To Develop Anti Snake Venom Sera (ASV) Against Four Deadly Venomous Snake Species and Study the Efficacy of Oral Adjuvant for Russeel Viper Snake Venom Immunization in Rabbits

Abstract

Background Snake venoms are known to be complex mixtures of diverse proteins that include the neurotoxins, enzymes and peptides. Besides the proteins they have carbohydrates, lipid and trace elements. The poisonous moiety of venomous proteins is poorly understood and especially their immunological responses on the immunization of horses and rodents like rabbits. Objectives 1. To develop anti snake venom sera (ASV) against four deadly venomous snake species. 2. To study the efficacy of oral adjuvant for Russel Viper snake venom immunization in rabbits. Methods Four poisonous snakes namely Cobras, Kraits, Russell Vipers and Saw Scales were collected from different districts of Sindh, a Province of Pakistan. Snake venoms were collected by manually compressing the venom glands on the rim of individual glass bottles. Venoms were centrifuged to remove the oral mucous material and preserved immediately at –20oC in a freezer and later lyophilized for the future use. Estimation of snake venom proteins, Immuno diffusion test, Enzyme Linked Immunosorbent Assay (ELISA), animal neutralization test and western blotting tests were performed using the standard protocols or with some modifications. Horses were injected with single venom to produce antibody specific to that venom. They were also immunized with combination of venoms to illicit antibody production against all their toxic moieties. These anti-sera were developed using a mixture of venoms and adjuvants with use of two different types of adjuvants (injectable and oral) and four different types of venomous proteins against Cobra, Krait, R. Viper and Saw Scale. ii Results Monovalent antibody developed in horses were found to be highly specific against all four species and comparable results were observed in divalent antibody where the animals were injected with a combination of two snake venoms. The horses did not produce trivalent & quadrivalent antibodies when they were injected with a combination of three or four venoms. Adverse reactions were observed in animals injected with combination of venoms as compared to the toxicity with single venom. Snake venoms combined with or without oral adjuvant were administered into rabbits in three doses. The antiserum produced against venom only was devoid of the neutralizing antibody whereas oral adjuvant produced antibody that manifested the neutralizing effect. The short duration of oral adjuvant immunization was highly beneficial as compared to injectable adjuvant immunization. SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel) and Western blotting (immunoblots) showed presence of highly specific proteins in the 14 and 64Kd region. ELISA and animal neutralization test showed that the oral immunological response was significantly higher than injectable route with three doses. Conclusion Newly developed Anti snake venoms produced by oral adjuvant against four venomous snakes were effective in neutralizing the corresponding venoms. Monovalent antibodies were highly specific, divalent of Cobra and Russell Viper were also effective. Trivalent and quadrivalent venom doses failed to produce significant antibodies. The oral adjuvant preparation successfully boosted the production of antibody in both animal species (horses and rabbits). Effect was certainly beneficial such as short duration of immunization safety and ease of administration. Key words