URI to anti-HRl and 1IR2 lo anti-HR2. lias iIs own imnninological

specifieity; 3) that formation of loxin-anliloxin eomplexes of lhe Iwo systems

occurs at lhe constanl molecular ratio of toxin to anliloxin irrespeetive of lhe

2 3

5 6

11 12 13 14 15

Mom. Inst. Butantan

Simp. Internac.

3S(l):331-337, 1966

AKIRA OI ISA KA, I1ISASIII KONDO, SATORU KONDO,

MASAMI KUROKAWA and RYOSUKE MURATa

335

coneenlration of lhe loxin; and 4) lhat lhe dissocialion constanls of lhe two

neulralizalion reactions are relatively small and of approximately the same

magnitude.

The first assumption lias heen verified hy the results of our experimenta (5).

The results shown in Fig. 1 seem to support the second assumption. The third

and fourth assumptions also seem lo be valid since the two neulralizalion re-

aetions follovved essentially the “J.aw of Multiple Proportion ’ * as shown in l'ig. 1,

where the slope of eaeh neutralization curve is approximately a unity.

For further verifieation of lhe model, an experiment was conducted. In rabie

3, antivenines No. 7. 9 and 23 were titrated separately against HR1 and HR2.

The ratios of anti-HRl to anti-HR2 in these antivenines were calculated lo be

2.73:1, 4.55:1 and 14.5:1, respectively. When the anti-hemorrhagic potencies of

lhe three antivenines are titrated against the test toxins containing HR1 and HR2

at varying ratios, antihemorrhagic activily against the individual hemorrhagic

principies should be determined as predicted in 1 able 3, if our model (rabie 2)

holds true.

TABLE 2 — A MODEL FOR THE MECIIANISM INVOLVED

IN TITRATION OF ANTIVENINE POTENCY IN THE RABBIT

SKIN IN THE PRESENCE OF TWO HEMORRHAGIC

PRINCIPI.ES AND THE CORRESPONDING ANTIBODIES

Proportion of the two hemorrhagic

principies in test toxin

HR 1

HR 2

Test toxin

O O

o o

• ©

Proportion of the two corresponding

antibodies in test antivenine

Specific antihemor¬

rhagic activity to

Anti-HRl

Anti - H R 2

be titrated

Antivenine A

Anti-HR 1

Anlivenine B

® ®

Anti-HR 2

A whito circle (O) represents a cortam quantity of toxin (HRI ) molecules ímmuno-

logically equivalent to that of the corresponding antibody (anti-HR I) molecules

os repro9onted by a double circle ((§)), A filled circle(^) for HR 2 and a circle

with a dark spot((§)) for onti-HR2 represent quantlties immunologically

equivalent to each other.

The “Law of Multiple Proportion” implies that “if a certain amount of antitoxin

neutralizes a certain quantity of toxin, then to neutralize a multiplum of the quantity

of toxin the same multiplum of the amount of antitoxin is requireri” (Jerne, 1951(6)).

SciELO

10 11 12 13 14 15

OOG PROBLEMS TN DETERMINATION OF ANTH1EMORRHAGIC POTENCY OF IIABU

' ,,,n (TRIMERESURVS FLAVOVIRIMfí ) ANTIVENINE IN THE PRESENCE OF

MULTIPLE IIEMORRIIAGIC PRINCIPLES AND THEIR ANTÍBODIES

The prediction sliown in Talile 3 was in exaet accordance wilh lhe aelual

results of lhe experiment, except for anlivenine No. 23 in Section 7. The experi-

ment lias heen puhlished elsewhere (5).

TABLE 3 THE NEUTRALIZATION REACTIONS DETERMINING THE END POINTS

OF TITRATIONS IN THE RABBIT SKIN PREDICTED FROM THE PROPOSED MODEL

(TABLE 1)

Section

Test toxin

Antivenine ( and the ratio of anti-HR 1 to

anti-HR2 in the antivenine)

The rotios of HR 1 to

HR 2

No 7

( 2.73:1)

No .9

(4.55H )

No. 23

( 14.5:1 )

60:40(1.5:0

HR 2*

HR 2

75:251 3:1)

HR 1*

HR 2

83: 1 714.9:1)

HR 1

H R 2

95: 5(1 9:1)

HR 1

* HR I is the obbreviation for tbe neutralijofion reoction of HR1 to onti-HRI; HR2 is for that of

HR2 to onti-HR 2.

CONCI.USION

We conclude that the antihemorrhagic polcncy of Hahu antivenine relativo

to a standard antivenine can only he determined when tlie two hemorrhagic

principies (HR1 and HR2) are used separately as test loxins instead of a crude

venom.

To generalize, all the toxie components in a venom indistinguishable in res-

pect to a hiological response should he separated and each eomponent should be

nsed as a test loxin (3,5). Tliis general conclusion has heen fortified hy our

experiments on determination of antilethal potency of Hahn antivenine. (7)

The reasoning tinis eonfirmed is applicahle to the assay of antitoxic potency

of a polyvalent antivenine. Unless each toxie eomponent in venoms is separated

and availablc as a tesl loxin, monovalent antivenines should not he comhined he-

fore determining the poteneies of lhe individual antivenines, or immunization wilh

mixed venoms should he avoided.

It is worth mentioning the suggestion made hy Iguclii (3) in 1940 ahout lhe

possible presence of multiple lelhal loxic components in culture fillrate of Cl.

welchii ( prrjringens ) lype A and the corresponding antihodies in different pro-

porlions in antitoxin preparalions; he ohserved varying antilethal poteneies for an

antitoxin preparation depending on the test loxin used.

As pointed out hy Llewellyn Smith (9) in 1938, lhe assay of the potency

of tetanus antitoxin is also influeneed hy the test loxin used. Based on similar

observalions, Relrie(lO) in 1943 suggested the multiplieily of composition of

tetanus loxin and lhe eonsequent multiplieily of antihodies in lhe antitoxin

preparalions.

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10 11 12 13 14 15

AKIRA OIISAKA, HISASI1I KONDO, SATORU KONDO,

MASAMI KUROKAWA and RYOSUKE MURATA

337

Mem. Insl. Butantan

S8a‘)'-^l t ^7 aC Í966 MASAMI KUROKAWA and RYOSUKE MURATA

The reasouing descrihed in tliis presenlalion should nalurally he applicahle

lo lhe assay of anliloxic aetivities of various haelerial anlitoxins inchiding Cl.

welchii type A antiloxin and Cl. te.tani anlitoxin.

Acknowledgment — We wish to express our gratitude to lhe Division of Public

Health, Kagoshima Prefecture, Japan, for their generous gifts of Hahu venom.

2 .

References

Ohsaka, A.. Ikezawa, H„ Kondo, II., Kondo, S. & Uchida, N. — Haemorrhagic

aetivities of Habu snake venom, and their relations to lethal toxicity, proteo-

lytic aetivities and other pathological aetivities. Brit. J. Exper. Path., 41:478-

486, 1960.

Kondo, H., Kondo, S„ Ikezawa, II., Murata, II. & Ohsaka, A. — Studies on

the quantitative method for the determination of hemorrhagie activity of Habu

snake venom. Japan. J. Med. Sc. & Biol., 13:43-51, 1960.

3. Ohsaka, A. & Kondo, II. — Biochemistry of snake venoms. Recent Advances

in Medicai Science and, Biology, 1:269-322, 1960.

4. Ohsaka, A., Omori-Satoh, T., Kondo, II., Kondo, S. & Murata, R. —- Bio-

chemical and pathological aspects of hemorrhagie principies in snake venoms

with special reference to Habu ( Trimeresurus flavoviridis ) venom. Mem. Inst.

Butantan, Sirny. Internac., 33(1) :193-205, 1966.

5. Kondo, II., Kondo, S., Sadahiro, S., Yamauchi, K„ Ohsaka, A. & Murata, II.

— Standardization of antivenine. II. A method for determination of antihe-

morrhagic potency of Habu antivenine in the presence of two hemorrhagie

principies and their antibodies. Japan. J. Med. Sc. & Biol., 18:127-141, 1965.

6. Jerne, N. K. — A study of avidity. Acta Pathol. Microbiol. Scand., Suppl., 87:

1-183, 1951.

7. Kondo, H., Kondo, S., Sadahiro, S„ Yamauchi, K., Ohsaka, A. & Murata, R.

— Standardization of antivenine. I. A method for determination of antilethal

potency of Habu antivenine. Japan. J. Med. Sc. & Biol., 18:101-110, 1965.

8. Iguchi, M. — Studies on the titration of the potency of Cl. welchii ( perfrin-

gens) type A antitoxin with special reference to variation in the potency de-

pending on the test toxin used. Japan. J. Buct., No. 536:615-631, 1940.

9. Llewellyn Smith, M. — The standardization of tetanus antitoxin: Factors in-

fluencing the assay. Quart. Buli. Health Organization of League of Nations,

7:739-769, 1938.

10. Petrie, G. F. — Observations on the variable interactions cf tetanus toxins

and antitoxins.

113-143, 1943.

Quart. Buli. Health Organization of League of Nations, 10:

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10 11 12 13 14 15

Mem. Inst. Butantan PINYA COIIEN and EDWARD B. SELIGMANN, JR.

Simp. Internar,

as(1):339-347, 1966

IMMUNOLOCIC STUDIFS OF COKAI. SNAKF VFNOM

PINYA COHEN and EDWARD B. SELIGMANN, JR.

National Institutes of Health, Bethesda, Maryland, U.S.A.

INTKOIHJCTION

Studics ou coral snakc venom were iniliated in 1961 as part of a program

The problcm oí coral snakc bile is not so great in the United States as it

is in Central and South America. Only Ivvo speeies of coral snakes occur in the

United States, and they have a relatively limited geographic range. The most

common of the two, Micrurus fulvius, is found in the southeast and westward

into lhe states hodering the Culf of México. A subspecies, M. fulvius tenere, is

found west of the Mississippi liiver. Most of the specimens of M. fulvius that

were collected lo provide venom for these studies were taken in Florida. The

other coral snakc, Micruroides euryxanthus, represents a monotypic genus. lis

range in lhe United States is limited to the Southern desert regions of Arizona

and New México, however, the snakc is also found in México. !l is considered

rare in the United States.

LD 50 and dose-response relationship of Micrurus fulvius venom

M. fulvius venom was obtained from the Miami Serpentarium, Miami, Flo¬

rida. It was prepared hy freeze-drying pooled. fresh venom from numerous milk-

ings of a large number of snakes.

The LD r> „ for M. fulvius venom was determined using 16-18 gm, albino, mice

of a randomly bred strain. Six mice were injected intraperitoneally witli physio-

logic saline Solutions of the appropriate venom concentration. The results were

recorded afler 18 hours and the LI)-,» calculated according to the method of

Keed and Muench(l). The dose-response relationship is shown in Figure 1.

The slope of the curve is fairly steep and the average LI).-,» is 13.0 pg (0.77

pg/gram of hody weight).

Antibody response to M. fulvius venom in goats

Two Togenherg goats were immunized willi suhcutaneous injections of /!/.

fulvius venom slerilized hy filtralion and mixed with an equal volume of Ain-

phojel * (nlutninum hydroxide gel). The development of neutralizing antibody

,! Wyeth Laboratories, Phlladelphia, Pennsylvanla, U.S.A.

2 3

5 6

10 11 12 13 14 15

340

IMMUNOLOGIC STUDIIÍS OF CORAL SNAKK VENOM

Fig. 1 — Dose-response relationship of Micrurus fulvius venom in

mice.

was followed weekly. The procedure previously described (2) to determine neu-

tralizing antihody in rahhit serum was also used for liie goal serum. Figure 2

shows the immunization schedule and the results of antibody titrations conducted

on pools of serum from the two goals. Neutralizing antihody was initially de-

tected during the sixlh week of immunization. The highest titer ohtained was

105 mouse LD 5I , (1.4 rng of venom) neutralized/ml of antiserum. Earlier work

in rahbits yielded an antiserum with a maximum neutralizing polency, in vitro,

of 38 mouse LD r , c , or approximately 0.5 mg of M. fulvius venom per ml (2).

The results in goats suggest that considerahle fluetuation of antibody leveis oc-

curs. However, once the goals have been well stimulated and develop high anti¬

body leveis, lliese high leveis can be regained rapidly if regular venom doses

are administered. The goats did not exhihit any obvious adverse systemic or

local reaetions during the eourse of immunization. They gained weiglit and lhe

hematocrit value remained normal during periodie lesls.

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10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

33(1): 339-347, 19(1(5

RINYA COIIEN anil EDWARD 15. SELIGMANN, JR.

341

12444468 10-2468--89-I0 -689-555-555555

VENOM (mgs)

Fig. 2 — Productlon of neutralizing antibody against Micrurus fulvius venom in goats.

EFFECTS OF M. fulvius VENOM ON WASHED KED CEIJ.S

Coral snake venom, like most of lhe vcnoms of lhe elapid grou|), has. neuro-

loxic activity. Limited information is available on its other properties. During

experiments with mice vve observed lbat when lethal doses of M. fulvius venom

werc given either intraperitoneally, intravenously or intramuscularly there was

evidence of cilher intravascular bemolysis, or damagc lo lhe vascular bed usually

seen in lhe form of bloody urine although hemorrhaging through lhe nostrils has

occurred occasionally. These observations led lo testing lhe effects of M. fulvius

venom on vvashed red cells of various animal species.

Three lo five day old red cells were washed three times in physiologic saline

and resuspended to two percent. Venom was dissolved in physiologic saline in

a concentration of 200 jug/ml and 0.5 ml of venom added to 2.5 ml of the red

cell suspension. The tubes were incubated at 37"C and periodically observed for

bemolysis of the red cells. Table 1 shows that red cells of the guinea-pig, dog,

mouse, and chicken were lysed but sheep, rabbit, monkey and human cells were

unaffected. Guinea-pig red cells were the most sensilive to the effects of the

venom. Tliis direct hemolytic activity, found in various snake vcnoms (3-5), is

distinct from the lytie action of phospholipase A which is also widely found in

snake venoms, including coral snake venom. Phospholipase A does not lyse washed

red cells but acts indirectly by catalyzing the reaction in which phospholipids,

such as lecithin, are converted lo lytie suhstances, like lysolecithin, which cause

bemolysis. Hoth the direct hemolytic factor and phospholipase A were found in

M. fulvius venom. Although washed rabbit and human red cells were unaffected

by lliis venom, in the presence of egg yolk lecithin these red cells were hemolyzed.

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10 11 12 13 14 15

342

IMMUNOLOGIC STUDIES OF CORAI, SNAKE VENOM

TABLE 1

DIRECT HEMOLYTIC ACTION OF MICRURUS FULVIUS

VENOM ON RED CELLS OF VARIOUS ANIMALS

Time (Hours)

Speci es

Guinea Pig

+++

Dog

—

+++

+ + +

+++

Mouse

—

+++

^mcKen

—

Sheep \

Monkey f

Rabbit ?

Man /

—

DEGREE OF HEMOLYSIS

_ - None + + = Moderate

+ = Slight

+ + + = Complete

INHIBITION OF DIRECT HEMOLYTIC FACTOR RY SERUM

lf antibodies specific for lhe hemolytic factor are produced, theu anliserum

should inhihit hemolytic activily. Both rabhit coral snake anliserum and normal

rahhit serum vvere inactivated at 56°C for 30 minutes. Equal volumes of venom

in a concentration of 320 jug/ml and serum were mixed and incubated willi

0.5 ml of 2 c /c washed guinea-pig red cells al 37°C. Both normal serum and anli¬

serum inhibited hemolysis for 24 hours. However, in lhe control mixturc of only

venom and red cells hemolysis occurred in 30 minutes. The results indicate

inhihilion is nonspecific; il is probably not due lo antibody since normal serum

produced lhe same resull. Normal human serum produced lhe same inhibitory

activity.

TiTRATION OF THE SERUM INHIBITOR

To determine lhe liter of lhe faclor in normal serum responsihle for inhihilion

of hemolysis hy lhe venom, Iwo-fold dilutions of inactivated, normal rabhit serum

vvere prepared. Equal volumes of venom in a concentration of 200 g,g/ml vvere

mixed vvith lhe serum dilutions. The venom-serum mixtures vvere then added

to equal volumes of 2'% guinea-pig red cells and incubated at 37"(i. Figure 3

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10 11 12 13 14 15

SERUM D1LUTI0N

Mem. Inst. Butantan

Simp. Internac.

33(1):339-347, 1966

PINYA COIIEN and EDWARD B. SELIGMANN, JR.

343

shows thc results of tliis litration. All serum dilutions represent initial dilutions

of serum prior to addition of other reagents. I he rate of hemolysis varied de-

pending upon the concentration of serum. Hemolysis occurred between 10 and

24 liours at the 1:4 dilution. There was complete inhibition of hemolysis wilh

undiluted serum and the 1 :2 dilution of serum after 24 hours.

1: 16,384 r

1:4,096

1:1,024

1 : 256

1 = 64

J L

4 6 8 10

TIME OF HEMOLYSIS (hours)

-v , L

Fig. 3 - Inhibition of thc direct hemolytic factor in Micrurus fulvius vcnom by normal

rabbit serum.

I.NHIUITOHY ACTIV1TY OF SREC1FIC SERUM FKACTIONS

Since cuide serum inhihited the direct hemolytic factor in the venom, experi-

ments were condueted lo determine whieh serum fraetions were associated with

inhibitory aetivity. Gamma-globulins (Human Fraetion II), heta-lipoproteins

(Human Fraetion III-O), alpha and hota-globulins (Human Fraetion IV-1) and

bovine alhumin were ohtained from a commereial source. Saline Solutions of

these fraetions were prepared in eoneentrations of 10 mg/ml. Two-fold dilutions

of the fraetions were mixed wilh equal volumes of venom in a concentration of

200 /J.g/ml and this mixture was ineuhated with guineas-pig red eells and observed

for 24 hours. Normal human and rahhit sera were tested with these two fraetions.

The results indieate lliat lhe alpha and heta-glohulins did not preveni hemolysis,

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10 11 12 13 14 15

344

1MMUNOLOGIC STUDIES OF CORAL SNAKE VENOM

liul at (li 1 iiliotis of 1 :2 and I :4 hemolysis was incomplele. Allmmin completely

inhibited hemolysis at dilulions of 1:2 ihrough 1:8. Both of these fractions

produced hemolysis when undiluted. The reason for Ihis is nuclear. However,

it may he due to an osmotic effect or the interaction of venom wilh the fractions

resulting in the release of a lytic agent. The Controls of serum fractions plus

cells withoul venom produced slighlly darkened cells hut no hemolysis.

TABLE 2

C0MPARAT1VE EFFECTS OF M1CRURUS FULVIUS AND

MICRUROIDES EURYXANTHUS VENOMS IN MICE

Micrurus fulvius

Micruroides euryxanthus

I.P.

I.V.

I.P.

I.V.

ld 50

13,<g

7 ,í g

26 ,;g

18 /< g

Onset of

Symptoms

5 min.

3 min.

15 min.

3 min.

Types of

Symptoms

Slight, labored movement

Immobile — Rear legs

appear paralyzed

Skin color

near normal

Skin

deep red

Skin color

near normal

Skin

deep red

Deatli within

4-12 hours

at LD 50

Death within

3-6 hours

at LD 50

Death within

1-2 hours

at LD 50

Death within

45 minutes

at LD 50

Bloody urine

No bloody urine

Prolongcd, labored

respirat ion

Rapid respiration;

less labored

General propekties of M. fulvius venom

The lieat stahility of lhe venom was detcrmined l>y placing tuhes conlaining

M. fulvius venom at a concentralion of 100 pg/ml of salinc in a boiling water

hatli. Tuhes vvere removcd at various times and 0.5 ml (50 pg) injected intra-

peritoneally into 16-18 g mice. All mice which received venom boiled for 20

minutes died. Mice which received venom boiled as long as 1 hours developed

slight signs of distress hui reeovered. There results indicate that the lethal coin-

ponent in M. fulvius venom is highly resistant lo heat.

On the assumption that the lethal component is primarily a protein the venom

was trealcd with trypsin. Venom was used in a concentralion of 100 pg/ml and

crude try|>sin was added to the venom lo give a final concentralion of 1% trypsin.

The mixture was incubated 1 hour at 37"C and mice received 0.5 ml (50 pg)

SciELO

10 11 12 13 14 15

Mem. Inst. Butantan

Simp. Internac.

:«(1):339-347, 19GÜ

PINYA COIIEN and EDWARD B. SELIGMANN, JR.

345

intraperiloneally. Six oí seven mice which

survived. Control mice which rcccivcd only

trypsin survived.

rcccivcd lhe trypsin-treated vcnoin

venom dicd and lhose receivina

COMPARATIVE EFFECTS IN MICE AND SEROLOGIC RELATIONSHIF OF

Micrurus fulvius (MF) and Micruroides euryxanthus (ME) VENOMS

Me venom was obtained from cxtractions performed in our laboratory and

from Dr. James R. Dixon, New Mcxico Slate University. The comparative ef-

fects of lhe two venoms in mice are summarized in Table 2. MF venom gave

lower id) r ,o values although mice which received ME venom died sooner. The

symptoms produced hy thc two venoms diffcred indicating possible diffcrenccs in

lhe lethal components oí the venoms.

Fig. 4 — Gel-diffusion reaetion oí 1 Micrurus fulvius and Micruroides

euryxanthus venom. Anti Elap = Sôro Antielapídico. Anti M. f. = Anti

M. fulvius serum. M. e. — Micruroides euryxanthus venom. M. f.

Micrurus fulvius venom.

2 3 4

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10 11 12 13 14 15

IMMUNOLOGIC STUDXKS OF CORAL SNAKE VENOM

A cross-neutralizalion lest vvas performed lo determine if rabbil anti-MF

seruni woulcl neutralize MK venom. The lest procedures used were previously do-

scribed (2). Both venoms were used in concenlrations of 7.7 LDao/ml. Neilher

neulralization nor preeipitation occurred in lhe helerologous reaclion allhough holh

occurred in lhe homologous MK control reaclion. Insuffieient MK venom lias

been colleeled lo produee anliserurn and perforrn lhe reciprocai cross-neitlraliza-

lion lest.

The relationship of lhe two venorns was also examined by gel-dif fnsion lesls.

The venoms were reacted against pooled, rabbil anti-MF serurn and against Sôro

Antielapídieo (Insliluto Bnlanlan) prodneed wilh Sonlh American coral snake

venoms. Figure 4 is a pholograph of lhe residis of lhe reactions after 3 days.

The MF venom was used in a concentration of 100 ftg/ml and lhe MK venom

was freshly colleeled on a filler paper disc and eliited immedialely wilh saline;

lhe concentration was unknown. The venoms have at least Iwo eommon anligens.

Th is is based on lhe strong reactions of identity formed by holh venoms wilh

Sôro Antielapídieo and lhe faet that MK venom prodneed at least two fainl bands

wilh MF antiserum. One of these fainl bands appears to have formed a reaclion

of partial identity with one of lhe bands prodneed in the homologous MF reaclion.

This reaclion of partial identity suggests lhe two venoms have some eommon

determinants although there are olhei antigenio differences. The strong reaclions

of both venoms with Sôro Antielapídieo indicatc a serologic relationship helween

the coral snake venoms of North and South America. The formalion of scveral

bands helween MK venom and Sôro Antielapídieo serurn and lhe presenee of

only one hand in the reaclion of MF with lliis serurn may be doe to concentration

sinee the aetual amount of MK venom used was unknown. However, il may

aetually indicatc that MK venom is more closely related, serologically, lo lhe

venoms of Soulh American coral snakes than MF venom.

SUMMAHY

tmmunologic and serologic sludies were conducled on venoms of lhe two

speeies of coral snakes foiind in lhe United States. Antiserum produeed in goats

gainst M. fulvius venom was eapahle of neutralizing 105 mouse Ll)., (l /ml: M. ful-

vius venom contains a direet hemolytic agent. Of the serurn fraetions examined.

inliibitory aetivity was found in the alplia and beta-glohulins and alhumin fraetions.

The lethal component in M. fulvius venom is heat stable and suseeptihle lo the

aelion of trypsin. M. fulvius and Micruroidcs curyxanllius venoms have at least

two anligenic components in eommon and are serologically related to Soulh

American coral snake venoms. However, Micruroidcs curyxanllius venom was not

neulralizcd by antiserum speeifie for M. fulvius venom. /I/. fulvius venom was

more toxie and caused m viro hernolysis iu miee. Micruroidcs curyxuullius \enoin

did not produee hernolysis but killed mice more rapidly at the LI) „ dose.

Acknowledyments — The authors gi'atefully acknowledge the able technical as-

sistance o£ Mr. William H. Berkeley throughout the course of these sludies. Ap-

preciation is expressed to Dr. Raymond D. Zinn, Head, N.I.II. Farm Animal Unit,

and Mr. Lconard D. Stuart for maintenance and care of the goats used in

immunizations and for periodic collection of blood samples.

2 3

5 6

11 12 13 14 15