Use of irradiated elapid and viperid venoms for antivenom production in small and large animals.

This work evaluated the feasibility of using toxoids obtained by gamma radiation in the production of antivenoms in small and large animals. Mixtures of African snake venoms from viperids or elapids were used. The viperid mixture contained the crude venom of five species of the genera Echis and Bitis, while the elapid mixture contained the crude venom of six species of the genera Naja and Dendroaspis. The viperid mixture had an LD50 of 1.25 mg/kg in mice, and the elapid mixture had an LD50 of 0.46 mg/kg. Both viper and elapid aqueous mixtures were subjected to Cobalt-60 gamma irradiation in three physical states: lyophilized, frozen and liquid. Radiation doses ranged from 0.5 to 100 kGy. The LD50s of the lyophilized and frozen mixtures of both viperid and elapid mixtures remained unaltered with radiation doses as high as 100 kGy; nevertheless, in the liquid state, doses of 3.5 and 5.5 kGy reduced the venom toxicity of both the viperid and elapid mixtures to 7.25 mg/kg and 1.74 mg/kg; less toxic by factors of 5.8 and 3.8, respectively. Groups of four rabbits and three horses were immunized with either irradiated or non-irradiated mixtures. In vitro and in vivo analysis of the rabbit and horse sera revealed that neutralizing antibodies were produced against both irradiated (toxoids) and native venom mixtures. None of the animals used in this study, either immunized with native venom or toxoids, developed severe local effects due to the application of venoms mixtures. Gamma-irradiated detoxified venoms mixtures, under well-controlled and studied conditions, could be a practical alternative for the production of polyvalent equine serum with high neutralization potency against snake venoms.

Irradiated Cerastes cerastes venom as a novel tool for immunotherapy.

Immunotherapy is the most effective treatment for the snake bites. The antivenoms are commonly obtained by hyperimmunization of animals that suffer from venom toxicity. The present report describes gamma irradiation effects on Cerastes cerastes venom. Doses of 1 kGy and 2 kGy gamma radiations were used for venom detoxification. These treated venoms did not have any residual lethal effects until 10 LD(50). Immunological analysis of sera raised against native and irradiated venoms, showed that elicited antibodies to irradiated venoms were able to recognize native venom. Anti-2 kGy irradiated venom had more protective ability than anti-native venom, as tested in mice.

Evaluation of the effect of gamma rays on the venom of Vipera lebetina by biochemical study.

Snake bites represent a serious public health problem in many areas of the world. In Algeria, two widespread snakes are Vipera lebetina and Cerastes cerastes. Vipera lebetina venom causes local hemorrhage and necrosis, and it may lead to permanent limb loss. The principal causes of mortality after snakebites are acute renal failure and hemorrhage, which occur not only locally, at the site of the bite, but also systemically, contributing to the cardiovascular shock characteristic of severe envenomation. Gamma radiation has been shown to be effective for attenuating venom toxicity. Vipera lebetina venom was irradiated with two doses of gamma rays (1 and 2 kGy) from a 60Co source, and the venom's toxic, enzymatic, and structural properties were analyzed. Intraperitoneal injection of the irradiated venoms (100-500 microg/20 g mouse body mass) revealed a significant decrease of the toxicity. Irradiated venoms with 1 and 2 kGy doses were four and nine times less toxic, respectively, than the native venom. A biochemical characterization of in vitro enzymatic activities was performed. Vipera lebetina displayed in vitro caseinolytic, amidolytic, esterasic, coagulant, and phospholipase A2 activities. Caseinolytic, amidolytic, esterasic, and coagulative activities were reduced for the irradiated venoms; only phospholipase A2 activity was abolished in the irradiated venom with a dose of 2 kGy. The native and irradiated venoms were separated by gel filtration and electrophoresis. Chromatographic and electrophoretic profiles were drastically changed as compared with the native venom. Vipera lebetina venom detoxified by gamma rays was used for active immunization, and the presence of antibody in the immune sera was detected by ELISA. The immunogenic properties were preserved and the antisera obtained with the irradiated venoms could cross-react. Antisera were able to neutralize the toxic effect of V. lebetina native venom. These results indicate that irradiation of V. lebetina venom with a dose of 2 kGy can promote a significant detoxification, keeping the immunological properties intact.

Effects of 60Co gamma radiation on toxicity and hemorrhagic, myonecrotic, and edema-forming activities of Cerastes cerastes venom.

Antisera are used as effective antidotes against the local effects of snake bites. To improve antisera production and extend the life of surrogates used to produce antibodies, the chronic effects of venom toxicity must be reduced. The present study evaluated the effectiveness of gamma irradiation to reduce the local effects associated with viperid snake bites by evaluating in NMRI mice the toxicity and edematic, hemorrhagic, and myonecrotic activities of native and irradiated Cerastes cerastes venoms. These results indicated that the toxicity of irradiated venoms (1 and 2 kGy) decreased as compared with that of native venom. The edematic and hemorrhagic activities were also reduced in the detoxified samples, particularly with the 2-kGy radiation dose. Furthermore, the creatine phosphokinase (CPK) activity was significantly increased in the serum and decreased in the myocardium after envenomation with native venom, but no significant enzymatic changes were observed in mice envenomated with irradiated venom. Histopathologic evaluation showed that native venom caused severe degenerative changes in the myocardium. In the case of 2-kGy-irradiated venom, no tissue alterations were observed. These results indicate that irradiation of venom with a 2-kGy dose may offer an effective method for reducing the chronic toxic effects of venom in immunized animals.

Neutralization of pathophysiological manifestations of Russell's viper envenoming by antivenom raised against gamma-irradiated toxoid.

Rabbits were immunized against gamma-irradiated (100 krads) Russell's viper venom toxoid adsorbed to aluminium phosphate gel. The antivenom (0.1 ml) neutralized 5 LD50, 8 minimum hemorrhagic doses (MHD) and 14 minimum necrotic doses (MND) of venom. The coagulant and protease activities of the viper venom were neutralized more effectively than phospholipase A activity, by the toxoid antivenom.

Active immunization of rabbit with gamma irradiated Russell's viper venom toxoid.

Russell's viper venom detoxified by gamma (gamma)-radiation (100 kR or 200 kR) was used as a toxoid for active immunization of rabbits following a short or long schedule of immunization without any adjuvant. Effective neutralization of venom toxin by immune sera of rabbits was observed with both schedules. Sera of rabbits immunized with 100 kR irradiated venom toxoid (100 kR toxoid antisera) were more potent than 200 kR toxoid antisera. The presence of antibody in the immune sera was detected by immunoelectrophoresis. The effect of gamma-radiation on some enzymes and venom protein profiles was studied. Phosphodiesterase, protease and hyaluronidase were inhibited by radiation though phospholipase A activity remained unaffected. Radiation did not produce any gross change in the protein profile of crude viper venom. Phosphodiesterase and protease activities of viper venom were neutralized more effectively by 100 kR toxoid antisera in the short schedule than in the long schedule of immunization.