Toxicological, enzymatic, and immunochemical characterization of Bothrops asper (Serpentes: Viperidae) reference venom from Panama / Caracterización toxicológica, enzimática e inmunoquímica del veneno de referencia de Bothrops asper (Serpentes: Viperidae) de Panamá

Introduction: It is estimated that 2 000 snakebites occur in Panama every year, 70 % of which are inflicted by Bothrops asper. Objective: To determine the biochemical and toxicologic effects and to assess the immunochemical characteristics of a reference pool of B. asper venom representative of Panama. Methods: The reference venom was prepared as a homogeneous mixture of the venoms obtained from 78 adult snakes collected in four geographic areas of Panama. Enzymatic and toxicological activities were assessed. The electrophoretic pattern was studied by SDS-PAGE. Immunoreactivity of various antivenoms was analyzed by Western blot. Results: B. asper reference venom has lethal, hemorrhagic, myotoxic, edema-forming, coagulant, defibrinating, proteinase and phospholipase A2 activities. SDS-PAGE showed the presence of protein bands with molecular weights ranging from 8 to 70 kDa, with the presence of predominant bands at ≈ 15 kDa and ≈ 30 to 66 kDa, which likely correspond to phospholipases A2 and metalloproteinases, respectively. Immunoblotting showed a high degree of recognition by various antivenoms, especially by antivenoms from Colombia and Costa Rica. Conclusions: Following recommendations by the World Health Organization, this reference venom of B. asper of Panama will become a useful tool for the preclinical evaluation of antivenoms distributed in this country.

Introducción: Se estima que 2 000 mordeduras de serpiente ocurren en Panamá cada año, el 70 % de las cuales son infligidas por Bothrops asper. Objetivo: Determinar los efectos bioquímicos y toxicológicos y evaluar las características inmunoquímicas del veneno de referencia de B. asper representativo de Panamá. Métodos: El veneno de referencia se preparó como una mezcla homogénea de los venenos obtenidos de 78 serpientes adultas recolectadas en cuatro áreas geográficas de Panamá. Se evaluaron las actividades enzimáticas y toxicológicas. El patrón electroforético se estudió mediante SDS-PAGE. La inmunoreactividad de varios antivenenos se analizó mediante transferencia de Western. Resultados: El veneno de referencia de B. asper tiene actividades letales, hemorrágicas, miotóxicas, formadoras de edema, coagulantes, desfibrinante, proteolítica y de fosfolipasa A2. El análisis de SDS-PAGE mostró la presencia de bandas de proteínas con pesos moleculares que varían de 8 a 70 kDa, con la presencia de bandas predominantes a ≈ 15 kDa y ≈ 30 a 66 kDa, que probablemente corresponden a fosfolipasas A2 y metaloproteinasas, respectivamente. La inmunotransferencia mostró un alto grado de reconocimiento por varios antivenenos, especialmente por antivenenos de Colombia y de Costa Rica. Conclusiones: Siguiendo las recomendaciones de la Organización Mundial de la Salud, este veneno de referencia de B. asper de Panamá se convertirá en una herramienta útil para la evaluación preclínica de antivenenos distribuidos en este país.

Neutralization of bitis parviocula (Ethiopian mountain adder) venom by the south african institute of medical research (SAIMR) antivenom / Neutralização do veneno de Bitis parviocula (serpente da Montanha da Etiópia) pelo antiveneno do Instituto Africano de Pesquisa Médica (SAIMR)

BACKGROUND: The Ethiopian mountain adder (Bitis parviocula) is a viperid known only from a few locations in southwestern Ethiopia. METHODS: a total of 30 µg of B. arietans and B. parviocula venoms were run on a 10-20 percent Tricine gel. To assay lethality dose fifty (LD50), five groups of eight mice for each venom were used. Hemorrhagic activity for crude venom was tested. Fibrinogenolytic activity of crude venom was measured using (2.5 mg/mL) of fibrinogen solution and (0.03 mg/mL) of crude venom. Gelatinase activity of the venom was tested on a Kodak X-OMAT TM film. Crude venoms of B. parviocula and B. arietans were tested for their abilities to affect clotting time, clotting rate and platelet function on whole human blood. RESULTS: The (SAIMR) antivenom was confirmed in this study to neutralize the lethal activity of venom from Bitis parviocula. The ED50s of SAIMR antivenom on B. parviocula and B. arietans neutralized half of 18.2 and 66.7 mg of venom, respectively. The hemorrhagic activities (MHDs) of B. parviocula and B. arietans were 0.88 and 1.7 µg, respectively. Bitis arietans and B. parviocula venoms degradated α and β chains at different times. The γ chains remained unaffected. Bitis parviocula venom did not exhibit gelatinase activity, while B. arietans had a MGD of 6.9 µg. At 3 mg/mL, the crude venoms of B. parviocula and B. arietans did not significantly affect clotting time or clotting rate. CONCLUSIONS: The SAIMR antivenom is very effective in neutralizing the venom of B. parviocula and should be considered in treating envenomations by these snakes.

BACKGROUND: Serpente das Montanhas da Etiópia (Bitis parviocula) é um viperídeo conhecido somente em poucas localizações do sudoeste da Etiópia. MÉTODOS: Um total de 30 µg de veneno de B. arietans e B. parviocula foram corridos em gel de 10 a 20 por cento de tricina. Para se estabelecer a quinquagésima dose de letalidade (LD50) foram usados cinco grupos de oito camundongos para cada veneno. A atividade hemorrágica para o veneno cru foi testada. A atividade fibrogenolítica do veneno cru foi medida usando 2,5 mg/mL de solução de fibrinogênio e 0,03 mg/mL de veneno cru. A atividade de gelatinase do veneno foi testada em um filme KODAK X-OMAT TM. Venenos crus de B. parviocula e B. arietans foram testados no que diz respeito à sua capacidade de afetar o tempo de coagulação, a velocidade de coagulação e a função plaquetogênica em sangue humano total. RESULTADO: o antiveneno SAIMR foi confirmado neste estudo no que diz respeito à neutralização da atividade letal do veneno de Bitis parviocula. ED50s do antiveneno SAIMR sobre a B. parviocula e B. arietans neutralizou metade de 18,2 e 66,7 mg respectivamente do veneno. As atividades hemorrágicas (MHDs) de B. parviocula e B. arietans foram respectivamente 0,88 e 1,7 µg. Os venenos de B. arietans e B. parviocula degradaram cadeias α e β em tempos diferentes. A cadeia Γ permaneceu não afetada. O veneno da B. parviocula não mostrou atividade de gelatinase, enquanto o de B. arietans teve um MGD de 6,9 µg. A nível de 3 mg/mL os venenos crus de B. parviocula e B. arietans não afetaram significantemente o tempo e a velocidade de coagulação. CONCLUSÕES: O antiveneno SAIMR é bastante efetivo para neutralizar o veneno da B. parviocula e deveria ser considerado para o tratamento de envenenamentos por estas serpentes.

Anti-venom potential of aqueous extract of stem bark of Mangifera indica L. against Daboia russellii (Russell’s viper) venom.

Several plant extracts rich in pharmacologically active compounds have shown to antagonize venom of several species. Mangifera indica has been used against snakebite by the traditional healers, However, there is paucity of scientific data in support. In this study, we evaluated the antivenom potential of aqueous extract of stem bark of M. indica against D. russellii venom-induced pharmacological effects such as life myotoxicity, edema, LD50 etc. The extract inhibited the phospholipase, protease, hyaluronidase, 5`nucleotidase, ATPase and alkaline phosphomonoesterase activities with varying IC50 values. It significantly inhibited both metalloproteases and serine proteases activities. Further, the extract significantly reduced the myotoxicity of the venom, as evident by the reduction of serum creatin kinase and lactate dehydrogenase activities. Though the extract completely inhibited in vitro PLA2 activity, it was unable to completely inhibit in situ hemolytic and in vivo edema-inducing activities, usually brought about by PLA2s. In lethality studies, co-injection of the venom preincubated with the extract showed higher protection than the independent injection of venom, followed by the extract in the mice. However, in both the cases the extract -a cocktail of inhibitors significantly increased the survival time, when compared to that of mice injected (i.p) with the venom alone. These results encourage further studies on the potential use of cocktail of inhibitors in improving the treatment of snake envenomation. Further, this study substantiates the use of M. indica as an antidote against snakebite by the traditional healers.

Inhibition of local effects of Indian Daboia/Vipera russelli venom by the methanolic extract of grape (Vitis vinifera L.) seeds.

Although anti-venom therapy is available for the treatment of fatal bite by snakes, it offers less or no protection against the local effects such as dermo- and myonecrosis, edema, hemorrhage and inflammation at the bitten region. The viper species are known for their violent local effects and such effects have been commonly treated with plant extracts without any scientific validation in rural India. In this investigation, the methanolic extract of grapes (Vitis vinifera L.) seed was studied against the Indian Daboia/Vipera russelli venom-induced local effects. The extract abolished the proteolytic and hyaluronidase activities and also efficiently neutralized the hemorrhage, edema-inducing and myonecrotic properties of the venom. In addition, the extract also inhibited partially the pro-coagulant activity of the venom and abolished the degradation of Aα and Bβ chains of human fibrinogen. Thus, the extract possesses potent anti-snake venom property, especially against the local effects of viper bites.

Effects of antithrombin III and antivenom on procoagulant activity of Russell's viper venom in a whole blood model.

The procoagulant activities of Russell's viper venom were assessed in an in vitro whole blood model. Sequential samplings showed that the generation of fibrinopeptide A (FPA), a marker of thrombin activity, and platelet factor 4 (PF4), a marker of platelet activity, exhibited bi-phasic kinetics with an initial slow phase followed by a rapid phase of secretion. In the presence of Russell's viper venom, the generation of both FPA and PF4 was accelerated with the bi-phasic kinetics of PF4 being maintained while that of FPA completely disappeared. Administration of either antivenom (1,600 ng) or 10 IU antithrombin III (AT-III) had no antagonistic effect against the venom but combination of both resulted in a significant prolongation of both FPA and PF4 release (p < 0.05). High dose AT-III (20 IU) resulted in normalization of both FPA and PF4 kinetics and serial levels of both parameters were lower than those treated with the combined regimen, although these were not statistically significant. Unlike the untreated venom activated whole blood, there was no clot formation following treatment with either the combined regimen or high dose AT-III. The results of this study suggested that the effect of Russell's viper venom on the clotting cascade is more potent and direct than that on platelet activity. There were complementary effects between antivenom and AT-III is controlling of both FPA and PF4 release induced by the venom. Furthermore, in this in vitro experiment, AT-III alone when administered in a sufficient dose, abolished the procoagulant effects of Russell's viper venom.

Neutralization potency of Russell's viper venom toxoid antivenom, as compared with standard antivenom.

Rabbits were immunized with gamma (gamma) irradiated Russell's viper venom toxoid, adsorbed to aluminium phosphate adjuvant. Antibody (raised against toxoid inoculation) titer was compared to a commercial antivenom on the basis of its ability to neutralize hemorrhagic, necrotic and lethal effects of viper venom. Toxoid immunization (on day 0, 15 and 30) produced antivenom which showed approximately one-third antilethal, half antihemorrhagic and antinecrotic titers as compared to those of the commercial hyperimmunized, concentrated horse antivenom.

Lethal and haemorrhagic activity of Russell's Viper venom--neutralization by polyvalent, monovalent and toxoid antiserum.

Lethal and haemorrhagic activity of Russell's Viper venom was compared against polyvalent bivalent commercial antiserum and monovalent antiserum raised in rabbit. Formaldehyde-detoxified venom offered 7-fold protection against lethal activity and 12.5-fold against haemorrhagic activity of the venom. Whole venom and formaldehyde-detoxified venom along with Freund's complete adjuvant, injected in rabbits produced high titre antiserum. Amongst all the six antiserum tested, the monovalent antiserum raised in rabbit, showed maximum precipitating bands in immunogeldiffusion and immunoelectrophoresis. The toxoid-antiserum offered maximum protection against the venom-induced lethality and the monovalent antiserum offered maximum protection against haemorrhagic activity.