R.D.G. Theakston

MICRO-ELISA FOR DETECTING AND ASSAYING SNAKE VENOM AND VENOM-ANTIBODY

Enzyme-linked immunosorbent assay (ELISA) can detect venom levels down to 1-5 ng/ml. The assay is specific; no cross-reaction of clinical importance was found in a range of 14 different types of venom. Both venom and venom-antibody levels were monitored in mice. Specific antibody was detected in human serum more than 2 years after an accidental bite by Echis carinatus. ELISA should clarify many basic problems of envenoming in man.

Insights into the mechanism of haemorrhage caused by snake venom metalloproteinases.

Local and systemic haemorrhage are common consequences of crotaline and viperine envenoming. Several studies carried out using purified toxins have indicated that local haemorrhage can be attributed to a distinct class of venom metalloproteinases. Analyses of their cDNAs predict multi-domain enzymes, with an N-terminal metalloproteinase domain, a disintegrin-like domain and a Cys-rich C-terminus. Haemorrhagic metalloproteinases are responsible for degrading proteins of the extracellular matrix and they also have cytotoxic effects on endothelial cells. However, to date very few investigations have been carried out on the effects of venom haemorrhagic metalloproteinases on components of the haemostatic system. We describe here the effects of a high molecular weight haemorrhagic metalloproteinase, jararhagin, from the venom of a South American pit viper Bothrops jararaca, on platelet and plasma components involved in haemostasis. Jararhagin, which is not inhibited in plasma, causes the loss of the platelet collagen receptor alpha 2 beta 1 integrin (gpIa/IIa or VLA-2) and degrades the adhesive plasma protein von Willebrand factor. Alterations of these haemostatic components are known to result in bleeding. This suggests that venom haemorrhagic metalloproteinases, in addition to causing local bleeding, may also contribute to systemic haemorrhage.

Approximate LD50 determinations of snake venoms using eight to ten experimental animals

A method is described for the assessment of lethal toxicity of venoms using a modified LD50 assay. With this test it is possible to obtain an LD50 using only 8-10 experimental animals, instead of 30 or more. It is suggested for ethical, scientific and economic reasons that this method be tested in laboratories involved in screening of venoms for lethality and, if found satisfactory, it should replace the classical LD50 assay.

Combining mitochondrial DNA sequences and morphological data to infer species boundaries: phylogeography of lanceheaded pitvipers in the Brazilian Atlantic forest, and the status of Bothrops pradoi (Squamata: Serpentes: Viperidae)

Phylogeographic studies using mitochondrial DNA sequence information are frequently used as the principal source of evidence to infer species boundaries. However, a critical analysis of further evidence is essential to test whether different haplotype clades identify different species. We demonstrate a hypothesis‐testing approach, using a combination of phylogeographic methods, multivariate morphometrics and matrix association tests, to investigate species boundaries in eastern Brazilian pitvipers conventionally assigned to the species Bothrops leucurus and B. pradoi. Two basal haplotype clades with partly overlapping geographical distributions are identified, which could either represent two partly sympatric species, or multiple haplotypes within one organismal lineage. We use partial Mantel matrix association tests to verify whether generalized morphology, or any of four supposedly diagnostic characters for the two species, show any association with mtDNA variation. Negative results lead to the conclusion that the haplotype clades do not denote independently evolving organismal lineages, and do not constitute separate species under any criterion.

Incidence and mortality of snake bite in savanna Nigeria.

Snake bite is a major medical problem among farming communities in the Benue and Niger Valley savanna region of Nigeria. Surveys in the more densely populated northern savanna areas such as Malumfashi (incidence 48 per 100 000 population, mortality 5.1%) show that the spitting cobra, Naja nigricollis, was the predominant species of medical importance. In the less cultivated Benue Valley area the annual snake-bite incidence was estimated to be 497 per 100 000 population, with a 12.2% mortality due mainly to the carpet viper, Echis carinatus. From these findings we estimate a yearly total of almost 10 000 deaths from snake bite in savanna Nigeria and about 23 000 deaths in West Africa. To rectify this serious health problem, concerted efforts are urgently needed.

The application of immunoassay techniques, including enzyme-linked immunosorbent assay (ELISA), to snake venom research

The development and application of immunoassay techniques in relation to snake venom research is reviewed. Enzyme linked immunosorbent assay (ELISA) is compared with radioimmunoassay, immunodiffusion, immunofluorescence, haemagglutination and immunoelectrophoresis. It is concluded that ELISA is the most versatile immunoassay technique so far applied to the field of venom research, its main advantages over other methods including relatively high levels of sensitivity and specificity, reproducibility, simplicity and ease of sample collection. It can also be readily modified into kit form and is easily adapted for use in large scale epidemiological studies and for accurate retrospective diagnosis of snake bite. None of the other assay systems considered fulfil these criteria to the same extent. ELISA is helping to advance epidemiological knowledge of snake bite, in exploring the role of active immunisation and in the compilation of accurate clinical patterns of envenoming. Other applications of the test include its use for potency screening of both new and developed commercially available antivenoms and for the detection of monoclonal antibodies which should eventually result in increased specificity of the assay system by eliminating cross reactions between venoms and antibodies of closely related species.

Envenoming by the common krait (Bungarus caeruleus) and Sri Lankan cobra (Naja naja naja): efficacy and complications of therapy with Haffkine antivenom

In Anuradhapura, Sri Lanka, 5 patients proved to have been bitten by common kraits (Bungarus caeruelus) and 2 by Sri Lankan cobras (Naja naja naja) were investigated. In all the cases of krait bite the patients were bitten while they were asleep: local signs were negligible but 4 developed symptoms of systemic envenoming including paralysis, muscle pain and tenderness and abdominal pain. Mild myoglobinaemia was found in one case. Of the 2 patients bitten by cobras, one developed severe local swelling which progressed to necrosis and the other local swelling and respiratory paralysis. Response to polyspecific antivenom (Haffkine, India) was neither rapid nor convincing. Venom antigenaemia became undetectable within 2 h of the start of antivenom treatment, but recurred 25 and 65 h later in 2 cases. Among a group of 27 patients treated with this antivenom (including 21 bitten by Russells vipers), the incidence of early anaphylactic and pyrogenic reactions was high at 52% and 65% respectively. Anticholinesterase did not improve paralysis in 2 patients bitten by kraits. The respiratory failure in 2 patients was successfully treated by mechanical ventilation for 8 and 30 h. These observations confirm the importance of neurotoxic symptoms following bites by these species but also suggest a contributory role of generalized rhabdomyolysis in krait victims and emphasize the problem of severe local tissue necrosis in cobra victims. There is a need for safer and more potent antivenoms for use in Sri Lanka.

Severe neurotoxic envenoming by the Malayan krait Bungarus candidus (Linnaeus): response to antivenom and anticholinesterase.

Five patients were bitten by the Malayan krait Bungarus candidus (Linnaeus) in eastern Thailand or north western Malaya. Two patients were not envenomed but the other three developed generalised paralysis which progressed to respiratory paralysis in two cases, one of which ended fatally. One patient showed parasympathetic abnormalities. Anticholinesterase produced a dramatic improvement in one patient. Another patient probably benefited from paraspecific antivenom. The efficacy of antivenoms and adjuvants such as anticholinesterases in patients with neurotoxic envenoming requires further study.

Reliability of the simple 20 minute whole blood clotting test (WBCT20) as an indicator of low plasma fibrinogen concentration in patients envenomed by Bothrops snakes

Reliability of the simple 20 minute whole blood clotting test (WBCT20) as an indicator of low plasma fibrinogen concentration in patients envenomed by Bothrops snakes. Toxicon 32, 1045-1050, 1994.--A simple whole blood clotting test (WBCT20) was assessed for its efficacy in determination of severe defibrinogenation in patients envenomed by Bothrops snakes in Brazil. There was a close relationship between the results of the WBCT20 and plasma fibrinogen levels in 69 moderately envenomed patients. The advantage of the WBCT20 over estimation of plasma fibrinogen concentrations in patients is that it is a simpler, faster and more reliable test. It is also of use in assessing the effectiveness of antivenom therapy in relation to the restoration of blood coagulability.

Evolution of Disintegrin Cysteine-Rich and Mammalian Matrix-Degrading Metalloproteinases: Gene Duplication and Divergence of a Common Ancestor Rather Than Convergent Evolution

The evolution of the Metalloproteinase Disintegrin Cysteine-rich (MDC) gene family and that of the mammalian Matrix-degrading Metalloproteinases (MMPs) are compared. The alignment of snake venom and mammalian MDC and MMP precursor sequences generated a phylogenetic tree that grouped these proteins mainly according to their function. Based on this observation, a common ancestry is suggested for mammalian and snake venom MDCs; it is also possible that gene duplication of the already-assembled domain structure, followed by divergence of the copies, may have significantly contributed to the evolution of the functionally diverse MDC proteins. The data also suggest that the structural resemblance of the zinc-binding motif of venom MDCs and MMPs may best be explained by common ancestry and conservation of the proteolytic motifs during the divergence of the proteins rather than through convergent evolution.