Development of dot-ELISA for the detection of venoms of major Indian venomous snakes.

India remained an epicenter for the snakebite-related mortality and morbidities due to widespread agricultural activities across the country and a considerable number of snakebites offended by Indian cobra (Naja naja), common krait (Bungarus caeruleus), Russell's viper (Daboia russelii), and saw-scaled viper (Echis carinatus). Presently, there is no selective test available for the detection of snake envenomation in India before the administration of snake antivenin. Therefore, the present study aimed to develop rapid, sensitive assay for the management of snakebite, which can detect venom, responsible snake species and serve as a tool for the reasonable administration of snake antivenin, which have scarcity across the world. The selective envenomation detection assay needs venom specific antibodies (VSAbs) for that monovalent antisera was prepared by hyperimmunization of rabbits with specific venom. However, obtained antibodies exhibit maximum activity towards homologous venom as well as quantifiable degree of cross-reactivity with heterologous venoms. Use of these antibodies for development of selective envenomation detection assay may create ambiguity in results, therefore needs to isolate VSAbs from monovalent antisera. The cross-reacting antibodies were specifically removed by immunoaffinity chromatography to obtain VSAbs. For the development of venom detection ELISA test (VDET), two different species of antibodies were used that offers enhanced sensitivity along with selective identification of the venoms of the responsible snakes. In conclusion, the developed VDET is rapid, specific, yet sensitive to detect venoms of offending snake species, and its venom concentration down to 1.0 ng/ml. However, the device observed with lowest venom concentration detection ability in the range <1.0 ng/ml from experimentally envenomated samples. The implementation of VDET will help in avoiding unnecessary usage and adverse reactions of snake antivenin. The test has all the merits to become a choice of method in envenomation diagnosis from medically important snakes of India.

Assessment of Cultivable Oral Bacterial Flora from Important Venomous Snakes of India and Their Antibiotic Susceptibilities.

Snakebite is a common, frequently devastating, occupational, socio-economic hazard, and it has a great impact on the rural population of India. Snakebite is a major cause of the human morbidity and mortality since ancient times, as it not only affects the victim by systemic envenomation but also by wound infections originating from deadly pathogenic microorganisms from the oral cavity of the offending snake. The pathogens from the oral cavity of the snake tend to initiate an infection, resulting in gas gangrene, soft tissue necrosis, and permanent physical disabilities. In light of this, the present study is designed to evaluate the oral microbiota of venomous snakes commonly found in India and assessment of their antibiotic susceptibilities. Oral cavity swabs of twenty snakes representing the Indian cobra, Russell's viper, Saw-scaled viper, and Common krait were selected for the study. These materials were enriched using microbiological media to facilitate the growth of bacteria and their subsequent isolation to assess the antibiotic susceptibilities. A total 205 strains were isolated from the oropharyngeal cavity of snakes, which represent the common pathogens, especially Morganella morganii, Escherichia coli, Aeromonas hydrophila, Pseudomonas aeruginosa, coagulase-negative Staphylococcus aureus, Bacillus species, Micrococcus species, and some anaerobes including Clostridium perfringens. The study can conclude that the oral cavity of the snakes has a diversity of Gram-positive and Gram-negative bacteria are susceptible to several antibiotics. The Gram-negative microorganisms showed 100% susceptibility to imipenem and levofloxacin, whereas Gram-positive microorganisms to azithromycin and amoxicillin/clavulanic acid.

Rapid and selective detection of experimental snake envenomation - Use of gold nanoparticle based lateral flow assay.

In this study, we have developed a gold nanoparticle based simple, rapid lateral flow assay (LFA) for detection of Indian Cobra venom (CV) and Russell's viper venom (RV). Presently, there is no rapid, reliable, and field diagnostic test available in India, where snake bite cases are rampant. Therefore, this test has an immense potential from the public health point of view. The test is based on the principle of the paper immunochromatography assay for detection of two snake venom species using polyvalent antisnake venom antibodies (ASVA) raised in equines and species-specific antibodies (SSAbs) against venoms raised in rabbits for conjugation and impregnation respectively. The developed, snake envenomation detection immunoassay (SEDIA) was rapid, selective, and sensitive to detect venom concentrations up to 0.1 ng/ml. The functionality of SEDIA strips was confirmed by experimental envenomation in mice and the results obtained were specific for the corresponding venom. The SEDIA has a potential to be a field diagnostic test to detect snake envenomation and assist in saving lives of snakebite victims.