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.     

Neutralization, by a monospecific Bothrops lanceolatus antivenom, of toxic activities induced by homologous and heterologous Bothírops snake venoms.

A monospecific Bothrops lanceolatus antivenom, currently used in Martinique, was tested for its efficacy in the neutralization of several toxic and enzymatic activities of the venoms of B. lanceolatus, B. atrox and B. asper. When tested by the i.p. route in mice, B. lanceolatus venom had an LD50 of 12.8 microg/g. In addition, it induced local tissue damage (hemorrhage, edema and myotoxicity) and showed indirect hemolytic activity, but was devoid of coagulant effect on human plasma in vitro and of defibrinating activity in mice. Antivenom was fully effective in the neutralization of lethal, hemorrhagic, edema-forming, myotoxic and indirect hemolytic effects of B. lanceolatus venom in assays involving preincubation of venom and antivenom. When tested against the venoms of B. asper and B. atrox, the antivenom completely neutralized the lethal, hemorrhagic, myotoxic and indirect hemolytic effects, and was partially effective in neutralizing edema-forming activity. In contrast, the antivenom was ineffective in the neutralization of in vitro coagulant and in vivo defibrinating effects induced by these two venoms.     

Preclinical assessment of the ability of polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced in Costa Rica to neutralize the venoms of North American snakes.

Polyvalent (Crotalinae) and anticoral (Elapidae) antivenoms produced by Instituto Clodomiro Picado, Costa Rica, were assessed for their ability to neutralize various toxic activities of the venoms of North American snakes of the genera Crotalus, Agkistrodon and Micrurus, in assays involving preincubation of venom and antivenom. When the intraperitoneal route of injection was utilized, polyvalent (Crotalinae) antivenom was effective in the neutralization of the venoms of Crotalus atrox, Crotalus adamanteus, Crotalus viridis viridis, Crotalus horridus atricaudatus, Agkistrodon contortrix contortrix and Agkistrodon piscivorus piscivorus, whereas the venom of Crotalus scutulatus was not neutralized. When the intravenous route was used, results differed depending on the "challenge dose" of venom employed. Polyvalent antivenom neutralized all venoms when mice were challenged with 2 LD(50)s of venom. When 5 LD(50)s were used, antivenom neutralized the venoms of C. atrox, C. adamanteus, C. v. viridis and C. h. atricaudatus, being ineffective in the neutralization of C. scutulatus, A. c. contortrix and A. p. piscivorus. Polyvalent antivenom was effective in the neutralization of hemorrhagic and myotoxic activities of all venoms studied. It also neutralized coagulant activity of C. adamanteus venom, whereas most of the venoms were devoid of clotting activity on plasma in vitro. Moreover, it neutralized defibrinating activity of the only three venoms that induced this effect (i.e. C. adamanteus, A. c. contortrix and A. p. piscivorus). Anticoral (Elapidae) antivenom neutralized lethality induced by the venom of Micrurus fulvius, using either the intravenous or the intraperitoneal routes of injection. Moreover, it neutralized myotoxic effect of this venom as well. It is concluded that polyvalent antivenom neutralizes lethality and other activities of most of the crotaline venoms tested. However, since it is ineffective in neutralizing the lethal effect of C. scutulatus venom, it is suggested that a venom containing presynaptically-active neurotoxic phospholipases A(2) related to "mojave toxin" needs to be introduced in the immunizing mixture in order to increase the neutralizing scope of this product in North America. Anticoral antivenom is highly effective in the neutralization of the venom of M. fulvius.     

Antiserum to myotoxin from prairie rattlesnake (Crotalus viridis viridis) venom.

C. L. Ownby, W. M. Woods and G. V. Odell. Antiserum to myotoxin from prairie rattlesnake (Crotalus viridis viridis) venom. Toxicon17, 373–380. 1979.—A myotoxic component was isolated from rattlesnake (Crotalus viridis viridis) venom and an antibody to it was produced in rabbits. Gel-filtration and cation exchange chromatography were used to fractionate the crude venom and two components were shown to be locally myotoxic using an in vivo assay. One of these components, fraction 3 of the Sephadex cation exchange column, was shown to be homogeneous by electrophoresis. This purified myotoxin was injected into rabbits, and when the resulting antiserum was reacted with the myotoxin in agar-gel double-diffusion plates, one precipitin line was formed. Anti-myotoxin serum reacted with only 2 of 14 crude venoms forming one precipitin line to both C. v. viridis and C. durissus terrificus. The line which formed against these two crude venoms was identical to the one that formed against myotoxin indicating the presence in C. d. terrificus venom of a component immunologically very similar to myotoxin from C. v. viridis venom. It is postulated that this component might be crotamine, a known myotoxic component in C. d. terrificus venom. Antibodies to myotoxin could not be detected in Wyeth's polyvalent Crotalidae antivenin although at least one antibody to crude C. v. viridis venom was present. It is possible that antiserum to pure myotoxin could prevent the local myonecrosis induced by the pure myotoxin, crude C. v. viridis venom and perhaps by other crotalid venoms.     

Observations on white and yellow venoms from an individual southern Pacific rattlesnake (Crotalus viridis helleri)

Biochemical differences in white and yellow venoms produced in the separate venom glands of an individual southern Pacific rattlesnake (Crotalus viridis helleri) were investigated. Compared to the yellow venom, the white venom contained fewer low molecular weight components and was considerably less toxic. Although the exact LD50 was not determined, the white venom did not produce toxic effects in mice when injected i.v. at concentrations up to 10 mg/kg. The i.v. LD50 of the yellow venom was approximately 1.6 mg/kg. Both white and yellow venoms had hemorrhagic activity, but the white venom caused less intradermal hemorrhage in mice. No L-amino acid oxidase activity was measured in the white venom and protease and phospholipase A2 activities of the white venom were much less than in the yellow venom. The white and yellow venoms both produced myonecrosis at 1, 3 and 24 hr after i.m. injection into mice, however, there were some qualitative differences in the myonecrosis produced. When the venom samples were reacted against Wyeth's polyvalent (Crotalidae) antivenom using immunodiffusion, three precipitin bands formed against the yellow venom, whereas only one formed against the white venom. When reacted against an antiserum to myotoxin alpha from C. viridis viridis venom, both the white and yellow venoms produced one precipitin band each.     

Enzyme-linked immunosorbant assay (ELISA) of size-selected crotalid venom antigens by Wyeth's polyvalent antivenom

The binding of Antivenom (Crotalidae) Polyvalent to fractions from crude venoms of eight crotalid and one viperid snake, obtained by high performance size-exclusion chromatography, was determined with an indirect enzyme-linked immunosorbent assay (ELISA). Most of the large (greater than 30,000 mol. wt) molecular mass crotalid venom fractions were associated with high (greater than 0.7 absorbance units) ELISA values. Similarly, the medium (13,000-30,000 mol. wt) and small (less than 14,000 mol. wt) molecular mass crotalid venom fractions were coincident with moderate (0.3-0.7 absorbance units) and low (less than 0.3 absorbance units) ELISA levels. Some variability in this pattern was seen with individual venom fractions. A distinctly different pattern of ELISA values were observed with two rattlesnake venoms: the South American (Crotalus durissus terrificus) and Mojave desert (Crotalus scutulatus scutulatus) rattlesnakes. The elution profile from these venoms showed a progression of low to moderate ELISA values within the large molecular mass fractions. This pattern was followed by a decline to low ELISA values throughout the remainder of the elution profile. When saw scaled viper (Echis carinatus leucogaster) venom fractions were tested, only background ELISA values were detected with antivenom. Similarly, background ELISA values were associated with the small molecular mass fractions of all venoms tested. In addition, the elution position for the basic peptides of southern Pacific (Crotalus viridis helleri) and timber (Crotalus h. horridus) rattlesnake venoms showed minimal ELISA values. These data support the view that except for the venom of C. durissus terrificus and C. s. scutulatus, most antivenom antibodies bind large (greater than 30,000 mol. wt) venom fractions. Thus, antivenom contains minimal levels of antibodies to the basic peptides in these venoms.     

Detection of antibodies to myotoxin a and prairie rattlesnake (Crotalus viridis viridis) venom in three antisera using enzyme-linked immunosorbent assay and immunodiffusion

Immunodiffusion and enzyme-linked immunosorbent assay (ELISA) were used to compare three antisera for their content of antibodies against myotoxin a and C. v. virdis venom. No antibodies were detected in Wyeth's polyvalent (Crotalidae) antivenin against myotoxin a using immunodiffusion, whereas ELISA indicated a low titer of such antibodies. However, antimyotoxin a serum and anti-C. v. viridis venom both had higher titers than antivenin when tested against myotoxin a and crude venom in the ELISA. These results correlate well with previous data which indicated that antiserum to myotoxin a was more effective than antivenin in neutralizing the myotoxicity of C. v. viridis venom. The high content of antibodies to myotoxin a in anti-C. v. viridis venom supports the hypothesis that this monovalent antiserum might be effective in neutralizing rattlesnake (C. v. viridis) venom-induced myonecrosis.     

Neutralization of edema, hemorrhage and myonecrosis induced by North American crotalid venoms in simulated first-aid treatments.

Venoms of the broad-banded copperhead (Agkistrodon contortrix laticinctus, ACL) and the prairie rattlesnake (Crotalus viridis viridis, CVV), like other crotalid venoms, cause severe local tissue damage such as edema, hemorrhage and myonecrosis. Antivenom therapy is not very effective in neutralizing this local tissue damage, and such observations support the need for an effective first-aid regimen aimed at minimizing local tissue reactions. Some of the local tissue damage induced by these venoms is due to phospholipase A2 myotoxins, and since para-bromophenacyl bromide (p-BPB), an inhibitor of PLA2 catalytic activity, has been shown to inhibit the myotoxic action of two PLA2 myotoxins, we hypothesized that this compound would inhibit part of the myotoxic activity of these crude venoms. For in vitro neutralization experiments, venoms were mixed with combinations of either p-BPB, antivenom or both prior to injection into the muscles of the lower hindlimb of mice. For in vivo neutralization experiments, mice were injected with venom followed by either topical DMSO containing p-BPB or intramuscular injection with saline containing p-BPB. A final set of mice received these same injections followed by i.p. infusions of antivenom to simulate experimental first-aid followed by hospital treatment. In the in vitro neutralization tests, edema was significantly reduced when both antagonists were used together, and there was a highly significant neutralization of ACL- and CVV-generated myonecrosis. In the in vivo neutralization experiments, hemorrhage was significantly reduced when injection of ACL venom was followed by topical DMSO-p-BPB, and myonecrosis was reduced when injection of ACL venom was followed by intramuscular injection of saline-p-BPB. Antivenom significantly reduced edema, hemorrhage and myonecrosis induced by CVV venom, but reduced only myonecrosis induced by ACL venom. Taken together, these results suggest a role for pBPB in the first-aid treatment of snakebite especially when followed by hospital treatment with antivenom.     

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.     

Biological and immunochemical characterization of Micrurus altirostris venom and serum neutralization of its toxic activities.

Micrurus altirostris venom from Rio Grande do Sul State, Brazil, was characterized by its biological activities, immunochemical properties and electrophoretic pattern. The results showed a high edematogenic activity, whose peak was observed after 30min of venom injection, as well as a high indirect hemolytic activity. This venom was myotoxic, as shown by a peak of CK release at 6h after injection, and also by the appearance of muscular lesions characterized by necrosis, loss of striated muscle fibers, and the presence of vacuolization, edema and inflammatory infiltrate. This venom showed minimum proteolytic activity and no hemorrhagic, dermonecrotic or coagulant activities. Nonetheless, M. altirostris venom presented high lethal activity. Electrophoretic patterns of Micrurus frontalis and M. altirostris venoms showed different protein bands. Anti-elapidic serum could recognize M. frontalis (homologous) and M. altirostris (heterologous) venoms by Western blotting, and both venoms presented similar titers when assayed by ELISA. The results observed on neutralization tests showed that the anti-elapidic serum produced by Instituto Butantan neutralized myotoxic and hemolytic activities. However, this antivenom could not neutralize the lethal activity of M. altirostris venom. Thus, these data suggest that M. altirostris venom presents different biological, enzymatic and immunological characteristics from other Micrurus venoms, and some activities are not neutralized by the commercial anti-elapidic serum produced in Brazil.     

The co-purification of a lectin (BJcuL) with phospholipases A2 from Bothrops jararacussu snake venom by immunoaffinity chromatography with antibodies to crotoxin

Antigens of Bothrops jararacussu snake venom cross-reacting with specific antibodies against crotoxin, an Asp49 PLA₂-containing heterodimeric complex from Crotalus durissus terrificus snake venom, were purified by two steps of immunoaffinity chromatography. The resulting fraction (Bj-F) was shown to be non-toxic (to mice and rabbits) and immunogenic to rabbits. Antibodies raised against Bj-F were able to protect mice against the lethal effect of both B. jararacussu and Crotalus durissus terrificus snake venoms. Then, the procedure developed showed to be useful for the rapid preparation of an antigen able to elicit neutralizing antibodies against the lethal activities of both venoms. Further fractionation of Bj-F revealed the concomitant presence of two major components: BJcuL, a lectin present in B. jararacussu venom, and BthTX-I, a Lys49 PLA₂ homolog, besides other molecules in minor amounts. Our data are discussed and raise the point that the presence of unrelated molecules may be taken into account when immuno-based methods are considered for purification purposes.     

Neutralizing potency of horse antibothropic Brazilian antivenom against Bothrops snake venoms from the Amazonian rain forest

Neutralization of lethal toxicity (50% effective dose; ED₅₀), hemorrhagic (minimum hemorrhagic dose; MHD) and hemolytic activity (PLA₂) and levels of antibodies, measured by enzyme-linked immunosorbent assay (ELISA), were investigated to test the potency of horse antibothropic serum (ABS) against Bothrops venoms from the Amazonian rain forest. ABS neutralized the lethal activity with a potency (mg of venom neutralized per 1 ml of antivenom) of 5.5, 3.7, 1.6, 1.3 and 6.5, respectively, for B. jararaca (reference venom for assessing the ABS potency in Brazil), B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms. The volume of antivenom (μl) that neutralized one MHD of B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms was 5, 7.71, 7.76, 8.3 and 5, respectively. ABS neutralized the PLA₂ activity with a potency of 6.2, 3.2, 1.4, 2.6 and 5 respectively, for B. jararaca, B. atrox, B. brazili, B. bilineatus smaragdinus and B. taeniatus venoms. ELISA reactivity of ABS against the separate venoms was found to be quite variable. The reactivity against B. jararaca venom was higher than against other Bothrops venoms. In conclusion, the assays described here suggest that Brazilian Bothrops polyspecific antivenom is not very efficient in neutralizing the effects of venom from some Amazonian Bothrops species.     

Comparative study of the venoms of three subspecies of Lachesis muta (bushmaster) from Brazil, Colombia and Costa Rica

A comparative study was performed on the pharmacology and biochemistry of venoms from three subspecies of Lachesis muta (L. m. stenophrys, L. m. muta and L. m. rhombeata) from Brazil, Colombia and Costa Rica. All venoms induced lethal, hemorrhagic, edema-forming, myotoxic, coagulant and defibrinating effects, showing also proteolytic and indirect hemolytic activities. The venoms of L. m. stenophrys from Costa Rica and L. m. muta from Cascalheira, Brazil, had the highest lethal and hemorrhagic activities and the venom of L. m. rhombeata showed the highest coagulant activity, whereas no significant differences were observed in myotoxic and edema-forming activities at most of the time intervals studied. In addition, venoms showed similar electrophoretic patterns on SDS–polyacrylamide gel electrophoresis. In conclusion, despite quantitative differences in toxic and enzymatic activities, together with subtle variations in electrophoretic patterns, our results indicate that experimental envenomation by these venoms induce a qualitatively similar pathophysiological profile.     

Antigenic relationships of fractionated western diamondback rattlesnake (Crotalus atrox) hemorrhagic toxins and other rattlesnake venoms as indicated by monoclonal antibodies

Seven hemorrhagic factors have been isolated from Crotalus atrox venom, but their antigenic relationships have not been well studied. In this study, two different monoclonal antibodies, C. atrox peak 8 (CA-P-8) and C. atrox subclone 5 (CA-5+), were produced against two C. atrox venom hemorrhagic fractions and used in an ELISA (enzyme-linked immunosorbent assay) to determine if the hemorrhagic factors in C. atrox venom are antigenically related. The same ELISA test was used to determine cross-reactivity of seven other crude Crotalidae venoms. The two monoclonal antibodies were tested for their ability to neutralize each hemorrhagic HPLC fraction separated from C. atrox venom. C. atrox venom was fractionated into 22 fractions using HPLC analytical DEAE ion exchange. Fractions 4-17 were hemorrhagic. The CA-P-8 monoclonal antibody reacted strongly with hemorrhagic fraction 8; CA-5+ had a broader reactivity and reacted with several HPLC hemorrhagic and non-hemorrhagic fractions. Crude venoms of C. adamanteus, C. scutulatus scutulatus and C. viridis lutosus reacted with CA-P-8, while C. viridis lutosus, C. viridis oreganus, C. scutulatus scutulatus and C. horridus horridus reacted with CA-5+. C. molossus molossus and C. lepidus lepidus did not react with CA-P-8 and CA-5+. Hemorrhagic HPLC fractions 6, 7, 8, were completely neutralized by monoclonal antibody CA-P-8; fraction 9 was partially neutralized. The present study indicated that some C. atrox venom HPLC hemorrhagic fractions have both common and unique epitopes. Antigenic determinants were also found to be shared among different Crotalus species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of Two Myotoxic Proteins from Venom of Crotalus viridis concolor

Abstract

The fractionation of the previously unstudied venom for Crotalus viridis concolor (midget faded rattlesnake) provided a protein fraction (FD-1) that generated myotoxic responses from envenomated mice. Highly basic homologous myotoxic proteins have been purified from the protein fraction, FD-1. The two major components, designated myotoxin I and myotoxin II, have considerable sequence homology with crotamine from C. d. terrificus, myotoxin a from C. v. viridis and peptide c from C. v. helleri. The amino acid sequences of the toxins from C. v. concolor contain at least two more residues of amino acid than previously described homologous toxins and myotoxin II contains a sequence region with microheterogeneity. The myotoxins cause extensive damage to the sarcoplasmic reticulum as evidenced by electron microscopic studies of envenomated muscle. The destruction is comparable to that observed with the homologous toxins. Studies with isolated sarcoplasmic reticulum vesicles failed to demonstrate any significant changes in Ca²⁺-Mg²⁺-ATPase or in uptake or release of Ca²⁺. Current evidence indicates that a closely related group of homologous toxins are present in rattlesnake venoms but the biochemical mode of action of these proteins remains obscure.     

Ability of a polyvalent antivenom to neutralize the venom of Lachesis muta melanocephala, a new Costa Rican subspecies of the bushmaster

Several toxic and enzymatic activities of the venom of L. m. melanocephala were studied. This venom has many similarities with that of L. m. stenophrys, although there are quantitative differences in venom activities, as well as in the immunodiffusion patterns of these venoms when reacted against polyvalent antivenom. This antivenom was tested for its ability to neutralize a series of toxic and enzymatic effects of L. m. melanocephala venom. A new method to study myonecrosis, based on the quantitation of residual creatine kinase in injected muscle, was used. Antivenom was highly effective in neutralizing lethal, hemorrhagic, myotoxic, edema-forming, defibrinating, caseinolytic and fibrinolytic activities when venom and antivenom were incubated prior to the test or, in the case of edema-forming activity, when antivenom was administered before venom injection. On the other hand, when antivenom was injected i.v. at different time intervals after venom injection neutralization of lethality was good, although neutralization of local effects, i.e. hemorrhage and edema, was poor. These results indicate that polyvalent antivenom contains antibodies capable of neutralizing toxic and enzymatic activities of L. m. melanocephala venom. Moreover, the partial inability of antivenom to neutralize local effects when administered after venom injection is probably due to the rapid development of these effects once venom is injected.     

Ability of antiserum to myotoxin a from prairie rattlesnake (Crotalus viridis viridis) venom to neutralize local myotoxicity and lethal effects of myotoxin a and homologous crude venom

Antiserum to myotoxin f isolated from prairie rattlesnake (Crotalus viridis viridis) venom was tested for its ability to neutralize the local myotoxic and lethal activities of myotoxin a and of crude C. v. viridis venom. Wyeth's polyvalent (Crotalidae) antivenin was also tested for its ability to neutralize the crude venom. Using a light microscopic method to quantitate myonecrosis, the effect of myotoxin a, i.e. vacuolation, could be distinguished from total myonecrosis induced by whole venom. The results indicate that anti-myotoxin a serum is more effective in neutralizing local myonecrosis, but polyvalent antivenin is more effective in neutralizing lethality when they are mixed with crude prairie rattlesnake venom prior to injection.     

Snake venom hemorrhagins: neutralization by commercial antivenoms

One monovalent (habu-antivenom) and five polyvalent antivenoms (Crotalidae; Orient, North, Central and South Africa) were tested for their ability to neutralize the hemorrhagic activity of 12 snake venoms (Agkistrodon, Bothrops, Crotalus, Sistrurus, Trimeresurus, Bitis, Echis spp.) when mixed prior to injection into the hind leg of mice. Considerable cross-neutralization was observed: antivenoms prepared against African snake venoms were equally or more potent in neutralizing the hemorrhagic activity of Crotalidae venoms. The same applies to Crotalidae antivenom which neutralized the activity of African snake venoms. Anti-hemorrhagic antibodies were isolated from a polyvalent antivenom by affinity chromatography using purified hemorrhagins from Bitis arietans and Crotalus adamanteus venom as ligands. These antibodies neutralized the activity of both hemorrhagins indicating common antigenic determinants in these molecules.     

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.     

Characterization of the major metalloprotease isolated from the venom of the northern Pacific rattlesnake, Crotalus viridis oreganus

Rattlesnake venoms typically contain several different metalloproteases, some of which are hemorrhagic toxins. Metallo-proteases contribute significantly to the often severe necrotic changes in tissues following envenomation, and these prominent components are important to the predigestive role of venoms. Venom of the northern Pacific rattlesnake (Crotalus viridis oreganus) contains at least five distinct metalloproteases, and the dominant protease (trivial name, CVO protease V) has been isolated and characterized as being a single polypeptide chain acidic protein with a molecular mass of 61 kDa and a pH optimum of 9.0. It catalyzes the hydrolysis of several protein substrates, including casein, and is inhibited by metal chelators such as EDTA, EGTA and 1,10-phenanthroline but not by serine protease inhibitors such as PMSF. Calcium is present at a molar ratio of approximately 1:1, but, unlike other described venom metalloproteases, this protease does not appear to contain zinc. Caseinolytic activity is not significantly inhibited by citrate (at pH 9.0) at levels up to 2.0 mM; at 100 mM citrate (at pH 9.0) more than 65% of activity is retained. It is partially inhibited by nanomolar concentrations of ATP, but higher amounts (micromolar) do not result in further inhibition of activity. The protease shows fibrinolytic and fibrinogenolytic activity, but is only weakly hemorrhagic in rats. When stored in solution for long periods it undergoes autolytic degradation. This protease or a homolog appears to be present in venoms from several rattlesnake species but is not present in venoms from juvenile C. v. oreganus. The presence of this component in venoms from adult Pacific rattlesnakes is responsible for the age-related increase in metalloprotease activity of the crude venom.