Comparison of the immunogenicity and antigenic composition of several venoms of snakes in the family Crotalidae

Crude venoms from the prairie rattlesnake (Crotalus viridis viridis), the western diamondback rattlesnake (Crotalus atrox), the eastern diamondback rattlesnake (Crotalus adamanteus) and the timber rattlesnake (Crotalus horridus horridus) were used to prepare monovalent antivenoms in rabbits. Each of these four monovalent antivenoms was reacted against six different venoms using the technique of immunoblotting (Western blot) to determine the relative immunogenicity of the four venoms and to compare the antigenic composition of six venoms. In addition to the four venoms listed above, venoms from the South American rattlesnake (Crotalus durissus terrificus) and the fer-de-lance (Bothrops atrox) were tested. SDS-PAGE showed that C. v. viridis venom contains the greatest number of components with 20, and the greatest number (7) less than 15,000 in mol. wt. C. durissus terrificus venom contains the least number of components, having 11. Immunoblotting experiments showed that the greatest reaction between venom and antivenom is not always obtained with the homologous system although the two greatest reactions obtained in this study were for two homologous reactions: that between monovalent anti-C. v. viridis venom and C. v. viridis venom, and that between monovalent anti-C. atrox venom and C. atrox venom. For antivenoms made to C. h. horridus and C. adamanteus venoms, the greatest reaction was obtained with C. atrox venom. There appeared to be no difference in immunogenicity between high-medium mol. wt (greater than 15,000) components and low mol. wt (less than 15,000) components in all systems tested except for C. atrox venom where two low mol. wt components gave a stronger reaction with the antivenom than would have been predicted based on their relative content in the venom as indicated by SDS-PAGE. If the immunoblots are scanned with a densitometer, both the qualitative (number of bands) and the quantitative (density of bands) reactions between venom and antivenoms can be taken into consideration by using a Reactivity Index (number of bands x density of bands). By comparing Reactivity Indexes of the various reactions obtained, the most cross-reactive antivenom tested was the monovalent antivenom to C. v. viridis venom, followed by anti-C. adamanteus, anti-C. atrox and anti-C. h. horridus in order of decreasing reactivity. The Reactivity Index can also be used to estimate the reactivity of a single antivenom with different venoms. The major limitation of this approach is the difficulty in standardizing the detection procedure using silver enhanced Protein A gold.     

Evaluation of four different immunogens for the production of snake antivenoms.

Four different immunogens were used to produce polyvalent antivenom in rabbits to the venoms of Bothrops atrox, Crotalus atrox, Crotalus adamanteus and Crotalus durissus terrificus. The immunogens were: (1) unfractionated mixture of the four crude venoms, and three fractions of the mixture as follows, (2) HPLC gel filtration high (> 50,000) mol. wt fraction, (3) HPLC gel filtration medium (14,000-50,000) mol. wt fraction, and (4) HPLC gel filtration low (< 14,000) mol. wt fraction. The resultant immune sera were compared with commercial antivenom (Wyeth, polyvalent Crotalidae) for total IgG content, ELISA reactivities, patterns of Western blots and ability to neutralize lethal and local hemorrhagic activities of the four venoms. The results indicate that the rabbit antivenoms had significantly higher ELISA reactivity and blotting signals than Wyeth antivenom. However, neither ELISA nor Western blotting signals correlated with the ability of the antivenoms to neutralize the lethal or hemorrhagic activities of the venoms. The protective ability of the antivenoms varied considerably. In general, antivenoms generated by using fractionated venoms as immunogens exhibited greater protective ability than antivenom produced by using the mixture of four venoms as immunogen. Some of the antivenoms provided greater or comparable protective ability for certain venoms when compared to Wyeth antivenom. It appears that the use of certain venom fractions as immunogens is a promising alternative for production of effective antivenoms.     

Antivenomics and venom phenotyping: A marriage of convenience to address the performance and range of clinical use of antivenoms

Toxins from the same protein family present in venoms from snakes belonging to different genera often share antigenic determinants. A practical consequence of this circumstance is that it might be possible to formulate on an immunologically sound basis a mixture of venoms for generating antivenoms against a wide range of species. A deep insight into the inter- and intraspecific variation of the antigenic constituents of venoms from snakes of different geographic origin represents the key for designing novel polyvalent pan-generic antivenoms. This review illustrates how proteomic protocols (‘venomics’ and ‘antivenomics’) can aid in assessing the crossreactivity of antivenoms against homologous and heterologous venoms, establishing thus the range of clinical application. Recent work showing how the knowledge of evolutionary trends along with venom phenotyping may have an impact in designing a mixture of venoms for immunization aimed to produce a pan-American anti-crotalic antivenom is discussed.     

Enzymatic and immunochemical characterization of Bothrops insularis venom and its neutralization by polyspecific Bothrops antivenom.

Herein we compared the biological activities of Bothrops insularis and Bothrops jararaca venoms as well as their neutralization by polyspecific Bothrops antivenom (PBA). On account of that, we investigated their antigenic cross-reactivity and the neutralization of lethal, myotoxic and defibrinating activities by polyspecific and species-specific antivenoms. Silver-stained SDS-PAGE gels evidenced many common bands particularly above 47 kDa between B. jararaca and B. insularis venoms. However, some protein bands between 46 and 28 kDa were observed exclusively in B. jararaca venom. Both venoms presented gelatinolytic, caseinolytic, fibrinogenolytic and phospholipase A(2) activities. No hyaluronidase activity was detected in both venoms by zymography. Polyspecific and species-specific antivenoms showed similar titers to B. jararaca and B. insularis venoms by ELISA, and recognized similar components by immunoblotting. The PBA was effective in neutralizing the lethal, myotoxic and defibrinating activities of both venoms as well as to abrogate microcirculatory disturbances induced by B. insularis venom. No statistically significant difference was observed for minimal hemorrhagic doses between both venoms. Antigenic cross-reactivity was evident between both venoms. Since toxic and enzymatic activities were similar, we speculate that B. insularis venoms can induce a local damage in humans comparable to that observed in other Bothrops venoms. Besides, the PBA was effective in neutralizing the toxic activities of B. insularis venom.     

Antigenic cross-reactivity of venoms obtained from snakes of genus Bothrops

Antigenic cross-reactivity was studied among the components of venoms from nine species of the genus Bothrops using species-specific antivenoms. Sera titration by DOT-ELISA detected similar levels of antibody when either homologous or heterologous antigens were used. Transblotted antigens, after SDS-PAGE fractionation, were also revealed by homologous and heterologous antivenoms. Antigens with mol. wt greater than 30,000 seemed to be the most cross-reactive. Antigens of about 24,000 mol. wt were poorly immunogenic. Antigens between 14-18,000 mol. wt cross-reacted only with B. moojeni, B. jararacussu, B. neuwiedi and B. pradoi venoms. Neutralization of the lethality of B. jararaca venom was observed by homologous and heterologous antivenoms.     

Comparison of the effect of Crotalus simus and Crotalus durissus ruruima venoms on the equine antibody response towards Bothrops asper venom: Implications for the production of polyspecific snake antivenoms

Antivenoms are preparations of immunoglobulins purified from the plasma of animals immunized with snake venoms. Depending on the number of venoms used during the immunization, antivenoms can be monospecific (if venom from a single species is used) or polyspecific (if venoms from several species are used). In turn, polyspecific antivenoms can be prepared by purifying antibodies from the plasma of animals immunized with a mixture of venoms, or by mixing antibodies purified from the plasma of animals immunized separately with single venom. The suitability of these strategies to produce polyspecific antibothropic-crotalic antivenoms was assessed using as models the venoms of Bothrops asper, Crotalus simus and Crotalus durissus ruruima. It was demonstrated that, when used as co-immunogen, C. simus and C. durissus ruruima venoms exert a deleterious effect on the antibody response towards different components of B. asper venom and in the neutralization of hemorrhagic and coagulant effect of this venom when compared with a monospecific B. asper antivenom. Polyspecific antivenoms produced by purifying immunoglobulins from the plasma of animals immunized with venom mixtures showed higher antibody titers and neutralizing capacity than those produced by mixing antibodies purified from the plasma of animals immunized separately with single venom. Thus, despite the deleterious effect of Crotalus sp venoms on the immune response against B. asper venom, the use of venom mixtures is more effective than the immunization with separate venoms for the preparation of polyspecific bothropic-crotalic antivenoms.     

Enzymatic characterization, antigenic cross-reactivity and neutralization of dermonecrotic activity of five Loxosceles spider venoms of medical importance in the Americas.

Loxosceles spiders have a wide distribution in the temperate and tropical regions of the world. Loxoscelism is characterized by necrotic skin ulceration at the bite site and, less commonly, a systemic illness that may be fatal. The purpose of this study was to characterize and compare aspects of the major medically important Loxosceles spider venoms in a standardized manner, particularly considering their neutralization by two Brazilian antivenoms. By SDS-PAGE (12% acrylamide), Loxosceles deserta, Loxosceles gaucho, Loxosceles intermedia, Loxosceles laeta and Loxosceles reclusa venoms had similar electrophoretic profiles, with the major protein bands of 32-35 kDa. All venoms exhibited gelatinolytic, caseinolytic and fibrinogenolytic activities in vitro with a large array of proteases, mainly between 18.1 and 31.8 kDa. Most of these enzymes were metalloproteases as this activity was abolished by 1,10-phenanthroline. Hyaluronidase activity was detected in a protein band of approximately 44 kDa in all venoms. Sphingomyelinase activity was demonstrated in all five venoms. Antigenic cross-reactivity, by Western blotting, was also observed among all venoms studied using commercial equine antivenoms produced in Brazil (Institute Butantan and CPPI). These antivenoms recognized mainly components between 25 and 40 kDa in all venoms with several minor components of >89 kDa. Strong cross-reactivity was also seen among all venoms through the ELISA technique (titre range: 64,000-512,000). All venoms (5 microg doses) induced a similar local reaction when injected intradermally into the flank of rabbits, demonstrating dermonecrosis, hemorrhage, vasoconstriction, edema, and erythema. However, no reaction was observed when each venom was pre-incubated (1 h, 37 degrees C) with Brazilian commercial sera prior to injection. The antivenoms also abolished the sphingomyelinase activity in vitro, suggesting the venoms of the major medically important Loxosceles spider species have generally similar toxic and enzymatic characteristics. Thus, as Brazilian commercial antivenoms are able to neutralize the dermonecrosis induced by Loxosceles venoms of diverse geographical origin, clinical studies should be undertaken on the potential for a single global Loxosceles antivenom.     

Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica.

Horse IgG isotypes and cross-neutralization of two snake antivenoms produced in Brazil and Costa Rica. Toxicon 000-000. This work compared the specificity, ELISA titers and IgG subclass content of the polyvalent antivenom (anti-Bothrops asper, Crotalus durissus durissus and Lachesis muta stenophrys) of Instituto Clodomiro Picado (Costa Rica) and the bothropic antivenom (anti-Bothrops jararaca, B. jararacussu, B. moojeni, B. neuwiedi and B. alternatus) of Instituto Butantan (Brazil). The role of IgG(T) and IgGa subclasses in neutralization of some venom toxic activities and the cross neutralization of the antivenoms against B. jararaca and B. asper venoms were also evaluated. Both antivenoms were able to recognize B. asper and B. jararaca venoms by immunoblotting and presented similar antibody titers when assayed by ELISA. IgG(T) was highest, followed by IgGa, IgGb and IgGc. IgGa and IgG(T) isotypes isolated from both antivenoms by affinity chromatography were tested for neutralization of lethal, hemorrhagic, coagulant and phospholipase A2 activities of the homologous venoms. In both antivenoms, IgG(T) was the major isotype responsible for neutralization of all the tested activities, followed by IgGa. These results suggest that Instituto Butantan and Instituto Clodomiro Picado antivenoms have the same IgG profile and their neutralizing ability is due mostly to the IgG(T) isotype. Also, they neutralize lethality in mice induced by homologous and heterologous venoms, the bothropic antivenom of Instituto Butantan being more effective.     

Differences in distribution of myotoxic proteins in venoms from different Bothrops species

Antigens with high myotoxic activity were isolated from Bothrops jararacussu venom by Sephadex G-75 and SP-Sephadex C-25. These antigens were recognized using western blotting by B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi antivenoms, and weakly by B. jararaca antivenom. B. alternatus, B. atrox, B. cotiara and B. erythromelas antivenoms failed to recognize these antigens. Antisera raised against these antigens recognized bands with mol. wt around 18,000 in the venoms of B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi and reacted in ELISA with non-denaturated B. jararaca venom. However it failed to react in ELISA with nondenatured B. alternatus, B. atrox, B. cotiara and B. erythromelas venoms. The myotoxicity induced by these crude venoms confirmed that these antigens are possibly the only major myotoxin as the levels of creatine phosphokinase activity in mice serum released by intramuscular injection of B. jararacussu, B. moojeni, B. neuwiedi and B. pradoi venoms (myotoxin +) were five to eight-fold higher than those obtained with B. alternatus, B. atrox, B. cotiara, B. erythromelas and B. jararaca venoms. Using the double immunodiffusion technique the myotoxins of B. jararacussu, B. neuwiedi and B. pradoi showed total identity while B. moojeni myotoxin behaved as a partially identical antigen.     

In-vitro Neurotoxicity of Two Malaysian Krait Species (Bungarus candidus and Bungarus fasciatus) Venoms: Neutralization by Monovalent and Polyvalent Antivenoms from Thailand

Bungarus candidus and Bungarus fasciatus are two species of krait found in Southeast Asia. Envenoming by these snakes is often characterized by neurotoxicity and, without treatment, causes considerable morbidity and mortality. In this study, the in vitro neurotoxicity of each species, and the effectiveness of two monovalent antivenoms and a polyvalent antivenom, against the neurotoxic effects of the venoms, were examined in a skeletal muscle preparation. Both venoms caused concentration-dependent inhibition of indirect twitches, and attenuated responses to exogenous nicotinic receptor agonists, in the chick biventer preparation, with B. candidus venom being more potent than B. fasciatus venom. SDS-PAGE and western blot analysis indicated different profiles between the venoms. Despite these differences, most proteins bands were recognized by all three antivenoms. Antivenom, added prior to the venoms, attenuated the neurotoxic effect of the venoms. Interestingly, the respective monovalent antivenoms did not neutralize the effects of the venom from the other Bungarus species indicating a relative absence of cross-neutralization. Addition of a high concentration of polyvalent antivenom, at the t₉₀ time point after addition of venom, partially reversed the neurotoxicity of B. fasciatus venom but not B. candidus venom. The monovalent antivenoms had no significant effect when added at the t₉₀ time point. This study showed that B. candidus and B. fasciatus venoms display marked in vitro neurotoxicity in the chick biventer preparation and administration of antivenoms at high dose is necessary to prevent or reverse neurotoxicity.     

Cross reactivity of mono- and polyvalent antivenoms with Viperidae and Crotalidae snake venoms

The lethal, local and defibrinating effects of two African Viperidae and four Crotalidae were assayed. Two commercial antivenoms (Behringwerke Nord Africa and Wyeth anticrotalic) and gamma-globulins from hyperimmune rabbit serum were tested for their ability to neutralize the toxic effects of the venoms. Cross neutralization of the lethal effect (antiviperidae antivenom against Crotalidae venom and vice versa) was evident with Behringwerke against Crotalidae as well as with Wyeth against Viperidae. No cross-reactivity was observed with monovalent antivenoms. Practically complete cross-reactivity was observed with polyvalent, but less with monovalent antivenoms in the neutralization of skin hemorrhage. The local effect was neutralized to 0-20% if the antivenom was mixed with the venom. An i.v. injection of antivenom had no effect after intradermally injected venom. Only Echis and Bothrops venoms cause defibrination in vivo. The ability of the two polyvalent antivenoms to neutralize defibrination was very weak. One half milliliter of antivenom neutralized 1 microgram (Echis carinatus) or 10 micrograms (Bothrops asper) of defibrinating venom if injected simultaneously. If antivenom was administered 3 hr after the venom, the fibrinogen level increased to about 50% of the normal values after 24 hr. Cross-reactivity in the neutralization of the defibrinating activity was observed with both polyvalent antivenoms.     

Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol.

The purpose of this study was to prepare a potent polyvalent antivenom against three elapids namely, the Thai cobra (Naja kaouthia, NK), the King cobra (Ophiophagus hannah, OH) and the banded krait (Bungarus fasciatus, BF). Two groups of horses were immunized. Group 1, comprising five horses, was immunized twice with a mixture of postsynaptic neurotoxins followed by an additional six immunizations with a mixture of crude venoms of the three elapids. Group 2, comprising four horses, was immunized with a mixture of crude venoms throughout the course. For the first immunization, the immunogens were emulsified in Complete Freund's adjuvant and injected using a low dose, low volume multi-site immunization protocol previously developed in this laboratory (Pratanaphon, R., Akesowan, S., Khow, O., Sriprapat, S. and Ratanabanangkoon, K. (1997) Production of highly potent horse antivenom against the Thai cobra (Naja kaouthia). Vaccine 15, 1523-1528). The second immunization was carried out with the immunogens in Incomplete Freund's adjuvant. Blood was drawn to assay the antibody titer by ELISA. Sera at the peak of ELISA titers were pooled and assayed for the median effective dose (ED(50)). The ED(50)'s of antivenom from Group 1 horses against NK, OH and BF venoms were 1.44, 0.22 and 0.23 ml serum/mg venom, respectively, while those from Group 2 horse sera were 0.88, 0.20 and 0.49 ml serum/mg venom, respectively. The potency of sera from Group 2 against BF venom was significantly higher, while the potencies against NK and OH venoms were comparable to those of the corresponding monovalent antivenoms produced under the same protocol. This potent, truly polyvalent antivenom should be useful in saving lives of victims envenomed by these elapids and the immunization protocol should be useful in the production of potent polyvalent antivenoms against other medically important elapids.     

Effectiveness of two common antivenoms for North, Central, and South American Micrurus envenomations

Micrurus snakes (coral snakes) may produce severe envenomation that can lead to death by peripheral respiratory paralysis. Only few laboratories produce specific antivenoms, and despite the cross-reactivity found in some Micrurus species venoms, the treatment is not always effective. To test two therapeutic antivenoms against the venom of four species of Micrurus from Southern America, North of South America, Central America, and North America, the determination of the lethal potency of the venoms, the study of some biochemical and immunochemical characteristics, and the determination of the neutralizing activity of both antivenoms were studied. North American and South American antivenoms neutralized well venoms from Micrurus species of the corresponding hemisphere but displayed lower effectiveness against venoms of species from different hemispheres. It was concluded that the neutralization of Micrurus venoms by regional antivenoms could be useful to treat the envenomation by some Micrurus snakes but is necessary to evaluate carefully the antivenoms to be used with the venoms from the snakes of the region. Also, considering the difficulties for coral snake antivenom production, the development of a polyvalent antivenom is useful to treat the envenomation by coral snakes from different regions is necessary.     

Lachesis muta muta venom: immunological differences compared with Bothrops atrox venom and importance of specific antivenom therapy.

Lachesis muta muta and Bothrops atrox snakes are responsible for most accidents occurring in the Amazon. The clinical features of the accidents are similar; however, there are still controversies about the efficacy of Bothrops antivenoms for treating L. m. muta accidents. In this work, we evaluated the antigenic cross-reactivity between these venoms using polyclonal and monoclonal antibodies and the efficacy of B. atrox and L. m. muta experimental antivenoms in cross-neutralizing the main toxic activities of each venom. Electrophoretic patterns differed consistently between the species. However, antigenic cross-reactivity was extensive except for a few bands. Several species-specific monoclonal antibodies were obtained by immunization of Balb/c mice with L. m. muta whole venom or B. atrox and L. m. muta specific antigens. The monoclonal antibodies specific to L. m. muta recognized different bands of this venom and the antibodies specific to B. atrox recognized a complex pattern on whole venom by Western blotting. These antibodies are important tools for developing an immunoassay able to discriminate patients bitten by these snakes. The experiments involving cross-neutralization of the main activities of the venoms showed that hemorrhage and blood incoagulability induced by B. atrox venom were similarly neutralized by both B. atrox and L. m. muta antivenoms. However, B. atrox antivenom partially neutralized the hemorrhage and completely failed in neutralizing coagulopathy induced by L. m. muta venom. Therefore, antigenic variation between B. atrox and L. m. muta venoms does occur and the use of specific antivenom is suggested for patients bitten by Lachesis snakes.     

An indirect haemolytic assay for assessing antivenoms

Dilutions of antivenom, venom, human erythrocytes and a phosphatidylcholine suspension, were incubated for 30 min at 37 degrees C. After centrifugation, the liberated haemoglobin was measured spectrophotometrically. The assay was used to assess an ovine antivenom against the venom from the South American rattlesnake, Crotalus durissus terrificus, and an equine Wyeth antivenin (Crotalidae, polyvalent). The ovine antivenom was more than five times as effective as the equine product. It also neutralized venoms from the Western diamondback rattlesnake, Crotalus atrox, and the fer-de-lance, Bothrops atrox. However, antivenoms raised against venoms from other Crotalus and Bothrops species provided little protection against the haemolytic activity of C. d. terrificus venom.     

Commercial monovalent antivenoms in Australia are polyvalent

Monovalent antivenoms have a lower volume of specific antibodies that may reduce reactions but require accurate snake identification to be used. Polyvalent antivenoms are larger volume and may have a higher reaction rate. However, they avoid the problem of snake identification and may be more cost-effective to manufacture. We have previously shown cross-neutralisation of two Australian elapid venoms, tiger snake (Notechis scutatus) and brown snake (Pseudonaja textilis) venoms, by their respective monovalent antivenoms. In this study enzyme immunoassays were used to quantify the amount of monovalent antivenom (quantity of monovalent antibodies to a specific snake venom) in vials of commercially produced antivenom in Australia. All antivenoms tested appeared to be polyvalent and contain varying amounts of all five terrestrial snake monovalent antibodies based on their binding to the five representative venoms. Redback spider antivenom did not have any measurable binding affinity for any of the five snake venoms, showing that the observed binding is not due to non-specific interactions with equine protein. The antivenoms had expiry dates over a 15 year period, suggesting that the antivenoms have been mixtures for at least this time. This study cannot be used to rationalise hospital stocks of antivenom in Australia because there is no guarantee that the antivenoms will remain as mixtures. However, it would be possible for the manufacturer to reduce the number of types of snake antivenoms available in Australia to two polyvalent antivenoms which would simplify treatment of snakebite.     

Antibacterial properties of KwaZulu natal snake venoms.

The objective was to ascertain whether local snake venoms have antibacterial properties. The venoms of the common night adder (Causus rhombeatus), gaboon adder (Bitis gabonica), puff adder (Bitis arietans), black mamba (Dendroaspis polylepis), eastern green mamba (Dendroaspis augusticeps), forest cobra (Naja melanoleuca), snouted cobra (Naja annulifera) and Mozambique spitting cobra (Naja mossambica) were collected and, by gel diffusion, tested against the bacteria Staphylococcus aureus, Escherichia coli, Pseudomonas aeriginosa, Bacteriodes fragilis, Bacteroides intermedius, Clostridium sordellii and Clostridium perfringens. All snake venoms showed antibacterial activity, with the adders showing most activity against the aerobes while the cobras showed lesser, but equal activity against the aerobes and anaerobes. Black mamba venom only showed activity against C. perfringens. In conclusion, local snake venoms have antibacterial properties which are dependent on the venom and bacterial type; and in the Naja spp., for anaerobic bacteria, diminish in winter. There is liable to be more than one toxin component responsible.     

Differences between the venoms of two sub-species of Russell's viper: Vipera russelli pulchella and Vipera russelli siamensis

Ion exchange chromatography was carried out using venoms obtained from two sub-species of Russell's viper; V. russelli siamensis from Burma and V. russelli pulchella from Sri Lanka. Differences were observed in the elution position of venom components having haemolytic and procoagulant activity but not those causing fibrinolysis. Only the V. russelli siamensis venom exhibited any platelet aggregating activity. The Indian (Haffkine) polyspecific and the Burmese (Burma Pharmaceutical Industries) monospecific antivenoms, when used in cross immunoelectrophoresis against the two venoms, revealed differences in the number and/or intensity of the precipitin bands present. An important functional consequence of this was that the Burmese antivenom did not neutralize the haemolytic activity of the V. russelli pulchella venom in vitro and would thus probably not be effective in treating this consequence of envenoming by Russell's viper in Sri Lanka. Differences in the composition and the clinical effects of the two venoms emphasizes the importance of using venom from the local snake for antivenom production if optimal clinical efficacy is to be achieved.     

Toxoids and Antivenoms of Venomous Snakes in Taiwan

Abstract

There are in Taiwan six major venomous snakes which can inflict severe bite on human victims. They are three hemorrhagic species i.e. the Taiwan habu (Trimeresurus mucrosquamatus), the green tree viper (Trimeresurus stejnegeri), and the hundred-pace snake (Deinagkistrodon acutus); two neurotoxic species, i.e. the cobra (Naja naja atra) and the krait (Bungarus multicinctus); and the Russell's viper (Daboia r. formosensis) whose venom is both coagulopathy and neurotoxic. Our aim has been the production of highly potent antivenoms for snake-bite treatment in this country. Among individual antivenoms for Taiwan venomous snakes, only those from the pitvipers show partial cross-neutralizing capacity with venoms of other pit vipers.

As all snake venoms are quite lethal to animals, it is important to tame or detoxify the crude venom before using it on the animal to obtain antivenoms. We have demonstrated that glutaraldehyde can be used successfully not only in the detoxification of snake venoms but also improving their immunogenecity. Protocols for toxoid preparation from the crude venoms in the process of manufacturing highly potent antisera have been improved in our institute. Two bivalent equine antivenoms specific for either the combined glutaraldehyde-treated venoms of N. n. atra and B. multicinctus or those of T. mucrosquamatus and T. stejnegeri were successfully produced and proven to be effective and useful. A tetravalent equine antivenom has been prepared likewise against the four major viperid venoms in Taiwan. Recently, we also developed a process to prepare an efficient hexavalent antivenom against all the six major venomous snakes.     

Specific identification of Lachesis muta muta snake venom using antibodies against the plasminogen activator enzyme, LV-PA.

Sandwich-type enzyme linked immunosorbent assays (ELISA) were developed to detect Lachesis muta muta (bushmaster) snake venom using antibodies against the plasminogen activator enzyme (LV-PA). Antibodies to LV-PA were obtained by immunization of one rabbit with the purified enzyme. The IgG fraction was purified from rabbit blood in a single step on a column of Sepharose-L. m. muta venom and used to coat the microtiter plates. The specificity of the assay was demonstrated by its capacity to correctly discriminate between the circulating antigens in mice that were experimentally inoculated with L. m. muta venom from those in mice inoculated with venoms from Bothrops atrox, B. brazili, B. castelnaudi, Bothriopsis taeniata, B. bilineata, Crotalus durissus ruruima and the antigenic Bothrops (AgB) and Crotalus (AgC) pools venoms used to produce Bothropic and Crotalic antivenoms at Fundacao Ezequiel Dias (FUNED). Measurable absorbance signals were obtained with 1.5 ng of venom per assay. The ELISA was used to follow the kinetic distribution of antigens in experimentally envenomed mice.