Kathleen Fernandes Grego

Snakebite incidents could occur even with deceased animals due to improper animal manipulation, since the venom may retain biochemical activity. However, it is unclear what alterations death may cause in snake venom, because even in well-preserved genera, such as Lachesis, venom is susceptible to variation due to several factors. Therefore, the purpose of this research is to compare Lachesis muta venom characteristics before and after its death. The investigation encompasses protein profile analyses, immune recognition by antivenom, and enzymatic and biological activities. Venom samples were collected from the same individual at three distinct time points: two samples while the specimen was alive and a sample post-mortem. In RP-HPLC, a shift in peak intensity was observed in the peptide and protease elution regions. The post-mortem sample presented more PLA₂ and LAAO enzymatic activities and a decrease in the coagulation capacity. All samples were well immuno-recognized by anti-Bothrops/Lachesis serum. Data suggest that L. muta venom maintains its functional and immunological integrity throughout senescence and following the animal’s death. The preservation of its biochemical properties supports the clinical relevance of accidents involving dead snakes and justifies the inclusion of such venoms in antivenom manufacturing protocols, providing a strategic alternative during supply shortages.

Despite coagulotoxicity being a primary weapon for prey capture by Bothrops species (lancehead pit vipers) and coagulopathy being a major lethal clinical effect, a genus-wide comparison has not been undertaken. To fill this knowledge gap, we used thromboelastography to compare 37 venoms, from across the full range of geography, taxonomy, and ecology, for their action upon whole plasma and isolated fibrinogen. Potent procoagulant toxicity was shown to be the main venom effect of most of the species tested. However, the most basal species (B. pictus) was strongly anticoagulant; this is consistent with procoagulant toxicity being a novel trait that evolved within Bothrops subsequent to their split from anticoagulant American pit vipers. Intriguingly, two of the arboreal species studied (B. bilineatus and B. taeniatus) lacked procoagulant venom, suggesting differential evolutionary selection pressures. Notably, some terrestrial species have secondarily lost the procoagulant venom trait: the Mogi Mirim, Brazil locality of B. alternatus; San Andres, Mexico locality of B. asper; B. diporus; and the São Roque of B. jararaca. Direct action on fibrinogen was extremely variable; this is consistent with previous hypotheses regarding it being evolutionary decoupled due to procoagulant toxicity being the primary prey-capture weapon. However, human patients live long enough for fibrinogen depletion to be clinically significant. The extreme variability may be reflective of antivenom variability, with these results thereby providing a foundation for such future work of clinical relevance. Similarly, the venom diversification trends relative to ecological niche will also be useful for integration with natural history data, to reconstruct the evolutionary pressures shaping the venoms of these fascinating snakes.

Snake venom is an expensive metabolic weapon used for digestion and defense. Detailed studies on the production of venoms are important for the manufacture of antivenoms and for the therapeutic management of snakebites. Bothrops leucurus is one of the snakes of medical importance responsible for a large number of accidents in Northeast Brazil. To establish the correlation between Bothrops leucurus venom (Blv) yield, under captive conditions, and the morphological characteristics (body mass and length), sex and age, 31 specimens were milked during one year, grouped by sex and age (juvenile, adult and long-lived), totaling 106 extractions in that period. We evaluated the electrophoretic profile (SDS-PAGE) under reducing conditions, the 50% lethal dose (LD50) and the minimum coagulant dose (DMC) of the extracted venoms. The body size was positively correlated with venom production in B. leucurus snakes. Regardless of sex and age, the venom showed no differences between liquid and solid composition or between right and left fang, however, the production of venom in females was twice the one found in males and more lethal. The clotting ability was lost as the animals aged, indicating that older snakes are not the best choice for venom pools in the production of antivenoms. These results are important for the choice of animals to antivenom production, and to understand the biological effects of snake venoms under captive conditions.

Maintenance of snakes at Butantan Institute started in the last century, intending to produce a different antivenom serum to reduce death caused by snakebites. Through a successful campaign coordinated by Vital Brazil, farmers sent venomous snakes to Butantan Institute by the railway lines with no cost. From 1908 to 1962, the snakes were kept in an outdoor serpentarium, where venom extraction was performed every 15 days. During this period, the snake average survival was 15 days. In 1963, the snakes were transferred to an adapted building, currently called Laboratory of Herpetology (LH), to be maintained in an intensive system. Although the periodicity of venom extraction remained the same, animal average survival increased to two months. With the severe serum crisis in 1983, the Ministry of Health financed remodeling for the three public antivenom producers, and with this support, the LH could be improved. Air conditioning and exhausting systems were installed in the rooms, besides the settlement of critical hygienic-sanitary managements to increase the welfare of snakes. In the early 1990s, snake survival was ten months. Over the years to the present day, several improvements have been made in the intensive serpentarium, as the establishment of two quarantines, feeding with thawed rodents, an interval of two months between venom extraction routines, and monitoring of snake health through laboratory tests. With these new protocols, average snake survival increased significantly, being eight years for the genus Bothrops, ten years for genus Crotalus and Lachesis, and four years for the genus Micrurus. Aiming the production of venoms of good quality, respect for good management practices is essential for the maintenance of snakes in captivity. New techniques and efficient management must always be sought to improve animal welfare, the quality of the venom produced, and the safety of those working directly with the venomous snakes.