Naja Venom Research Articles

Proteome analysis of toxic fractions of Moroccan cobra Naja haje venom using mass spectrometry

ABSTRACT Snake venoms constitute a complex blend of proteins and peptides, with their toxicity and pharmacological effects varying depending on their geographic origin. Naja haje, a venomous snake of significant medical importance in regions like Morocco and North Africa, can inflict severe pain, swelling, and neurotoxic symptoms upon biting. Without prompt medical intervention, these symptoms can rapidly worsen, potentially leading to fatal outcomes. Understanding the venom’s components is crucial for developing effective antivenom therapies. Specific antibodies that target the venom’s toxic elements are essential for neutralising its activity. Therefore, we conducted a proteomic analysis of these elements within Naja haje venom using gel filtration, HPLC, and mass spectrometry. Our findings identified eight fractions, among which fractions 5 and 6 exhibited notable toxicity, with fraction 5 resulting in mortality. Furthermore, we observed a prevalence of 3FTx, a diverse component in Naja venom, alongside short-chain neurotoxins such as short neurotoxin, cytotoxin, and muscarinic toxin-like proteins. Recognising these harmful elements is crucial for developing precise antivenom treatments. This investigation represents the first proteomic analysis of toxic fractions from Naja haje venom in Morocco, providing valuable insights into venom composition and aiding in the development of targeted therapies.

Effect of Egyptian spitting cobra Naja nubiae crude venom on immunogenic activity of rats

Snakes show defensive activities, often counting visual or auditory displays against an aggressor. The study observed what happens to rats administered subcutaneously sub-lethal doses of crude venom Naja nubiae. The pro-inflammatory cytokines, such as tumor necrosis alpha (TNF-α) and interleukin-6 (IL-6), as well as the anti-inflammatory cytokines such as interleukin-10 (IL-10), and inflammatory mediator's prostaglandin E-2 (PG-E2), were evaluated. Vascular permeability (VP) was employed to assess how leaky or permeable blood vessels are in various tissues and organs, including the rat peritoneal cavity and lymphoid organs. Lymphoid organs' histological alterations brought on by Nubiae venom. The study found that the two venom doses—1/4 and 1/2 LD50—induced high levels of inflammatory activity as evidenced by the production of inflammatory cytokines. These findings demonstrated that venom enhanced innate immunity through specifically increased T helper cells, IL-6, TNF-α, IL-10, and PG-E2. The results reveal whether the venom has an immunomodulatory effect and promotes inflammation. The data have a substantial impact on the development of new drugs and treatments for inflammatory conditions.

Comparison of the intrageneric neutralization scope of monospecific, bispecific/monogeneric and polyspecific/monogeneric antisera raised in horses immunized with sub-Saharan African snake venoms.

Snakebite envenomation inflicts a high burden of mortality and morbidity in sub-Saharan Africa. Antivenoms are the mainstay in the therapy of envenomation, and there is an urgent need to develop antivenoms of broad neutralizing efficacy for this region. The venoms used as immunogens to manufacture snake antivenoms are normally selected considering their medical importance and availability. Additionally, their ability to induce antibody responses with high neutralizing capability should be considered, an issue that involves the immunization scheme and the animal species being immunized. Using the lethality neutralization assay in mice, we compared the intrageneric neutralization scope of antisera generated by immunization of horses with monospecific, bispecific/monogeneric, and polyspecific/monogeneric immunogens formulated with venoms of Bitis spp., Echis spp., Dendroaspis spp., spitting Naja spp. or non-spitting Naja spp. It was found that the antisera raised by all the immunogens were able to neutralize the homologous venoms and, with a single exception, the heterologous congeneric venoms (considering spitting and non-spitting Naja separately). In general, the polyspecific antisera of Bitis spp, Echis spp, and Dendroaspis spp gave the best neutralization profile against venoms of these genera. For spitting Naja venoms, there were no significant differences in the neutralizing ability between monospecific, bispecific and polyspecific antisera. A similar result was obtained in the case of non-spitting Naja venoms, except that polyspecific antiserum was more effective against the venoms of N. melanoleuca and N. nivea as compared to the monospecific antiserum. The use of polyspecific immunogens is the best alternative to produce monogeneric antivenoms with wide neutralizing coverage against venoms of sub-Saharan African snakes of the Bitis, Echis, Naja (non-spitting) and Dendroaspis genera. On the other hand, a monospecific immunogen composed of venom of Naja nigricollis is suitable to produce a monogeneric antivenom with wide neutralizing coverage against venoms of spitting Naja spp. These findings can be used in the design of antivenoms of wide neutralizing scope for sub-Saharan Africa.

Inhibition of the Naja naja venom toxicity by polymeric nanoparticles loaded with Leucas aspera methanolic extract.

Snakebite is a neglected tropical disease that affects millions of people worldwide. Developing effective treatments can make a significant contribution to global health efforts and public health initiatives. To reduce mortality due to snakebite, there is an immediate need to explore novel and effective treatment methodologies. In that context, nanoparticle-based drug delivery is gaining a lot of attention. Hydrophilic nanoparticles are suitable for the delivery of therapeutic peptides, proteins, and antigens. The present investigation is aimed at evaluating the anti-ophidian potential of the methanolic extract of the ethno-medicinal herb Leucas aspera (Willd.) loaded within chitosan nanoparticles (CNP-LA), against the Indian cobra (Naja naja) venom enzymes. For this purpose, nanoparticles were prepared using the ionic gelation method to enhance the efficacy of the extract. The physicochemical and structural features of nanoparticles were investigated using dynamic light scattering (DLS), Fourier-transform Infrared (FTIR), field emission scanning electron microscopy (FE-SEM), and X-ray diffraction (XRD) techniques. It was found that CNP-LA has an average size of 260nm with a polydispersity index of 0.132 (PDI) and zeta potential of 34.7 mV, with an encapsulation efficiency of 92.46%. The in vitro release study was performed at pH 5.0 and 7.4. Furthermore, in vitro studies indicated that CNP-LA inhibited the phospholipase A2, hemolytic, and caseinolytic activities of Naja naja venom with the percentage inhibition of 92.5%, 83.9%, and 94.5%, respectively. This is the first report on the application of herbal methanolic extract loaded within chitosan nanoparticles for neutralizing snake venom enzymes with increased efficiency.

High-Voltage Toxin'Roll: Electrostatic Charge Repulsion as a Dynamic Venom Resistance Trait in Pythonid Snakes.

The evolutionary interplay between predator and prey has significantly shaped the development of snake venom, a critical adaptation for subduing prey. This arms race has spurred the diversification of the components of venom and the corresponding emergence of resistance mechanisms in the prey and predators of venomous snakes. Our study investigates the molecular basis of venom resistance in pythons, focusing on electrostatic charge repulsion as a defense against α-neurotoxins binding to the alpha-1 subunit of the postsynaptic nicotinic acetylcholine receptor. Through phylogenetic and bioactivity analyses of orthosteric site sequences from various python species, we explore the prevalence and evolution of amino acid substitutions that confer resistance by electrostatic repulsion, which initially evolved in response to predatory pressure by Naja (cobra) species (which occurs across Africa and Asia). The small African species Python regius retains the two resistance-conferring lysines (positions 189 and 191) of the ancestral Python genus, conferring resistance to sympatric Naja venoms. This differed from the giant African species Python sebae, which has secondarily lost one of these lysines, potentially due to its rapid growth out of the prey size range of sympatric Naja species. In contrast, the two Asian species Python brongersmai (small) and Python bivittatus (giant) share an identical orthosteric site, which exhibits the highest degree of resistance, attributed to three lysine residues in the orthosteric sites. One of these lysines (at orthosteric position 195) evolved in the last common ancestor of these two species, which may reflect an adaptive response to increased predation pressures from the sympatric α-neurotoxic snake-eating genus Ophiophagus (King Cobras) in Asia. All these terrestrial Python species, however, were less neurotoxin-susceptible than pythons in other genera which have evolved under different predatory pressure as: the Asian species Malayopython reticulatus which is arboreal as neonates and juveniles before rapidly reaching sizes as terrestrial adults too large for sympatric Ophiophagus species to consider as prey; and the terrestrial Australian species Aspidites melanocephalus which occupies a niche, devoid of selection pressure from α-neurotoxic predatory snakes. Our findings underline the importance of positive selection in the evolution of venom resistance and suggest a complex evolutionary history involving both conserved traits and secondary evolution. This study enhances our understanding of the molecular adaptations that enable pythons to survive in environments laden with venomous threats and offers insights into the ongoing co-evolution between venomous snakes and their prey.

Naja naja snake venom-induced local toxicities in mice is by inflammasome activation

Snake bite envenomation causes tissue damage resulting in acute and chronic inflammatory responses. Inflammasome activation is one of the factors involved in tissue damage in a mouse model of snake envenomation. The present study examines the potency of Indian Big Four snake venoms in the activation of inflammasome and its role in local and systemic tissue toxicity. Among Indian Big Four snake venoms, Naja naja venom activated NLRP3 inflammasome in mouse macrophages. Activation of NLRP3 inflammasome was also observed in mouse foot paw and thigh muscle upon administration of N. naja venom. Intraperitoneal administration of N. naja venom cause systemic lung damage showed activation of NLRP3 inflammasome. Treatment with MCC950, a selective NLRP3 inflammasome inhibitor effectively inhibited N. naja venom-induced activation of caspase-1 and liberation of IL-1β in macrophages. In mice, MCC950 partially inhibited the activation of NLRP3 inflammasome in N. naja venom administered foot paw and thigh muscle. In conclusion, the present data showed that inflammasome is one of the host responses involved in N. naja snake venom-induced toxicities. The inhibition of inflammasome activation will provide new insight into better management of snake bite-induced local tissue damage.

Recombinant human scFv antibody fragments against phospholipase A2 from Naja naja and Echis carinatus snake venoms: In vivo neutralization and mechanistic insights

Snake envenomation results in a range of clinical sequelae, and widely used animal-based conventional antivenoms exhibit several limitations including the adverse immunological effects in human snake bite victims. Therefore, human monoclonal anti-snake venom antibodies or fragments can be an alternate therapy for overcoming the existing limitations. We developed venom-neutralizing humanized scFv antibodies and analyzed biochemical mechanisms associated with the inhibition of toxicity. Tomlinson I and J human scFv antibody libraries were screened against Naja naja and Echis carinatus venoms, and seven unique scFv antibodies were obtained. Further, specific toxins of snake venom interacting with each of these scFvs were identified, and phospholipase A2 (PLA2) was found to be prominently captured by the phage-anchored scFv antibodies. Our study indicated PLA2 to be one of the abundant toxins in Naja naja and Echis carinatus venom samples. The scFvs binding to PLA2 were used to perform in vivo survival assay using the mouse model and in vitro toxin inhibition assays. scFv N194, which binds to acidic PLA2, protected 50% of mice treated with Naja naja venom. Significant prolongation of survival time and 16% survival were observed in Echis carinatus venom-challenged mice treated with scFv E113 and scFv E10, respectively. However, a combination comprised of an equal amount of two scFvs, E113 and E10, both interacting with basic PLA2, exhibited synergistically enhanced survival of 33% in Echis carinatus venom-challenged mice. No such synergistically enhanced survival was observed in the case of combinatorial treatment with anti-Naja naja scFvs, N194, and N248. These scFvs demonstrated partial inhibition of venom-induced myotoxicity, and E113 also inhibited hemolysis by 50%, which corroborates the enhanced survival during combinatorial treatment in Echis carinatus venom-challenged mice.

Effect of Microgravity on the Crystallization of Cardiotoxin from the Venom of Spectacled Cobra Naja naja

Effect of Microgravity on the Crystallization of Cardiotoxin from the Venom of Spectacled Cobra Naja naja

Effect of Microgravity on the Crystallization of Cardiotoxin from the Venom of Spectacled Cobra Naja naja

Cardiotoxins, which belong to the family of three-finger toxins, are the main components of cobra venom. They exhibit various types of biological activity, including antimicrobial and cytotoxic against cancer cells. Data on the minimal structural differences between individual toxins are necessary for understanding the molecular mechanisms of their action. This information can be obtained by high-resolution X-ray diffraction analysis. The influence of microgravity on the crystal packing and diffraction quality of crystals of cardiotoxin from cobra Naja naja has been investigated. Cardiotoxin crystals, which were grown on the International Space Station, provided maximally high resolution for the structure of this toxin. Protein crystallized extremely in the hexagonal space group, whereas more than half of crystals grown under laboratory conditions belonged to the orthorhombic system.

Evaluation of Neutralization Potential of Naja naja and Daboiarusselii Snake Venom by Root Extract of Cyanthillium cinereum.

One of the main reasons for the death due to snake bites is the non-availability of antivenoms in the regions where they are needed. The use of medicinal plants and plant-based natural products as an alternative to antivenom will become a milestone in snake bite envenomation. The present study investigates the in vitro antivenom properties of Cyanthillium cinereum root extracts. The C. cinereum root's aqueous extract was prepared by the Soxhlet extraction method, and phytochemical screening was performed to detect the presence of various bioactive compounds. Thin-layer chromatography (TLC) and gas chromatography-mass spectrometry (GC-MS) analysis were performed for the detection and identification of phytochemical constituents. In this study, an in vitro model is used to assess the antivenom capability of aqueous extract. Venom toxicity and neutralization assays were as follows: An in vitro pharmacological evaluation was performed by direct hemolysis assay, indirect hemolytic assay, proteolytic activity, neutralization of procoagulant activity, and gelatin liquefaction method. Qualitative analysis of phytochemicals by the standard method showed the presence of various phytochemical constituents. Also, GC-MS analysis showed the presence of three major compounds that possess antivenom activity from the obtained 60 bioactive compounds, and their chemical structures were also determined. Venom protein profiling was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The plant extract was able to neutralize the Naja naja (N. naja) and Daboia russelii (D. russelii) venom induced hemolysis and it was reduced below 50 and 40%, respectively and the extract was also able to reduce the hemolytic halo produced by venoms. Procoagulant activity and gelatin liquefaction assay showed that venom-induced clotting was neutralized by increasing the root extract concentration sufficiently. The aqueous extract of the root of C. cinereum showed potent in vitro venom-neutralizing activity, and it can be used as a formidable therapeutic agent against N. naja and D. russelii envenomation. Suji S, Dinesh MD, Keerthi KU, Anagha KP, Arya J, Anju KV. Evaluation of Neutralization Potential of Naja naja and Daboia russelii Snake Venom by Root Extract of Cyanthillium cinereum. Indian J Crit Care Med 2023;27(11):821-829.

The Royal Armoury: Venomics and antivenomics of king cobra (Ophiophagus hannah) from the Indian Western Ghats

Despite being famous as ‘the king’ of the snake world, the king cobra (Ophiophagus hannah) has remained a mysterious species, particularly with respect to its venom ecology. In contrast, venom research has largely focussed on the ‘big four’ snakes that are greatly responsible for the burden of snakebite in the Indian subcontinent. This study aims to bridge the current void in our understanding of the O. hannah venom by investigating its proteomic, biochemical, pharmacological, and toxinological profiles via interdisciplinary approaches. Considering their physical resemblance, the king cobra is often compared to the spectacled cobra (Naja naja). Comparative venomics of O. hannah and N. naja in this study provided interesting insights into their venom compositions, activities, and potencies. Our findings suggest that the O. hannah venom, despite being relatively less complex than the N. naja venom, is equally potent. Finally, our in vitro and in vivo assays revealed that both Indian polyvalent and Thai Red Cross monovalent antivenoms completely fail to neutralise the O. hannah venom. Our findings provide guidelines for the management of bites from this clinically important yet neglected snake species in India.

Multilevel Comparison of Indian Naja Venoms and Their Cross-Reactivity with Indian Polyvalent Antivenoms.

Snake envenoming is caused by many biological species, rather than a single infectious agent, each with a multiplicity of toxins in their venom. Hence, developing effective treatments is challenging, especially in biodiverse and biogeographically complex countries such as India. The present study represents the first genus-wide proteomics analysis of venom composition across Naja species (N. naja, N. oxiana, and N. kaouthia) found in mainland India. Venom proteomes were consistent between individuals from the same localities in terms of the toxin families present, but not in the relative abundance of those in the venom. There appears to be more compositional variation among N. naja from different locations than among N. kaouthia. Immunoblotting and in vitro neutralization assays indicated cross-reactivity with Indian polyvalent antivenom, in which antibodies raised against N. naja are present. However, we observed ineffective neutralization of PLA2 activities of N. naja venoms from locations distant from the source of immunizing venoms. Antivenom immunoprofiling by antivenomics revealed differential antigenicity of venoms from N. kaouthia and N. oxiana, and poor reactivity towards 3FTxs and PLA2s. Moreover, there was considerable variation between antivenoms from different manufacturers. These data indicate that improvements to antivenom manufacturing in India are highly desirable.

Comparative venom toxin analyses of Nigerian viperidae and elapidae snakes

Envenoming by snakebite is a serious health problem that maims and kills a large number of people, primarily in rural areas of developing African countries. The first comparative venom proteomic analyses of four snakes from the viperidae (E. ocellatus and B. arietans) and elapidae (N. haje and N. katiensis) families are presented in this study. Two-dimensional electrophoresis was combined with matrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze the venoms. Proteins were identified by comparing mass spectrometry spectra to those in the reviewed Uniprot-Serpentes database. A protein spot was considered differentially present between samples at a p-value of < 0.05 and a fold change of >2. Viper venoms contained cytotoxic-inducing proteins such as SVMPs, SVSPs, and cytotoxins, whereas elapid snake venoms contained neurotoxic proteins such as PLA2, 3-FTx, and neurotoxins. The PDQuest annotated protein spots on the 2-DE gels showed that the proteins in these snakes' venoms were differentially expressed between snake families and species. The elapid venoms were predominantly acidic (low pI) with low molecular masses, whereas the viperid venoms had high molecular masses and a pI in the region of 7. Venom phosphodiesterase, L-amino acid oxidase and cysteine-rich venom protein were common in the venoms of these snakes, while an uncommon protein actiflagelin was detected in the Naja venoms. Our findings show that there is significant variation in the toxin profiles of these snakes, both at the species and family levels. This has an impact on the clinical manifestations of envenomation. A thorough understanding of the various toxins found in venomous snakes may aid in the development of new and improved therapeutic strategies.

Antivenom Activity of Solanum xanthocarpum Hydroethanolic Root Extract Targeting the Enzymes Present in Naja Snake Venom

Background: The work reported in this paper targets the ethnobotanical role of one of the traditional medicinal plant Solanum xanthocarpum, whose medicinal value was reported in Ayurveda. Traditionally Solanum xanthocarpum was used as the antidote for treatment of snake bites by different tribal community of India, to explore and scientifically validate these concept this work was planned. Methods: We have prepared the hydro ethanolic root extract (HERE) of Solanum xanthocarpum and investigated its pharmacological activity both in vitro and in vivo, targeting the enzymes present in Naja snake venom. We have used the wistar albino rat as the animal model in our study. Result: It was inferred by our study that Solanum xanthocarpum is safe for wistar albino rats upto 2000 mg/kg body weight. Phospholipase A2 (PLA2) and anti-fibrinogenolytic activity are significantly neutralized by Solanum xanthocarpum. Dose dependent inhibition of human red blood cell (HRBC) lysis was also observed. In the future, bioactive compounds from HERE could be isolated and standardized to act as an antidote to Naja venom.

Potential of seaweed biomass: snake venom detoxifying action of brown seaweed Padina boergesenii against Naja naja venom

Potential of seaweed biomass: snake venom detoxifying action of brown seaweed Padina boergesenii against Naja naja venom

Monoclonal-Based Antivenomics Reveals Conserved Neutralizing Epitopes in Type I PLA2 Molecules from Coral Snakes.

For over a century, polyclonal antibodies have been used to treat snakebite envenoming and are still considered by the WHO as the only scientifically validated treatment for snakebites. Nevertheless, moderate innovations have been introduced to this immunotherapy. New strategies and approaches to understanding how antibodies recognize and neutralize snake toxins represent a challenge for next-generation antivenoms. The neurotoxic activity of Micrurus venom is mainly due to two distinct protein families, three-finger toxins (3FTx) and phospholipases A2 (PLA2). Structural conservation among protein family members may represent an opportunity to generate neutralizing monoclonal antibodies (mAbs) against family-conserved epitopes. In this work, we sought to produce a set of monoclonal antibodies against the most toxic components of M. altirostris venom. To this end, the crude venom was fractionated, and its major toxic proteins were identified and used to generate a panel of five mAbs. The specificity of these mAbs was characterized by ELISA and antivenomics approaches. Two of the generated mAbs recognized PLA2 epitopes. They inhibited PLA2 catalytic activity and showed paraspecific neutralization against the myotoxicity from the lethal effect of Micrurus and Naja venoms' PLA2s. Epitope conservation among venom PLA2 molecules suggests the possibility of generating pan-PLA2 neutralizing antibodies.

Naringenin isolated from Citrus reticulata blanco fruit peel inhibits the toxicity of snake venom proteins - An in vitro and in vivo study

Snake envenomation is a serious hazard in tropical countries. In the current study, the bioactive compound from the methanolic extract of Citrus reticulata was purified using polarity gradient chromatography and structurally characterized by UV-spectroscopy, NMR, and LC-MS. The purified metabolite, naringenin, inhibited PLA2 (51%) and hemolytic (59.8%) activity of the Naja naja venom. Histopathological examination and edema studies carried out using mice confirmed the inhibitory effects of naringenin. This is the first report on the inhibitory potential of naringenin isolated from Citrus reticulata. The study thus unravels the possibility for the use of Citrus reticulata metabolites as a supplement during the snake bite treatment.

Preclinical assessment of VPEAV, a new trivalent antivenom for elapid snakebite envenoming in the Philippines: Proteomics, immunoreactivity and toxicity neutralization

Snakebite envenoming is an important neglected tropical disease. Antivenom supply, however, remains limited in many parts of the world. This study aimed to examine the protein composition, immunoreactivity and neutralization efficacy of a new antivenom product (VINS Philippine Elapid Antivenoms, VPEAV) developed for the treatment of snakebite envenoming caused by the Philippine Cobra (Naja philippinensis), Samar Cobra (Naja samarensis) and King Cobra (Ophiophagus hannah). Size-exclusion chromatography, sodium-dodecyl sulfate-polyacrylamide gel electrophoresis and tandem mass spectrometry showed that VPEAV consisted of F(ab)’2 (∼90% of total antivenom proteins) with minimal protein impurities. Indirect ELISA showed varying immunoreactivity of VPEAV toward the different venoms (EC50 = 4–16 μg/ml), indicating distinct venom antigenicity between the species. In mice, the neutralization potency of VPEAV against the King Cobra venom was moderate (potency, P = 2.6 mg/ml, defined as the amount of venom completely neutralized per unit volume of antivenom). The potency was significantly lower against the N. philippinensis and N. samarensis venoms (P = 0.18–0.30 mg/ml), implying a higher dose may be needed for effective neutralization of the Naja venoms. Together, the findings suggest the potential and limitation of VPEAV in neutralizing the venom toxicity of the three Philippine elapid snakes.

Augmented rescue of macroglobulins by supplementation of anti-snake venom with methanolic extract of Andrographis paniculata in Naja naja envenomation

Proteins of the macroglobulin family are prime targets of venom enzymes in snake bite. A massive reduction in the active concentration of these multifunctional proteins in snake bite, makes the living system vulnerable to dysregulation. This study investigates the ability of Indian polyvalent anti-snake venom (ASV), methanolic extract of Andrographis paniculata (MAP) and their combination in rescuing human alpha 2-macroglobulin (A2MG) and its homologues in rat plasma, from inactivation by Naja naja (N.N) venom enzymes. In-vitro experiments were conducted with heparinized human plasma and in-vivo experiments with female Wistar rats. Along with appropriate controls, there were 3 test groups in in-vitro and 8 test groups in in-vivo experiments. The in-vitro test groups were exposed to N.N venom for zero, 30 or 90 min prior to incubation with ASV or MAP or reduced ASV supplemented with MAP and incubated for 16 h at 37 °C. Chymotrypsin-bound esterase (CTBE) activity of A2MG was estimated. Rats were administered the venom intramuscularly and treated with ASV/MAP/ASV + MAP. CTBE activity of macroglobulin homologues was measured on day 1, 7 and 14. Survival of animals was noted. In human plasma, addition of ASV or MAP or ASV + MAP prevented loss of A2MG activity maximally to the extent of 88–100% (p = 0.001). In rats, reduced concentration of ASV supplemented with MAP showed complete rescue of macroglobulin homologues and 90% survival. The compulsive evidence from this study, underscores the merits of using this multipronged strategy in rescuing the macroglobulins and improving survival in envenomation due to N.N.

Comparative Snake Venom Analysis for Facilitating Wildlife Forensics: A Pilot Study.

Confirm and authentic identification of species is required for the implementation of wildlife laws in cases of illegal trafficking of snake venoms. Illegally trafficked snake venom might be misidentified with other drugs of abuse, and sometimes, the species of venom-yielding snake cannot be verified. Snake venoms from medically important snake species, Naja naja and Daboia russelii, were procured from Irula Snake Catcher's Society, Tamil Nadu, India. Comparative analyses of both venoms were carried out using SDS-PAGE, LC-MS/MS, ICP-MS, and mtDNA analysis. The protein concentration of Naja naja and Daboia russelii venoms was 76.1% and 83.9%, respectively. SDS analysis showed a distinct banding pattern of both venoms. LC-MS/MS results showed proteins and toxins from 12 to 14 protein families in Naja naja and Daboia russelii venoms. Elemental analysis using ICP-MS showed a different profile of some elements in both venoms. mtDNA analysis of venoms using universal primers against Cyt b gene showed homology with sequence of Naja naja and Daboia russelii genes. The study proposed a template of various conventional and advanced molecular and instrumental techniques with their pros and cons. The template can be used by forensic science laboratories for detection, screening, and confirmatory analysis of suspected venoms of snakes. Clubbing of various techniques can be used to confirm the identification of species of snake from which the alleged venom was milked. The results can be helpful in framing charge-sheets against accused of illegal venom trafficking and can also be used to verify the purity and quality of commercially available snake venoms.