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The ability of serum from nine newborn specimens of Clelia clelia (Colubridae) to neutralize hemorrhagic action of Bothrops asper venom was tested. All serum samples neutralized completely the hemorrhagic effect of the venom in mice. This finding shows that the neutralizing ability of C. clelia serum towards the hemorrhagic activity of B. asper venom is innate.

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The coagulant, defibrinating, fibrino lytic and fibrinogenolytic activities of venoms from ten species of Costa Rican crotaline snakes were studied, together with the neutralization of these effects by a polyvalent antivenom. The venoms of Bothrops asper, B. schlegelii, B. nummifer, B. godmani, Lachesis muta and Crotalus durissus induced a coagulant effect in vitro, and all of them, with the exception of B. nummifer, also induced defibrination in vivo. The four non-coagulant venoms (B. lateralis, B. ophryomegas, B. nasuta and B. picadoi) induced a degradation of the alpha (A) chain of fibrinogen, thereby inhibiting coagulation. However, they did not induce defibrination upon i.v. injection. All of the venoms showed fibrinolytic activity in vitro. Polyvalent antivenom was effective in the neutralization of coagulant, defibrinating, fibrinolytic and fibrinogenolytic activities of these venoms, with the exception of coagulant effect induced by C. durissus venom. Since only three venoms are used in the immunization of horses, these results demonstrate the high degree of immunological cross reactivity between components affecting coagulation in Costa Rican crotaline snake venoms.

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A monovalent antivenom was produced by immunizing two horses with venom of the pit viper Bothrops asper (Ophidia: Viperidae). Although development of the immune response against four toxic and enzymatic activities of the venom was similar in both horses during the first two thirds of the immunization schedule, antibody response in one of the horses reached much higher levels in the last part of the immunization. Immunoelectrophoretic analysis indicates that there were precipitating antibodies in the sera of these horses during all the stages of immunization. However, immunoprecipitation did not correlate with the ability of sera to neutralize toxic activities of B. asper venom. Monovalent antivenom was more effective than the commercially available polyvalent antivenom in the neutralization of Bothrops asper venom. On the other hand, despite the fact that it neutralizes lethal and hemorrhagic activities of the venoms of Lachesia muta and Crotalus durissus durissus, it was less effective than polyvalent antivenom in these neutralizations. Moreover, it does not neutralize defibrinating activity induced by these two venoms, whereas it neutralizes this effect in the case of B. asper venom. It is proposed that monovalent antivenom may be highly effective in the case of envenomations induced by Bothrops asper venom; its use in treating accidents by L. muta and C. durissus would be indicated only if polyvalent antivenom is not available. Results also demonstrate that it is important to monitor antibody response individually in horses being immunized for antivenom production, due to the conspicuous variability in the response of different animals.

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The distribution, karyotype and morphological characteristics of Bothrops ophryomegas from Costa Rica are described. This species presents a conspicuous sexual dimorphism and dichromatism. It is distributed in the dry forest areas of Guanacaste and Puntarenas. Its karyotype is indistinguisable from those described for other crotaline species.

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In Costa Rica, rattlesnakes mate during the early dry season (December and January) and births occur in the early rainy season (May-July). Gestation is therefore about 6 months. The mean number of offspring is 22.9 and is significantly correlated with the size of the female. Newborn rattlesnakes are 27.5-43.0 cm in length and weight 11.4-46.3 g. They are relatively docile. Adult males are longer and heavier than females. Females seem to have their first litter when their size exceeds 120 cm.

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The coral snake Micrurus nigrocinctus has two reproductive patterns in Costa Rica. Specimens of the Pacific population (M. n. nigrocinctus) mate during the early dry season (November, January). Oviposition takes place in February and March; the mean number of eggs was 7.9 (5-14) in this population. Births occur between April and June after 47-81 days of incubation. The total length of neonates is 168-212 mm, and the weight is 1.2-2.0 g. Specimens of the Atlantic population (M. n. mosquitensis) seem to have an extended breeding season. Oviposition in this subspecies was observed in March and June; the mean number of eggs was 6.7 (5-8). Births take place in May and August, after two months of incubation. Neonates have 173-189 mm in total length and 1.9-2.4 g in mass. Adult females are longer than males, especially in M. n. mosquitensis.

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The ability of several batches of polyvalent antivenom to neutralize indirect hemolytic activity of Bothrops asper venom was studied using a sensitive plate test. All samples of antivenom tested effectively neutralized this activity. A highly significant correlation was observed between neutralization of indirect hemolysis and neutralization of lethal activity. This simple and sensitive in vitro test could be used to monitor antibody levels in horses immunized to produce polyvalent antivenom.

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We studied the ability of the polyvalent antivenom produced in Costa Rica to neutralize lethal, hemorrhagic, edema-forming, proteolytic, hemolytic, hyaluronidase and fibrinolytic activities of the venoms of Bothrops asper and B. nummifer from Honduras, and of Agkistrodon bilineatus and Crotalus durissus durissus from Guatemala. Neutralizing ability of antivenom was expressed as ED50 (effective dose 50%), defined as the antivenom/venom ratio at which the activity of the venom is reduced 50%. Antivenom is highly effective in the neutralization of lethal, hemorrhagic, hemolytic, hyaluronidase, and caseinolytic activities of B. asper, B. nummifer, and C. d. durissus venoms. In the case of B. nummifer venom, neutralization of fibrinolytic effect was only partial, whereas this activity was adequately neutralized when studying the venoms of B. asper and C. d. durissus. The venom of A. bilineatus was adequately neutralized by the antivenom, with the only exception of hemolytic effect that was reduced only partially. However, in quantitative terms, a relatively large volume of antivenom was required to neutralize some effects induced by A. bilineatus venom. Regarding edema-forming activity, antivenom neutralized efficiently the venoms of B. asper and A. bilineatus, whereas that of B. nummifer was neutralized only partially; on the other hand, edema induced by the venom of C. d. durissus was not neutralized at all. Immunochemical results indicate a close immunological relationship between venoms of B. asper, B. nummifer and C. d. durissus collected in Honduras and Guatemala with those of the same species collected in Costa Rica. Interspecies comparison, however, showed variation between venoms obtained from different species.(ABSTRACT TRUNCATED AT 250 WORDS)

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The time-course and composition of inflammatory infiltrate in mouse gastrocnemius injected with Bothrops asper venom was studied. The venom induced myonecrosis, and a prominent decrease in muscle levels of creatine kinase (CK) as early as 3 hr after envenomation. Inflammatory infiltrate was scarce by 6 hr. but increased markedly at 24, 48 and 72 hr. Samples of infiltrate obtained at 6 and 24 hr contained polymorphonuclear leucocytes as the predominant cell type, whereas at 48 hr and 72 hr the relative number of macrophages increased. Inflammatory cells were located within necrotic muscle cells, as well as in the interstitial space, but there were some necrotic areas devoid of inflammatory cells even one week after envenomation. When correlating the presence of inflammatory cells with degradation of myofibrillar proteins, it was observed that at 6 hr there was little muscle protein degradation. By 48 hr a decrease in "non collagen" proteins was observed, together with a reduction in some myofibrillar components, as judged by electrophoresis. Proteolytic enzymes of inflammatory cells may play an important role in myofibrillar protein degradation after myonecrosis induced by B. asper venom.

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Liquid and lyophilized samples of Lachesis muta venom were stored at different temperatures and for different periods of time, and analyzed by polyacrylamide gel electrophoresis (PAGE) and immunoelectrophoresis. Only slight variations were evident when three pools of freeze-dried venom, that had been kept at -30 degrees C for several months were compared with fresh venom. These results suggest that L. muta venom is not altered drastically when stored under these conditions.

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The polyvalent antivenom produced at the Instituto Clodomiro Picado, Costa Rica, was tested for its capacity to neutralize proteolytic and hemorrhagic activities of ten Costa Rican crotaline venoms. In experiments with preincubation of venom and antivenom, the latter efficiently neutralized proteolytic activities of nine venoms, with ED50 ranging from 50 to 300 microliters antivenom/mg venom. The venom of Bothrops nummifer was neutralized less efficiently (ED50 = 760 microliters/mg.) Antivenom was also very effective in neutralizing hemorrhagic activity, having its lowest neutralizing ability against the venom of B. picadoi (ED = 430 microliters/mg) and its highest towards the venom of B. asper (Pacific region) (ED50 = 47 microliters/mg). There was a significant correlation between the ability of antivenom to neutralize proteolytic and hemorrhagic effects. In spite of the ability of antivenom to neutralize hemorrhage when incubated with venom prior to injection, hemorrhage was only partially neutralized when antivenom was administered i.v. at different time periods after envenomation. This suggests that the rapid development of local hemorrhage, instead of the absence of antivenom antibodies, is the explanation for the poor neutralization observed in these types of experiments.

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The ability of a polyvalent antivenom produced in Costa Rica to neutralize hyaluronidase and indirect hemolytic activities of Costa Rican crotalid venoms was tested. Antivenom neutralized remarkably well hyaluronidase activity of all venoms tested, with ED50 doses ranging from 2 to 45 microliter antivenom/mg venom. Neutralization of indirect hemolytic activity was also achieved using the antivenom, with ED50 values ranging from 332 to 716 microliter antivenom/mg venom.

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Biochemically and pharmacologically, myotoxins isolated from snake venoms can be placed in four main groups: myotoxic phospholipases A, low molecular weight basic toxins, cardiotoxins, and hemorrhagic myotoxins. The myotoxic phospholipases A notexin, taipoxin, crotoxin, and Bothrops asper myotoxin induce muscle necrosis by first affecting the integrity of the plasma membrane, thereby inducing a calcium influx that culminates in cell death. The small basic myotoxin crotamine acts on the voltage-sensitive sodium channels of skeletal muscle sarcolemma, inducing a sodium influx which is responsible for depolarization and contraction of skeletal muscle, as well as for vacuolization of sarcoplasmic reticulum. Cardiotoxins are basic membrane-active polypeptides that disorganize the structure of membranes; the myotoxic activity of cardiotoxins results from their ability to disrupt skeletal muscle sarcolemma. Finally, two hemorrhagic toxins (hemorrhagic toxin b and viriditoxin) are myotoxic; apparently, they secondarily to ischemia which develops in muscular tissue as a consequence of the hemorrhagic action of these toxins.

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The karyotypes of five species of colubrid snakes from Costa Rica are as follows: Imantodes cenchoa and Drymobius margaritiferus have a diploid number of 36, with 16 macro- and 20 microchromosomes. The fourth pair is heteromorphic in females of I. cenchoa, with a metacentric Z and a submetacentric W chromosomes. Karyotypes of Erythrolampius bizonus and Leimadophis epinephalus have 28 chromosomes, without a clearcut separation between macro- and microchromosomes. In the case of E. bizomus, the fourth pair contains the sex chromosomes Z and W, both are submetacentric, but the W is smaller. Xenedon rabdocephalus has a diploid number of 34 chromosomes (22 macro- and 12 microchromosomes); pair 3 is heteromorphic in females, with a submetacentric Z and a smaller metacentric W. The karyotype of X. rabdocephalus may be derived from a primitive karyotype by means of reduction in the number of microchromosomes and centric fissions of two pairs of metacentric autosomes.

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The pathogenesis of local lesions induced by Bothrops picadoi venom was studied at the electron microscopic level. White mice were injected i.m. with 100 micrograms of venom. A strong hemorrhagic action was the earliest and most prominent local effect observed in this study. In skeletal muscle fibers, rupture of the sarcolemma was observed and myofibril structure was drastically affected, showing amorphous masses of myofilaments. Myelin layers in many axons were disorganized.

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This paper describes the species of venomous snakes found in Costa Rica as well as anti-venom sera prepared by the Instituto Clodomiro Picado: polyvalent sera for use in human or veterinary medicine, and anti-Micrurus and anti-Lachesis monovalent sera. These sera are prepared in horses, purified with ammonium sulphate, then concentrated and delivered either in liquid or freeze-dried form. Controls are in accordance with NIH recommendations. For cases of hypersensitivity to horse serum, a polyvalent anti-Bothrops and anti-Crotalus serum from sheep has been prepared.

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A multivalent coral snake antivenin was prepared in horses immunized with a mixture of venoms from the species Micrurus nigrocinctus, M. mipartitus, and M. frontalis, following immunization procedures previously reported (2). Plasma from the horses was fractionated with ammonium sulfate. The antivenin produced was then tested against venoms from ten species. The neutralization titers obtained indicate it would be useful in treating bites received from most of the important coral snake species in North and South America, namely: M. fulvius, M. alleni, M. carinicaudus dumerilii, M. corallinus, M. frontalis, M. lemniscatus, M. mipartitus, M. nigrocinctus, and M. spixii. The authors note that appropriate cooperation by scientists in various countries would make production of this antivenin an easy matter, and that the product could conveniently be distributed via PAHO or other international agencies. They also note that the antivenin showed no significant neutralizing effect against M. surinamensis venom. This situation would appear to justify preparing a monovalent antivenin against M. surinamensis, or else including M. surinamensis venom in an immunization formula for a multivalent antidote.

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