Bitis Research Articles

Investigating Snake-Venom-Induced Dermonecrosis and Inflammation Using an Ex Vivo Human Skin Model.

Snakebite envenoming is a neglected tropical disease that causes >100,000 deaths and >400,000 cases of morbidity annually. Despite the use of mouse models, severe local envenoming, defined by morbidity-causing local tissue necrosis, remains poorly understood, and human-tissue responses are ill-defined. Here, for the first time, an ex vivo, non-perfused human skin model was used to investigate temporal histopathological and immunological changes following subcutaneous injections of venoms from medically important African vipers (Echis ocellatus and Bitis arietans) and cobras (Naja nigricollis and N. haje). Histological analysis of venom-injected ex vivo human skin biopsies revealed morphological changes in the epidermis (ballooning degeneration, erosion, and ulceration) comparable to clinical signs of local envenoming. Immunostaining of these biopsies confirmed cell apoptosis consistent with the onset of necrosis. RNA sequencing, multiplex bead arrays, and ELISAs demonstrated that venom-injected human skin biopsies exhibited higher rates of transcription and expression of chemokines (CXCL5, MIP1-ALPHA, RANTES, MCP-1, and MIG), cytokines (IL-1β, IL-1RA, G-CSF/CSF-3, and GM-CSF), and growth factors (VEGF-A, FGF, and HGF) in comparison to non-injected biopsies. To investigate the efficacy of antivenom, SAIMR Echis monovalent or SAIMR polyvalent antivenom was injected one hour following E. ocellatus or N. nigricollis venom treatment, respectively, and although antivenom did not prevent venom-induced dermal tissue damage, it did reduce all pro-inflammatory chemokines, cytokines, and growth factors to normal levels after 48 h. This ex vivo skin model could be useful for studies evaluating the progression of local envenoming and the efficacy of snakebite treatments.

Comparing Traditional and Toxin-Oriented Approaches towards Antivenom Production against Bitis arietans Snake Venom.

Accidents with snakes are responsible for about 32,000 deaths annually in sub-Saharan Africa, caused mostly by snakes from the genus Bitis, in particular Bitis arietans. B. arietans venom is composed of a complex mixture of toxins, mainly metalloproteases, serine proteases, phospholipases, lectins, and disintegrins. In this work, we compared two approaches to anti-B. arietans antivenom production: immunization with crude snake venom ("traditional approach") and immunization with selected key toxins isolated from the snake venom ("toxin oriented" approach). Fractions from B. arietans venom were isolated by size exclusion chromatography. Crude venom and samples containing serine proteases or metalloproteases were selected for the immunization of BALB/c mice. Anti-B. arietans and anti-serine proteases plasmas showed a similar recognition profile and higher titers and affinity than the anti-metalloproteases plasma. Cross-recognition of other Bitis venoms was observed, but with low intensity. Although the plasma of all experimental groups inhibited the enzymatic activity of B. arietans venom in vitro, in vivo protection was not achieved. Our results have shown limitations in both approaches considered. Based on this, we proposed a model of polyclonal, species-specific, monovalent antivenoms that could be used as a base to produce customizable polyvalent sera for use in sub-Saharan Africa.

Diversification of the African legless skinks in the subfamily Acontinae (Family Scincidae)

Cladogenic diversification is often explained by referring to climatic oscillations and geomorphic shifts that cause allopatric speciation. In this regard, southern Africa retains a high level of landscape heterogeneity in vegetation, geology, and rainfall patterns. The legless skink subfamily Acontinae occurs broadly across the southern African subcontinent and therefore provides an ideal model group for investigating biogeographic patterns associated with the region. A robust phylogenetic study of the Acontinae with comprehensive coverage and adequate sampling of each taxon has been lacking up until now, resulting in unresolved questions regarding the subfamily’s biogeography and evolution. In this study, we used multi-locus genetic markers (three mitochondrial and two nuclear) with comprehensive taxon coverage (all currently recognized Acontinae species) and adequate sampling (multiple specimens for most taxa) of each taxon to infer a phylogeny for the subfamily. The phylogeny retrieved four well-supported clades in Acontias and supported the monophyly of Typhlosaurus. Following the General Lineage Concept (GLC), many long-standing phylogenetic enigmas within Acontias occidentalis and the A. kgalagadi, A. lineatus and A. meleagris species complexes, and within Typhlosaurus were resolved. Our species delimitation analyses suggest the existence of hidden taxa in the A. occidentalis, A. cregoi and A. meleagris species groups, but also suggest that some currently recognized species in the A. lineatus and A. meleagris species groups, and within Typhlosaurus, should be synonymised. We also possibly encountered “ghost introgression” in A. occidentalis. Our inferred species tree revealed a signal of gene flow, which implies possible cross-over in some groups. Fossil evidence calibration dating results showed that the divergence between Typhlosaurus and Acontias was likely influenced by cooling and increasing aridity along the southwest coast in the mid-Oligocene caused by the opening of the Drake Passage. Further cladogenesis observed in Typhlosaurus and Acontias was likely influenced by Miocene cooling, expansion of open habitat, uplifting of the eastern Great Escarpment (GE), and variation in rainfall patterns, together with the effect of the warm Agulhas Current since the early Miocene, the development of the cold Benguela Current since the late Miocene, and their co-effects. The biogeographic pattern of the Acontinae bears close resemblance to that of other herpetofauna (e.g., rain frogs and African vipers) in southern Africa.

A dwarf among giants: phylogenetic position of the elusive Angolan Adder (Bitis heraldica) and biogeographic affinities of Angolan Afromontane regions

ABSTRACT The Angolan Adder, Bitis heraldica (Bocage, 1889), is endemic to the Angolan central plateau and is one of the most poorly known and rarely observed species of African snakes. The phylogenetic placement of B. heraldica within the four subgenera of the genus Bitis (Bitis, Macrocerastes, Calechidna, Keniabitis) has been problematic. The few recent taxonomic and phylogenetic revisions dealing with African viperids and the genus Bitis have not included this species, resulting in the default acceptance of B. heraldica as member of the subgenus Calechidna, based on similar morphology and size. During a survey in Huambo Province, central Angola, we collected the first specimen of this species in the past 65 years, allowing us to examine its phylogenetic relationships to other Bitis using multigene Bayesian and maximum likelihood analyses of all species of Bitis, except B. harenna. Contrary to expectations that B. heraldica is a member of the subgenus Calechidna, our results unambiguously place B. heraldica among members of the subgenus Macrocerastes, which contains the largest-bodied species of the genus. The placement of B. heraldica within Macrocerastes raises interesting questions about body-size evolution in viperids and contributes to a better understanding of biogeographic patterns in south-western Africa, and particularly Angola.

Varespladib (LY315920) neutralises phospholipase A2 mediated prothrombinase-inhibition induced by Bitis snake venoms

Anticoagulant toxicity is a common function of venoms produced by species within the Bitis genus. Potent inhibition of the prothrombinase complex is an identified mechanism of action for the dwarf species B. cornuta and B. xeropaga, along with some localities of B. atropos and B. caudalis. Snake venom phospholipase A2 toxins that inhibit the prothrombinase complex have been identified in snake venom, including an isolated phospholipase A2 toxin from B. caudalis. Current research is investigating the ability of the drug varespladib to inhibit snake venom phospholipase A2 toxins and reduce their toxicity. In particular, varespladib is being investigated as a treatment that could be administered prior to hospital referral which is a major necessity for species such as those from the genus Bitis, due to envenomations often occurring in remote regions of Africa where antivenom is unavailable. Using previously validated coagulation assays, this study aimed to determine if the toxins responsible for inhibition of the prothrombinase complex in the venom of four Bitis species are phospholipase A2 toxins, and if varespladib is able to neutralise this anticoagulant activity. Our results demonstrate that varespladib strongly neutralises the prothrombinase-inhibiting effects of all venoms tested in this study, and that this prothrombinase-inhibiting mechanism of anticoagulant activity is driven by phospholipase A2 class toxins in these four species. This study extends previous reports demonstrating varespladib has broad efficacy for treatment of phospholipase A2 rich snake venoms, indicating it also inhibits their anticoagulant effects mediated by prothrombinase-inhibition.

Venomous Landmines: Clinical Implications of Extreme Coagulotoxic Diversification and Differential Neutralization by Antivenom of Venoms within the Viperid Snake Genus Bitis.

The genus Bitis comprises 17 snake species that inhabit Africa and the Arabian Peninsula. They are responsible for a significant proportion of snakebites in the region. The venoms of the two independent lineages of giant Bitis (B. arietans and again in the common ancestor of the clade consisting of B. gabonica, B. nasicornis, B. parviocula and B. rhinoceros) induce an array of debilitating effects including anticoagulation, hemorrhagic shock and cytotoxicity, whilst the dwarf species B. atropos is known to have strong neurotoxic effects. However, the venom effects of the other species within the genus have not been explored in detail. A series of coagulation assays were implemented to assess the coagulotoxic venom effects of fourteen species within the genus. This study identified procoagulant venom as the ancestral condition, retained only by the basal dwarf species B. worthingtoni, suggesting anticoagulant venom is a derived trait within the Bitis genus and has been secondarily amplified on at least four occasions. A wide range of anticoagulant mechanisms were identified, such as coagulant and destructive activities upon fibrinogen in both giant and dwarf Bitis and the action of inhibiting the prothrombinase complex, which is present in a clade of dwarf Bitis. Antivenom studies revealed that while the procoagulant effects of B. worthingtoni were poorly neutralized, and thus a cause for concern, the differential mechanisms of anticoagulation in other species were all well neutralized. Thus, this study concludes there is a wide range of coagulotoxic mechanisms which have evolved within the Bitis genus and that clinical management strategies are limited for the procoagulant effects of B. worthingtoni, but that anticoagulant effects of other species are readily treated by the South African polyvalent antivenom. These results therefore have direct, real-work implications for the treatment of envenomed patients.

Ancient habitat shifts and organismal diversification are decoupled in the African viper genus Bitis (Serpentes: Viperidae)

AbstractAimThe expansion of open habitats during the mid‐Miocene has been hypothesized as a driver of allopatric speciation for many African taxa. This habitat‐dependent mode of diversification has been implicated in the shift from C3 (e.g. forest/woodland) to C4 dominated systems (i.e. open savanna, grasslands) in a number of African squamates. We examined this hypothesis using a genus of African viperid snakes (Bitis) with both open habitat and forest‐dwelling representatives.LocationAfrica.MethodsA comprehensive multilocus dataset was used to generate a calibrated species tree using a multispecies coalescent model. Individual gene trees and patterns of nuclear allele sharing were used to assess species monophyly and isolation. To test the habitat‐dependent evolution hypothesis, we generated an ancestral character state reconstruction for open and closed habitats using the dated phylogeny. This was related to the timing of open habitat expansion and forest/woodland contraction in Africa.ResultsThe genus Bitis originated in the Oligocene, with species level diversification in the late Miocene/Pliocene. Four well‐supported clades correspond to the recognized subgenera Bitis, Keniabitis, Macrocerastes and Calechidna. Several previously unrecognized lineages potentially represent cryptic species.Main conclusionsHabitat‐dependent evolution does not appear to have been a main driver for generic level viperine diversification: the ancestral state for Bitis was open habitat and at least one clade moved into forest in the Miocene, long after forest had contracted and fragmented. Forest‐dependent species diversified only in the late Miocene, presumably as forest became further reduced in extent, fitting an allopatric model of speciation. Although our results do not favour a general pattern of habitat‐dependent diversification in Bitis, cladogenesis within the subgenus Calechidna for “arenicolous” species (Bitis caudalis complex) and “rupicolous” species (B. atropos‐cornuta complex), corresponds to the aridification of southwest Africa. This suggests there are subtleties not captured in the broad open habitat category, which are relevant for understanding the role of habitat‐dependent evolution.

Microstructure-mediated Optical Effects in Southern African Snakes

The scales of the African Viper Bitis arietans were tested for optical effects. Spectral intensity was recorded at incident angles over the visible spectrum for dark, pale, and ventral scale regions. The lowest spectral intensity recordings were associated with scales which have the greatest level of micro-structuring. Our results indicate that scale appearance in B. arietans is a product of microstructure-mediated optical effects. The optical effect may play a role in improving the ecological performance of the snake in its natural environment.

Scale-dependent Orientation in Movement Paths: A Case Study of an African Viper

Decisions relating to the orientation of movement by animals and how this translates into movement patterns can occur at multiple spatial scales simultaneously, but this interaction is poorly understood for many groups of animals. Using the tracks left by moving snakes in their sandy habitat, we studied the movement paths of the African snake Bitis schneideri (Namaqua dwarf adder) for evidence of broad-scale directional persistence and short-range avoidance of exposure. Although snakes clearly displayed directional persistence, they preferentially moved to nearby shrubs, thereby minimizing exposure to solar and thermal radiation and/or predation. Thus, snakes made decisions relating to orientation at a minimum of two scales, the interaction of which resulted in snakes moving ≈17% (mean straightness index = 0.85) further than the simple broad-scale straight-line distance. We assert that the actual path chosen by moving snakes represents a trade-off of various costs and risks that include risk of predation, exposure to the elements, time and energy expenditure. Our study highlights the need for cognizance of the possibility of the scale dependence of orientation and movement in studies of snake movement, and adds to a growing literature demonstrating previously unrecognized behavioural complexity in snakes.

African Viper Poly-His Tag Peptide Fragment Efficiently Binds Metal Ions and Is Folded into an α-Helical Structure.

Snake venoms are complex mixtures of toxic and often spectacularly biologically active components. Some African vipers contain polyhistidine and polyglycine peptides, which play a crucial role in the interaction with metal ions during the inhibition of snake metalloproteases. Polyhistidine peptide fragments, known as poly-His tags, play many important functions, e.g., in metal ion transport in bacterial chaperon proteins. In this paper, we report a detailed characterization of Cu(2+), Ni(2+), and Zn(2+) complexes with the EDDHHHHHHHHHG peptide fragment (pHG) derived from the venom of the rough scale bush viper (Atheris squamigera). In order to determine the thermodynamic properties, stoichiometry, binding sites, and structures of the metal-pHG complexes, we used a combination of experimental techniques (potentiometric titrations, electrospray ionization mass spectrometry, UV-vis spectroscopy, circular dichroism spectroscopy, and electron paramagnetic resonance spectroscopy) and extensive computational tools (molecular dynamics simulations and density functional theory calculations). The results showed that pHG has a high affinity toward metal ions. The numerous histidine residues located along this sequence are efficient metal ion chelators with high affinities toward Cu(2+), Ni(2+), and Zn(2+) ions. The formation of an α-helical structure induced by metal ion coordination and the occurrence of polymorphic binding states were observed. It is proposed that metal ions can "move along" the poly-His tag, which serves as a metal ion transport pathway. The coordination of Cu(2+), Ni(2+), and Zn(2+) ions to the histidine tag is very effective in comparison with other histidine-rich peptides. The stabilities of the metal-pHG complexes increase in the order Zn(2+) < Ni(2+)≪ Cu(2+).

African Adders: Partial Characterization of Snake Venoms from Three Bitis Species of Medical Importance and Their Neutralization by Experimental Equine Antivenoms

BackgroundAn alarming number of fatal accidents involving snakes are annually reported in Africa and most of the victims suffer from permanent local tissue damage and chronic disabilities. Envenomation by snakes belonging to the genus Bitis, Viperidae family, are common in Sub-Saharan Africa. The accidents are severe and the victims often have a poor prognosis due to the lack of effective specific therapies. In this study we have biochemically characterized venoms from three different species of Bitis, i.e., Bitis arietans, Bitis gabonica rhinoceros and Bitis nasicornis, involved in the majority of the human accidents in Africa, and analyzed the in vitro neutralizing ability of two experimental antivenoms.Methodology/Principal FindingsThe data indicate that all venoms presented phospholipase, hyaluronidase and fibrinogenolytic activities and cleaved efficiently the FRET substrate Abz-RPPGFSPFRQ-EDDnp and angiotensin I, generating angiotensin 1–7. Gelatinolytic activity was only observed in the venoms of B. arietans and B. nasicornis. The treatment of the venoms with protease inhibitors indicated that Bitis venoms possess metallo and serinoproteases enzymes, which may be involved in the different biological activities here evaluated. Experimental antivenoms produced against B. arietans venom or Bitis g. rhinoceros plus B. nasicornis venoms cross-reacted with the venoms from the three species and blocked, in different degrees, all the enzymatic activities in which they were tested.ConclusionThese results suggest that the venoms of the three Bitis species, involved in accidents with humans in the Sub-Saharan Africa, contain a mixture of various enzymes that may act in the generation and development of some of the clinical manifestations of the envenomations. We also demonstrated that horse antivenoms produced against B. arietans or B. g. rhinoceros plus B. nasicornis venoms can blocked some of the toxic activities of these venoms.

Recovering the evolutionary history of Africa’s most diverse viper genus: morphological and molecular phylogeny of Bitis (Reptilia: Squamata: Viperidae)

Assessing evolutionary relationships among wide-ranging species can be particularly beneficial to our understanding of speciation patterns and biogeography of taxa, with broad implications for conservation and applications for human health. Integrative phylogenetic analyses that incorporate multiple independent datasets (e.g., DNA, protein, phenotype) can resolve many problematic issues in systematics such as cryptic diversity and incongruence between datasets. Vipers in the genus Bitis are widely distributed throughout much of sub-Saharan Africa, filling a variety of ecological niches and presenting an important public health problem. However, evolutionary relationships among this medically and ecologically important genus have not been fully resolved due to inadequate taxon sampling and lack of informative characters. Here, we conduct the first phylogenetic study incorporating complete sampling of known species within the genus Bitis. Using morphological, molecular, and combined approaches under multiple criteria, we recovered many of the species groups detected by previous investigators, further validating four currently recognized subgenera. Bitis arietans and Bitis worthingtoni appear to be early-diverging, monotypic lineages, while the “big Bitis” group and the small southern African species form distinct clades. Although our study provides additional information regarding the interspecific relationships within Bitis, the placement of Bitis albanica, Bitis heraldica, and Bitis inornata remains problematic. This study enhances our understanding of the evolutionary history of species within the genus Bitis incorporating a combined evidence approach to phylogenetics.

The unusual binding mechanism of Cu(ii) ions to the poly-histidyl domain of a peptide found in the venom of an African viper

Copper complexes of a poly-His/poly-Gly peptide (EDDHHHHHHHHHGVGGGGGGGGGG-NH2), a natural component of a snake venom, were studied by means of both experimental (thermodynamic, spectroscopic and MS) techniques and molecular dynamics (MD) simulations and density functional theory (DFT) calculations. This peptide proved to be an exceptionally effective copper chelator, forming complexes which are thermodynamically more stable than those formed by both the albumin-like ATCUN motif and several other poly-histidine protein fragments. We show that, in a poly-histidine stretch, copper seems to prefer binding to residues separated by one amino acid and that a correlation between an α-helical structure of the predicted complexes and their thermodynamic stability is observed.

Hemostasis dynamics during coagulopathy resulting from Echis envenomation

This work provides a graphic description of the time course of hemostasis tests results during spontaneous evolution of Echis envenoming and correction of hemostasis disorders with antivenom therapy.The dynamics of fibrinogenemia (g L−1), prothrombin time (PT, %), activated partial thromboplastin time (aPTT, patient/normal ratio) and platelet count (Giga L−1) were collected from coagulopathic envenomed patients of a 12 years prospective study in Africa. Sixty patients were included. 47 of them (78%) received an antivenom (33 ± 12 ml) and 13 did not. Thirty patients (50%) presented bleeding. Only one patient died. The time for fibrinogen to be more than 1 g L−1 was 181 ± 116 h (7.5 days) in the spontaneous evolution group versus 40 ± 21 h in the antivenom group (p < 0.0001). The times for reaching a PT above 50% were 140 ± 64 min (5.8 days) versus 25 ± 15 h (p < 0.00001) and for reaching an aPTT less than 1.5 times the normal values, 116 ± 76 h (4.7 days) versus 10 ± 9 h respectively (p < 0.0002). Thrombopenia was not a common feature of Echis envenomation.This study is the first one to provide a chart of the evolution of the hemostatic tests during envenomation caused by Echis bites. The plots enable to estimate that, in Echis envenomation, in the absence of antivenom administration, hemostasis remains severely affected until the 8–10th day of evolution. On the contrary, efficient antivenom against African vipers corrects clotting functions within a few hours.

Anonymous nuclear markers for the African adders (Serpentes: Viperidae: Bitis)

We report five novel anonymous nuclear loci for the African viper genus Bitis, developed from a genomic library of a single individual puff adder (Bitis arietans). All loci amplify consistently and are variable among seven B. arietans sampled from disparate portions of the geographic range. Trials with nine congeneric species showed one locus amplifies across all species tested, three amplify in a subset of species and one only amplifies in B. arietans. Genetic divergences for these loci range from comparable, to more than double that of a commonly used nuclear protein-coding marker in Bitis. These loci will provide a valuable resource for future investigations of these snakes.

Snakebite admissions in Zimbabwe: pattern, clinical presentation and management.

To describe the toxicoepidemiology of snakebite admissions to eight major referral hospitals in Zimbabwe. Retrospective and descriptive case review. Four central hospitals (i.e., Harare, Parirenyatwa, Mpilo and United Bulawayo Hospitals) and four provincial hospitals (i,e., Gwanda, Bindura, Gweru and Mutare hospitals). Parameters assessed for included patient demographics e.g. age and gender, length of hospital stay, circumstances leading to snakebite, season and time of day of bite, culprit species, clinical presentation, management before and after admission, and outcome. There were a total of 273 admissions due to snakebite. Over 60% of bites were to the feet and about 36% were to the arms and hands. The type of snake involved was recorded in 14.6% (40) the cases with puff adders (Bitis arietans) being the most commonly implicated species. First aid and other pre-hospital treatments were recorded in 38 cases with tourniquets employed in 24 cases (63.2%) and 13 patients visiting a traditional medical practitioner before going to hospital. There were similar proportions of males to females admitted with snakebite and the case fatality rate in this study was 2.9%. Most bites from snakes in Zimbabwe occur to the feet and are probably from puff adders. The torniquet appears to be the most commonly employed first aid measure for snakebite in Zimbabwe, and snakebite victims still consult traditional healers.

First reported case of African viper bite treated with Indian polyvalent anti snake venom

Dear Editor, The compositional differences between venoms among different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. The polyvalent variety of anti snake venom (ASV) with local specificity remains the mainstay of therapy in most countries. ASV against the venom of one species occasionally may have partial cross neutralizing activity against other venoms, usually from closely related species, known as paraspecific activity. We managed a case of an African viper bite successfully using Indian polyvalent ASV as the specific African variety was not available to us. A 25 year old serving soldier of a foreign army contingent in UN mission in the Democratic Republic of Congo, was brought on 29 Sep 07 at 0025 hours, with a history of a mildly painful snake bite of about two hours duration on the left foot. There was no history suggestive of neurological involvement. Clinical examination revealed normal vital parameters and a normal general examination. Local exam revealed swelling and tenderness of left foot extending beyond the ankle to the distal leg. Two fang marks 1.5 cm apart were visible on dorsum of left foot along with a small patch of ecchymosis (Fig. 1). Fig. 1 Fang marks with swelling of foot. A bed side whole blood clotting time revealed no clotting at 30 minutes, with a clotting time of 35 minutes by capillary method. Bleeding time was in normal range. Since specific ASV for the regional area was not available, a decision to give Indian polyvalent ASV (which was available) was taken as an emergency life saving measure. A total of 150 ml of reconstituted ASV (15 vials of polyvalent Indian variety) was administered as per standard protocol over next nine hours in view of progressively increasing swelling in the leg and deranged clotting time in range of 15 to 21 minutes. The progression of the swelling of the limb halted and clotting parameters started improving (Fig. 2). Antibiotics and other supportive therapy were given. The patient had a haemoglobin drop from 16.3 to 13.6 gm per dl and mild thrombocytopenia in range of 1,20,000 to 1,40,000/mm2 during the initial few days which subsequently recovered. He also developed an uncomplicated resolving small abdominal wall hematoma (confirmed by ultrasound). He was discharged at three weeks with a complete and uneventful recovery. Fig. 2 Reduction of swelling of the limb by day three. The mortality after untreated viper bites like Crotalus durissus terrificus is said to have been up to 70 %, but this has been reduced to less than 12% by ASV treatment and in African varieties like Atractaspis and bush vipers (like those found in the Congo basin) the mortality ranges from 8-43% [1, 2]. “Specific” antivenom, implies that the antivenom has been raised against the venom of a particular snake that has bitten the patient (Monovalent or monospecific antivenom). Polyvalent antivenom neutralises the venoms of several different species of snakes. FAV AFRICA is the polyvalent equine ASV recommended for Sub-Saharan African snakes. The Indian ASV contains sera against the saw scaled viper, Russel's viper. Indian Cobra (Naja naja) and the banded krait. Analysis of Indian cobra (Naja naja) venom samples from eastern, western and southern India revealed differences in composition, biochemical, and pharmacological properties emphasizing regional variation: continents will logically be different [3]. There are only a handful of documented cases of snake bites managed with ASV pertaining to different geographical locations. Use of Tiger Snake ASV in Stephen's Banded Snake envenomation in Australia, pit viper envenomation (South American) treated with nonspecific ASV and two North American coral snake venoms treated with Mexican ASV have been described [4, 5, 6]. A study was performed to assess the ability of polyvalent anti snake venom VACSERA (produced by Egypt), to neutralize venoms of snakes not only from Egypt but also African and Middle Eastern habitats showed encouraging results [7]. There is a description of a severe systemic envenomation of a African bush viper managed successfully with Near Middle East ASV [8]. Ours is possibly the first reported case of an African viper bite successfully managed with Indian polyvalent ASV given in the backdrop of unavailability of specific antisera. We recommend that more studies be carried out on this paraspecific activity of the Indian ASV.

Efficacy of North American crotalid antivenom against the African viper Bitis gabonica (Gaboon viper).

Envenomations by exotic snakes occur from zoological collections and private individual collectors. Antivenoms to these snakes may not be readily available. The objective of this study is to determine the efficacy of North American crotalid antivenin in treating mice envenomated with venom of the African viper Bitis gabonica (Gaboon viper). The subjects of the study were Swiss Webster mice weighing approximately 30 g. The study was conducted in the University research laboratory. B. gabonica venom was obtained from Venom Supplies Pty Ltd (Tanunda, South Australia) and reconstituted in sterile water. North American Crotalid Fab2 antivenin (Anavyp, Instituto Bioclon, Mexico) was donated by the manufacturer. The experimental groups were: Group I received two times an intraperitoneal LD(50) dose of venom, 2.58 mg/kg. Group II received the same dose after incubation for 1 h with 10 mg of antivenin. Time to onset of toxicity defined as respiratory rate <10/min or absence of response to prodding. t test and Chi square with p < 0.05 considered significant. Time to onset of toxicity was 7.040 +/- 4.334 h in group I, and 20.665 +/- 2.074 in group II (p = 0.0064, 95% confidence interval of difference of means -22.694 to -4.556). Antivenin was efficacious to statistical significance at 4, 8, 12, and 16 h (p values of 0.062, 0.0067, 0.0067, and 0.0253, respectively). Improvement at 20 and 24 h (p values of 0.0673 and 0.0673, respectively) did not achieve statistical significance. North American Crotalid antivenin (Anavyp, Instituto Bioclon, Mexico) demonstrated efficacy in increasing time to onset of distress in mice poisoned with B. gabonica (Gaboon viper) venom. Based on this result, treatment of humans envenomated with B. gabonica with North American Croatlid antivenin could be considered for severe envenomations if specific B. gabonica antivenin is unavailable.

Characterization of a new polyvalent antivenom (Antivipmyn ® Africa) against African vipers and elapids

As a response to the antivenom shortage in Sub-Saharan Africa, evident for well over a decade, we developed a new polyvalent anti-ophidian antivenom (Antivipmyn ® Africa) designed for use in the region. We report a detailed characterization of its biochemical composition (protein content and profiling by size-exclusion chromatography and electrophoresis) as well as the specific and para-specific neutralization potencies (as median effective dose in the mouse lethality test). Additionally, we studied the neutralization of hemorrhagic, anti-hemostatic and necrotic activities of Echis ocellatus venom, responsible for a majority of severe envenomations in the continent according to existing epidemiological data. The antivenom is currently under production and has already been employed in the field in a pragmatic Phase III clinical trial in the Republic of Benin. It is a purified lyophilized polyvalent equine F(ab′) 2-based product obtained by immunization with the venoms of eleven species of African snakes of the Genera Echis, Bitis, Naja and Dendroaspis. The criteria for its design are discussed, particularly in terms of the implementation of realistic public health policies targeting mostly rural populations in the continent.

Snake Venom and Haemostasis – An Overview

Venomous snakebites are still a public health problem in tropical countries. Even in metropolitan areas, such as Bangkok, green pit viper (Cryptelytrops albolabris and C. macrops) bites are still prevalent. As we are invading their territory, they are adapting to live among humans in our backyards. With some exceptions, clinical syndromes of snakebites can be divided simply into two classes: elapid envenomation attacks neuromuscular junctions, causing muscular paralysis; viper envenomation targets the haemostatic system, resulting in bleeding disorders. Therefore, viper venoms are of great interest to haematologists. Vipers (Viperinae) are members of the snake family that develops the most advanced venom-injecting organs, the long sheathed fangs that can be folded like switchblades. Some vipers possess pit organs, infrared detectors for locating warm-blooded prey in the dark. This specialised subfamily is termed crotalidae (Pit viper), separating them from viperidae (true viper). Vipers found in the Americas are pit vipers, while African vipers are true vipers. Asia and Europe are homes to both subfamilies.