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The report describes successful management of 10 women in 2nd and 3rd pregnancy trimesters with EchiTab IgG antivenom after carpet viper (Echis ocellatus) envenoming. All women survived but foetal loss in a victim with delayed presentation and a case of mild hypersensitivity reaction were recorded. Excellent outcomes can be achieved in rural and semi-nomadic populations without specialized care and immediate access and provision of effective antivenoms is paramount in curtailing snakebite maternal morbidity, mortality and foetal loss. [ABSTRACT FROM AUTHOR]

In many cases of envenoming following snake bite, the snake responsible for the accident remains unidentified; this frequently results in difficulty deciding which antivenom to administer to the systemically-envenomed victim, especially when only monospecific antivenoms are available. Normally the specific diagnosis of snake bite can be conveniently made using clinical and laboratory methods. Where clinical diagnosis depends upon the recognition of specific signs of envenoming in the patient, laboratory diagnosis is based on the changes which occur in envenomed victims including the detection of abnormalities in blood parameters, presence/absence of myoglobinuria, changes in certain enzyme levels, presence/absence of neurotoxic signs and the detection in the blood of specific venom antigens using immunologically-based techniques, such as enzyme immunoassay. It is the latter which is the main subject of this review, together with the application of techniques currently used to objectively assess the effectiveness of new and existing antivenoms, to assess first aid measures, to investigate the possible use of such methods in epidemiological studies, and to detect individual venom components. With this in mind, we have discussed in some detail how such techniques were developed and how they have helped in the treatment of envenoming particularly and in venom research in general.

Venomic analysis of the venoms of Naja nigricollis, N. katiensis, N. nubiae, N. mossambica, and N. pallida revealed similar compositional trends. The high content of cytotoxins and PLA(2)s may account for the extensive tissue necrosis characteristic of the envenomings by these species. The high abundance of a type I α-neurotoxin in N. nubiae may be responsible for the high lethal toxicity of this venom (in rodents). The ability of EchiTAb-Plus-ICP antivenom to immunodeplete and neutralize the venoms of African spitting cobras was assessed by antivenomics and neutralization tests. It partially immunodepleted 3FTx and PLA(2)s and completely immunodepleted SVMPs and CRISPs in all venoms. The antivenom neutralized the dermonecrotic and PLA(2) activities of all African Naja venoms, whereas lethality was eliminated in the venoms of N. nigricollis, N. mossambica, and N. pallida but not in those of N. nubiae and N. katiensis. The lack of neutralization of lethality of N. nubiae venom may be of medical relevance only in relatively populous areas of the Saharan region. The impaired activity of EchiTAb-Plus-ICP against N. katiensis may not represent a major concern. This species is sympatric with N. nigricollis in many regions of Africa, although very few bites have been attributed to it.

Prospective studies of snake bite patients in Chittagong, Bangladesh, included five cases of bites by greater black kraits (Bungarus niger), proven by examination of the snakes that had been responsible. This species was previously known only from India, Nepal, Bhutan and Burma. The index case presented with descending flaccid paralysis typical of neurotoxic envenoming by all Bungarus species, but later developed generalized rhabdomyolysis (peak serum creatine kinase concentration 29,960 units/l) with myoglobinuria and acute renal failure from which he succumbed. Among the other four patients, one died of respiratory paralysis in a peripheral hospital and three recovered after developing paralysis, requiring mechanical ventilation in one patient. One patient suffered severe generalized myalgia and odynophagia associated with a modest increase in serum creatine kinase concentration. These are the first cases of Bungarus niger envenoming to be reported from any country. Generalized rhabdomyolysis has not been previously recognized as a feature of envenoming by any terrestrial Asian elapid snake, but a review of the literature suggests that venoms of some populations of Bungarus candidus and Bungarus multicinctus in Thailand and Vietnam may also have this effect in human victims. To investigate this unexpected property of Bungarus niger venom, venom from the snake responsible for one of the human cases of neuro-myotoxic envenoming was injected into one hind limb of rats and saline into the other under buprenorphine analgesia. All animals developed paralysis of the venom-injected limb within two hours. Twenty-four hours later, the soleus muscles were compared histopathologically and cytochemically. Results indicated a predominantly pre-synaptic action (β-bungarotoxins) of Bungarus niger venom at neuromuscular junctions, causing loss of synaptophysin and the degeneration of the terminal components of the motor innervation of rat skeletal muscle. There was oedema and necrosis of extrafusal muscle fibres in envenomed rat soleus muscles confirming the myotoxic effect of Bungarus niger venom, attributable to phospholipases A₂. This study has demonstrated that Bungarus niger is widely distributed in Bangladesh and confirms the risk of fatal neuro-myotoxic envenoming, especially as no specific antivenom is currently manufactured. The unexpected finding of rhabdomyolysis should prompt further investigation of the venom components responsible. The practical implications of having to treat patients with rhabdomyolysis and consequent acute renal failure, in addition to the more familiar respiratory failure associated with krait bite envenoming, should not be underestimated in a country that is poorly equipped to deal with such emergencies.

Background: In West Africa, envenoming by saw-scaled or carpet vipers (Echis ocellatus) causes great morbidity and mortality, but there is a crisis in supply of effective and affordable antivenom (ISRCTN01257358)., Methods: In a randomised, double-blind, controlled, non-inferiority trial, "EchiTAb Plus-ICP" (ET-Plus) equine antivenom made by Instituto Clodomiro Picado was compared to "EchiTAb G" (ET-G) ovine antivenom made by MicroPharm, which is the standard of care in Nigeria and was developed from the original EchiTAb-Fab introduced in 1998. Both are caprylic acid purified whole IgG antivenoms. ET-G is monospecific for Echis ocellatus antivenom (initial dose 1 vial) and ET-Plus is polyspecific for E. ocellatus, Naja nigricollis and Bitis arietans (initial dose 3 vials). Both had been screened by pre-clinical and preliminary clinical dose-finding and safety studies. Patients who presented with incoagulable blood, indicative of systemic envenoming by E. ocellatus, were recruited in Kaltungo, north-eastern Nigeria. Those eligible and consenting were randomly allocated with equal probability to receive ET-Plus or ET-G. The primary outcome was permanent restoration of blood coagulability 6 hours after the start of treatment, assessed by a simple whole blood clotting test repeated 6, 12, 18, 24 and 48 hr after treatment. Secondary (safety) outcomes were the incidences of anaphylactic, pyrogenic and late serum sickness-type antivenom reactions., Findings: Initial doses permanently restored blood coagulability at 6 hours in 161/194 (83.0%) of ET-Plus and 156/206 (75.7%) of ET-G treated patients (Relative Risk [RR] 1.10 one-sided 95% CI lower limit 1.01; P = 0.05). ET-Plus caused early reactions on more occasions than did ET-G [50/194 (25.8%) and 39/206 (18.9%) respectively RR (1.36 one-sided 95% CI 1.86 upper limit; P = 0.06). These reactions were classified as severe in 21 (10.8%) and 11 (5.3%) of patients, respectively., Conclusion: At these doses, ET-Plus was slightly more effective but ET-G was slightly safer. Both are recommended for treating E. ocellatus envenoming in Nigeria., Trial Registration: Current Controlled Trials ISRCTN01257358.

The immunoreactivity of EchiTAb-Plus-ICP, an antivenom developed for the treatment of snakebite envenoming in sub-Saharan Africa, to venoms of seven Echis and Bitis species, was assessed by "antivenomics." This proteomic approach is based on the ability of an antivenom to immunodeplete homologous or heterologous venom proteins. Our results show an extensive cross-reactivity of this antivenom against all Echis and Bitis venoms studied, as revealed by the complete immunodepletion of the majority of venom components, including metalloproteinases, serine proteinases, C-type lectin-like proteins, some phospholipases A(2) and L-amino acid oxidase. However, some phospholipases A(2), disintegrins and proteinase inhibitors were immunodepleted to only a partial extent. These results support the hypothesis that immunizing horses with a mixture of the venoms of Echis ocellatus, Bitis arietans, and Naja nigricollis generates antibodies capable of recognizing the majority of components of medically-relevant homologous and heterologous viperid venoms of the genera Bitis and Echis from sub-Saharan Africa.

The demographics, epidemiology, first aid, clinical management, treatment and outcome of snake bites causing no significant signs of systemic envenoming were documented in Chittagong Medical College Hospital, Bangladesh, between May 1999 and October 2002. Among 884 patients admitted, 350 were systemically envenomed and 534 were without signs of either systemic or significant local envenoming. The average age of patients with physical evidence of snake bite but no systemic envenoming was 26.4 years. Most had been bitten on their feet or hands. Ligatures had been applied proximal to the bite site in >95% of cases and the bite site had been incised in 13%. Patients were typically discharged at 24h. Those with clinical signs of systemic envenoming resembled the non-envenomed cases demographically and epidemiologically except that they arrived at hospital significantly later than non-envenomed patients, having spent longer with traditional healers. No non-envenomed patient was treated with antivenom and none went on to develop symptoms of systemic envenoming after discharge. The potential complications and confusing signs caused by ligatures and incision demand that all patients admitted with a history of snake bite be kept under observation for 24h after admission even if they have no signs of systemic envenoming., (Copyright 2010 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

The aim of this study was to identify candidate antivenoms with specific activity against the venom of the saw-scaled or carpet viper (Echis ocellatus) in northern Nigeria, where bites by this species cause great morbidity and mortality but where effective antivenoms have become scarce and unaffordable. Selected antivenoms were destined to be compared by randomised controlled clinical trials (RCTs). Standard pre-clinical neutralisation assays were carried out in rodents. We included two licensed antivenoms of established clinical efficacy and 6 candidate antivenoms. Although 6 of the tested antivenoms showed promising efficacy, all but 3 were excluded from further study because of inadequate pre-clinical efficacy or because they were unavailable or unaffordable for the anticipated RCTs. Median effective doses (ED(50)) of the remaining three candidate antivenoms suggested that the following doses might neutralise the maximum observed venom yield of 24.8 mg (dry weight) of venom milked from captive E. ocellatus: 10 ml of MicroPharm "EchiTAb G" (ET-G) antivenom; 30 ml of Instituto Clodomiro Picado "EchiTAb-Plus-ICP" (ET-Plus) antivenom; 50 ml of VacSera, Cairo "EgyVac" antivenom. A preliminary clinical dose-finding and safety study of these three antivenoms was carried out in 24 patients with incoagulable blood after E. ocellatus bites who were not severely envenomed. A 3+3 dose escalation design was employed. Initial doses of 10 ml ET-G and 30 ml ET-Plus restored blood coagulability in groups of 6 patients with early mild reactions (pruritus only) in not more than one third of them. EgyVac antivenom did not fulfil efficacy or safety criteria in 12 patients. On the basis of these results, ET-G and ET-Plus were selected for comparison in a RCT., (Copyright 2009 Elsevier Ltd. All rights reserved.)

A preclinical assessment was performed on the neutralizing efficacy of a whole IgG polyspecific antivenom (EchiTAb-Plus-ICP), designed for the treatment of snakebite envenomings in Nigeria. It was generated by immunizing horses with the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the most medically important species in Nigeria. Antivenom was tested against the venoms of E. ocellatus, Echis leucogaster, Echis pyramidum leakeyi, B. arietans, Bitis gabonica, Bitis rhinoceros and Bitis nasicornis. The neutralization of the venom toxins responsible for the lethal, hemorrhagic, coagulant and local necrotizing activities was assessed, since these are the most significant effects that characterize envenoming by these species. Echis sp venoms exerted lethal, hemorrhagic, coagulant and necrotizing effects, whereas the Bitis sp venoms tested induced lethality, hemorrhage and necrosis, but were devoid of coagulant activity. The antivenom was effective in the neutralization of all effects tested in all venoms. Highest neutralization was achieved against the venoms of E. ocellatus and B. arietans, and the lowest neutralizing potency was against the venom of B. nasicornis, a species that has a low clinical relevance. It is concluded that EchiTAb-Plus-ICP, whilst specifically designed for Nigeria, has a good preclinical neutralizing profile against homologous and heterologous viperid venoms from other sub-Saharan African locations. It therefore constitutes a promising therapeutic option for the treatment of snakebite envenoming in this region., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Objective: Serious morbidity and mortality following snakebite injuries are common in tropical regions of the world. Although antivenom administration is clinically effective, it carries an important risk of early anaphylactic reactions, ranging from relatively benign nausea, vomiting, and urticaria to life-threatening angioedema, bronchospasm and hypotension. Currently, no adequately powered study has demonstrated significant benefit from the use of any prophylactic drug. A high rate of anaphylactic reactions observed during a trial of three different antivenoms in Ecuador prompted adoption of premedication with intravenous (i.v.) hydrocortisone and diphenhydramine together with dilution and slower administration of antivenom., Design: In a rural mission hospital in Eastern Ecuador, 53 consecutive snakebite victims received a new antivenom regimen in 2004-2006, comprising prophylactic drugs and i.v. infusion of diluted antivenom over 60 min. They were compared to an historical control cohort of 76 patients treated in 1997-2002 without prophylactic drugs and with i.v. "push" injection of undiluted antivenom over 10 min. All these patients had incoagulable blood on admission and all were treated with Brazilian Instituto Butantan polyspecific antivenom., Results: Baseline characteristics of the historical control and premedicated groups were broadly similar. In the historical group, early reaction rates were as follows: 51% of patients had no reaction; 35% had mild reactions; 6% moderate; and 6% severe. In the premedicated/slow i.v. group, 98% of patients had no reaction; 0 mild; 0 moderate; and 2% severe. The difference in reaction rates was statistically significant (p<0.001)., Conclusions: Premedication with intravenous hydrocortisone and diphenhydramine together with dilution of antivenom and its administration by i.v. infusion over 60 min appeared to reduce both the frequency and severity of anaphylactic reactions. A randomized blinded controlled trial is needed to confirm these encouraging preliminary findings.

Of 860 snakes brought to 10 hospitals in Sri Lanka with the patients they had bitten, 762 (89%) were venomous. Russell's vipers (Daboia russelii) and hump-nosed pit vipers (Hypnale hypnale) were the most numerous and H. hypnale was the most widely distributed. Fifty-one (6%) were misidentified by hospital staff, causing inappropriate antivenom treatment of 13 patients. Distinctive clinical syndromes were identified to aid species diagnosis in most cases of snake bite in Sri Lanka where the biting species is unknown. Diagnostic sensitivities and specificities of these syndromes for envenoming were 78% and 96% by Naja naja, 66% and 100% by Bungarus caeruleus, 14% and 100% by Daboia russelii, and 10% and 97% by Hypnale hypnale, respectively. Although only polyspecific antivenoms are used in Sri Lanka, species diagnosis remains important to anticipate life-threatening complications such as local necrosis, hemorrhage and renal and respiratory failure and to identify likely victims of envenoming by H. hypnale who will not benefit from existing antivenoms. The technique of hospital-based collection, labeling and preservation of dead snakes brought by bitten patients is recommended for rapid assessment of a country's medically-important herpetofauna.

In a prospective study of snake bites involving 10 hospitals in Sri Lanka, 302 (35%) of 860 patients with bites by identified snakes proved to have been bitten by hump-nosed pit vipers (301 by Hypnale hypnale and 1 by H. nepa). Most victims were males aged between 11 years and 50 years who had been bitten on their feet or ankles while walking at night close to their homes. There was local swelling in 276 (91%) and local necrosis in 48 (16%). Eleven (4%) required amputation of fingers or toes and 12 (4%) received skin grafts. In 117 patients (39%) blood incoagulability was first detected between 15 min and 48 h after the bite, and in 116 of them this was present on admission to hospital. Spontaneous systemic bleeding was observed in 55 patients (18%). Acute renal failure developed in 10%, five of whom died to give an overall case fatality rate of 1.7%. Thus, bites by hump-nosed pit vipers can cause debilitating local and fatal systemic envenoming. In Sri Lanka and southwestern India where bites by these snakes are common, the only available antivenoms (raised against cobra, krait, Russell's viper and saw-scaled viper venoms) are ineffective and carry a high risk of reactions.

A prospective study was designed to define epidemiologic and clinical features of krait bites to improve diagnosis, management, and prevention. Among 762 cases of venomous snake bites admitted to 10 Sri Lankan hospitals in which the snake responsible was brought and identified, 88 (11.5%) were caused by common kraits (Bungarus caeruleus). Bites were: most frequent in September through November. Distinctive features of B. caeruleus bites (compared with bites by other species in parentheses) were bitten while sleeping on the ground, 100% (1%); indoors, 100% (49%); between 2300 and 0500 hours, 100% (3%). Only 13% of krait victims were bitten on their lower limbs (82%), only 9% had local swelling (in all cases mild) at the site of the bite (93%), 64% developed respiratory paralysis (2%), and 91% experienced (often severe) abdominal pain (10%). Case fatality was 6% (3%). This distinctive pattern of epidemiology and symptoms will aid clinical recognition (syndromic diagnosis) and prevention of krait bite envenoming.

Complete blood counts are used frequently by physicians to assess and manage the development of complications of diseases. We studied 100 patients bitten by Bothrops jararaca snakes, and correlated their haematological values with the severity of envenoming and the development of complications. Patients who developed both local and systemic bleeding showed a greater drop in packed cell volume, red blood cell (RBC) count and haemoglobin concentration than those with who did not bleed. No morphological changes in RBCs were seen in blood films. Total white blood cell (WBC) counts were significantly higher in the clinically "more severe" group than in the "less severe" group on admission. Neutrophilic leucocytosis with left shift was present on admission, concurrently with a decrease in eosinophil and lymphocyte counts. These changes tend to become more marked 6h after antivenom therapy, and are greatest in "more severe" envenoming. Thrombocytopenia on admission is positively associated with the development of systemic bleeding and the severity of envenoming. Thrombocytopenia may also be a useful prognostic indicator for the development of local complications, such as necrosis. The intensity of neutrophilia and eosinopenia might be used to follow the progression of necrosis in victims of snake bite.

Envenoming by the West African saw-scaled viper, Echis ocellatus resembles that of most vipers, in that it results in local blistering, necrosis and sometimes life-threatening systemic haemorrhage. While effective against systemic envenoming, current antivenoms have little or no effect against local tissue damage. The major mediators of local venom pathology are the zinc-dependant snake venom metalloproteinases (SVMPs). The high degree of structural and functional homology between SVMPs and their mammalian relatives the matrix metalloproteinases (MMPs) suggests that substrate/inhibitor interactions between these subfamilies are likely to be analogous. In this study, four recently developed MMP inhibitors (MMPIs) (Marimastat, AG-3340, CGS-270 23A and Bay-12 9566) are evaluated in addition to three metal ion chelators (EDTA, TPEN and BAPTA) for their ability to inhibit the haemorrhagic activities of the medically important E. ocellatus venom and one of its haemorrhagic SVMPs, EoVMP2. As expected, the metal ion chelators significantly inhibited the haemorrhagic activities of both whole E. ocellatus venom and EoVMP2, while the synthetic MMPIs show more variation in their efficacies. These variations suggest that individual MMPIs show specificity towards SVMPs and that their application to the neutralization of local haemorrhage may require a synthetic MMPI mixture, ensuring that a close structural component for each SVMP is represented.

The efficacy and safety of two whole IgG polyvalent antivenoms (A and B) were compared in a randomised, blinded clinical trial in 67 patients systemically envenomed by Bothrops asper in Colombia. Both antivenoms were fractionated by caprylic acid precipitation and had similar neutralising potencies, protein concentrations and aggregate contents. Antivenom B was additionally treated with beta-propiolactone to lower its anticomplementary activity. Analysing all treatment regimens together, there were no significant differences between the two antivenoms (A=34 patients; B=33 patients) in the time taken to reverse venom-induced bleeding and coagulopathy, to restore physiological fibrinogen concentrations and to clear serum venom antigenaemia. Blood coagulability was restored within 6-24 h in 97% of patients, all of whom had normal coagulation and plasma fibrinogen levels 48 h after the start of antivenom treatment. Two patients (3.0%) had recurrent coagulopathy and eight patients suffered recurrence of antigenaemia within 72 h of treatment. None of the dosage regimens of either antivenom used guaranteed resolution of venom-induced coagulopathy within 6 h, nor did they prevent recurrences. A further dose of antivenom at 6 h also did not guarantee resolution of coagulopathy within 12-24 h in all patients. The incidence of early adverse reactions (all mild) was similar for both antivenoms (15% and 24%; P>0.05).

Tx1 from the venom of the Brazilian spider, Phoneutria nigriventer, is a lethal neurotoxic polypeptide of M(r) 8600 Da with 14 cysteine residues. It is a novel sodium channel blocker which reversibly inhibits sodium currents in CHO cells expressing recombinant sodium (Nav1.2) channels. We cloned and expressed the Tx1 toxin as a thioredoxin fusion product in the cytoplasm of Escherichia coli. After semipurification by immobilized Ni-ion affinity chromatography, the recombinant Tx1 was purified by reverse phase chromatography and characterized. It displayed similar biochemical and pharmacological properties to the native toxin, and it should be useful for further investigation of structure-function relationship of Na channels.

Background: Snake venom is a potentially lethal and complex mixture of hundreds of functionally diverse proteins that are difficult to purify and hence difficult to characterize. These difficulties have inhibited the development of toxin-targeted therapy, and conventional antivenom is still generated from the sera of horses or sheep immunized with whole venom. Although life-saving, antivenoms contain an immunoglobulin pool of unknown antigen specificity and known redundancy, which necessitates the delivery of large volumes of heterologous immunoglobulin to the envenomed victim, thus increasing the risk of anaphylactoid and serum sickness adverse effects. Here we exploit recent molecular sequence analysis and DNA immunization tools to design more rational toxin-targeted antivenom., Methods and Findings: We developed a novel bioinformatic strategy that identified sequences encoding immunogenic and structurally significant epitopes from an expressed sequence tag database of a venom gland cDNA library of Echis ocellatus, the most medically important viper in Africa. Focusing upon snake venom metalloproteinases (SVMPs) that are responsible for the severe and frequently lethal hemorrhage in envenomed victims, we identified seven epitopes that we predicted would be represented in all isomers of this multimeric toxin and that we engineered into a single synthetic multiepitope DNA immunogen (epitope string). We compared the specificity and toxin-neutralizing efficacy of antiserum raised against the string to antisera raised against a single SVMP toxin (or domains) or antiserum raised by conventional (whole venom) immunization protocols. The SVMP string antiserum, as predicted in silico, contained antibody specificities to numerous SVMPs in E. ocellatus venom and venoms of several other African vipers. More significantly, the antiserum cross-specifically neutralized hemorrhage induced by E. ocellatus and Cerastes cerastes cerastes venoms., Conclusions: These data provide valuable sequence and structure/function information of viper venom hemorrhagins but, more importantly, a new opportunity to design toxin-specific antivenoms-the first major conceptual change in antivenom design after more than a century of production. Furthermore, this approach may be adapted to immunotherapy design in other cases where targets are numerous, diverse, and poorly characterized such as those generated by hypermutation or antigenic variation.

In this study, we isolated a novel prothrombin activator from the venom of Bothrops cotiara, a Brazilian lance-headed pit viper (Cotiara, Jararaca preta, Biocotiara), which we have designated "cotiaractivase" (prefix: cotiar- from B. cotiara; suffix: -activase, from prothrombin activating activity). Cotiaractivase was purified using a phenyl-Superose hydrophobic interaction column followed by a Mono-Q anion exchange column. It is a single-chain polypeptide with a molecular weight of 22,931 Da as measured by mass spectroscopy. Cotiaractivase generated active alpha-thrombin from purified human prothrombin in a Ca2+-dependent manner as assessed by S2238 chromogenic substrate assay and SDS-PAGE. Cotiaractivase cleaved prothrombin at positions Arg271-Thr272 and Arg320-Ile321, which are also cleaved by factor Xa. However, the rate of thrombin generation by cotiaractivase was approximately 60-fold less than factor Xa alone and 17 x 10(6)-fold less than the prothrombinase complex. The enzymatic activity of cotiaractivase was inhibited by the chelating agent EDTA, whereas the serine protease inhibitor PMSF had no effect on its activity, suggesting that it is a metalloproteinase. Interestingly, S2238 inhibited cotiaractivase activity non-competitively, suggesting that this toxin contains an exosite that allows it to bind prothrombin independently of its active site. Tandem mass spectrometry and N-terminal sequencing of purified cotiaractivase identified peptides that were identical to regions of the cysteine-rich and disintegrin-like domains of known snake venom metalloproteinases. Cotiaractivase is a unique low molecular weight snake venom prothrombin activator that likely belongs to the metalloproteinase family of proteins.

Envenoming by snakes results in severe systemic and local pathology. Intravenous administration of antivenom, prepared from IgG of venom immunised horses or sheep, is the only effective treatment of systemic envenoming. Conventional antivenoms, formulated as intact IgG, papain-cleaved (Fab) or pepsin-cleaved F(ab')2 fragments, are however ineffective against the local venom effects because of their inability to penetrate the blood/tissue barrier. We have embarked on a new research program to examine (i) whether the unusually small (15 kDa) antigen-binding fragment of camelid heavy chain IgG (V(H)H) can be exploited to neutralise the local effects of envenoming and (ii) whether a novel antivenom to treat both the systemic and local effects of envenoming can be formulated by combining anti-snake venom V(H)H and conventional F(ab')2. In this preliminary study, we demonstrate that camels and llamas respond to immunisation with Echis ocellatus venom with high antibody titres and broad antigen specificity. These encouraging immunological results were matched by the successful elimination of venom-induced haemorrhage by IgG from the venom-immunised camels and llamas. Unexpectedly, we report for the first time that camelid serum contains a non-IgG, highly potent inhibitor of venom-induced haemorrhage.

The snake venom rhodocytin has been reported to bind to integrin alpha2beta1 and glycoprotein (GP) Ibalpha on platelets, but it is also able to induce activation independent of the 2 receptors and of GPVI. Using rhodocytin affinity chromatography, we have identified a novel C-type lectin receptor, CLEC-2, in platelets that confers signaling responses to rhodocytin when expressed in a cell line. CLEC-2 has a single tyrosine residue in a YXXL motif in its cytosolic tail, which undergoes tyrosine phosphorylation upon platelet activation by rhodocytin or an antibody to CLEC-2, but not to collagen, thrombin receptor agonist peptide (TRAP), or convulxin. Tyrosine phosphorylation of CLEC-2 and other signaling proteins by rhodocytin is inhibited by the Src family kinase inhibitor PP2. Further, activation of murine platelets by rhodocytin is abolished in the absence of Syk and PLCgamma2, and partially reduced in the absence of LAT, SLP-76, and Vav1/Vav3. These findings define a novel signaling pathway in platelets whereby activation of CLEC-2 by rhodocytin leads to tyrosine phosphorylation of its cytosolic tail, binding of Syk and initiation of downstream tyrosine phosphorylation events, and activation of PLCgamma2. CLEC-2 is the first C-type lectin receptor to be found on platelets which signals through this novel pathway.

Many toxins from viperid venoms have been characterised as powerful activators of platelets. Here, the venom from the East African Lowland viper, Proatheris superciliaris, was investigated for its effect on platelets and the coagulation system. Whole P. superciliaris venom stimulated platelet shape change and aggregation; however, the stimulation of platelet activation was unaffected by the structurally distinct Src family kinase inhibitors PP1 and PD0173952, suggesting that platelet activation was mediated by a G protein-coupled receptor. A platelet reactive 34-kDa protein was isolated from P. superciliaris venom which we have designated proatherocytin. This protein induced Src kinase-independent aggregation of both human and mouse platelets that was inhibited by the serine protease inhibitor AEBSF. Proatherocytin did not clot bovine or human fibrinogen, degrade fibrinogen or hydrolyse the serine protease substrate benzoyl-FVR-pNA. It activated human PAR1 on stably transfected rat kidney epithelial cells, whereas no activation of the trypsin receptor PAR2 was shown. Surprisingly, Edman degradation of proatherocytin revealed sequence identity with existing disintegrin-like domains of snake venom metalloproteinases. These results suggest that proatherocytin is a highly selective PAR1 agonist that also causes mouse platelet aggregation, probably through cleavage of PAR4.

To assess the indirect effects of snake venom metalloproteinases (SVMP) on host tissue local necrosis, we investigated the effect of the SVMP jararhagin on the gene expression profiles of human fibroblasts in vitro and mouse tissue in vivo. Two functional classes of up-regulated proteins, cell death and inflammatory disease were identified as being significantly populated. The changes in gene expression observed by qRT-PCR on laser microdissected mouse muscle tissue treated with jararhagin were similar with significant up-regulation of proinflammatory transcripts such as IL-1 beta, IL-6, CXCL1, CXCL2, IL-8, and apoptosis, inflammation responsive transcripts such as TNF-alpha induced protein 6. Proteolytically inactive jararhagin had no effect on the gene expression profile of fibroblasts, indicating proteolysis as the primary mechanism affecting gene expression of cells and tissues resulting in a proinflammatory, pro-apoptotic host response which likely exacerbates the local necrosis frequently observed at the site of envenoming.

Two metalloproteinases, a 24-kDa P-I EoVMP1 and a 56-kDa P-III EoVMP2, have recently been isolated from the venom of the West African saw-scaled viper Echis ocellatus. We now reveal a new 65-kDa haemorrhagic group P-III metalloproteinase which we have designated EoVMP3. The aim of this study was to determine whether these three snake venom metalloproteinases (SVMPs) affect platelets and blood coagulation. EoVMP1 had no effect on the aggregation of washed human platelets, whereas EoVMP2 inhibited collagen-induced platelet aggregation. In contrast, EoVMP3 did not inhibit the aggregation of platelets by collagen but instead activated platelets in the absence of any additional co-factors. All three SVMPs were capable of activating prothrombin to varying degrees and can therefore be described as procoagulants. EoVMP1, EoVMP2 and EoVMP3 share sequence identity with other members of the reprolysin family, but differ greatly in their effects on some of the components that control haemostasis.

A polyspecific Pan-African antivenom has been produced from the plasma of horses immunized with a mixture of the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the three most medically important snakes in sub-Saharan Africa. The antivenom is a whole IgG preparation, obtained by caprylic acid precipitation of non-IgG plasma proteins. The antivenom effectively neutralizes the most important toxic activities of the three venoms used in the immunization in standard assays involving preincubation of venom and antivenom before testing. This antivenom compares favourably with other antivenoms designed for use in Africa with respect to neutralization of the toxins present in the venom of E. ocellatus. Caprylic acid fractionation of horse hyperimmune plasma is a simple, convenient and cheap protocol for the manufacture of high quality whole IgG antivenoms. It constitutes a potentially valuable technology for the alleviation of the critical shortage of antivenom in Africa.

The antigenic relationship between snake venom metalloproteinases (SVMPs) was analysed using rabbit antisera raised against the native forms of two SVMPs purified from Echis ocellatus venom. Using enzyme-linked immunosorbent assay (ELISA), western blotting and two-dimensional SDS-PAGE, our findings show that antibodies raised against EoVMP1, a non-haemorrhagic class P-I 24kDa SVMP, and EoVMP2, a haemorrhagic class P-III 56kDa SVMP, demonstrate cross-reactivities which relate to the domain hierarchy observed in class P-I to P-III/IV SVMPs. A third 65kDa P-III metalloproteinase (designated EoVMP3) was also isolated from E. ocellatus venom using hydrophobic interaction, size exclusion and anion exchange chromatography. In comparative immunoassays, EoVMP2 and EoVMP3 bound strongly to the commercial monovalent ovine Fab fragment antivenom EchiTAbtrade mark (raised against the same venom), but EoVMP1 showed no cross-reactivity. This could indicate that antivenoms may lack antibodies to potentially important venom components.

Objective: To compare the efficacy and safety of three polyspecific antivenoms for bites by pit vipers., Design: Randomised double blind comparative trial of three antivenoms., Setting: Shell, Pastaza, southeastern Ecuador., Participants: 210 patients with incoagulable blood were recruited from 221 consecutive patients admitted with snake bite between January 1997 and December 2001., Intervention: One of three antivenoms manufactured in Brazil, Colombia, and Ecuador, chosen for their preclinical potency against Ecuadorian venoms., Main Outcome Measures: Permanent restoration of blood coagulability after 6 and 24 hours., Results: The snakes responsible for the bites were identified in 187 cases: 109 patients (58%) were bitten by Bothrops atrox, 68 (36%) by B bilineatus, and 10 (5%) by B taeniatus, B brazili, or Lachesis muta. Eighty seven patients (41%) received Colombian antivenom, 82 (39%) received Brazilian antivenom, but only 41 (20%) received Ecuadorian antivenom because the supply was exhausted. Two patients died, and 10 developed local necrosis. All antivenoms achieved the primary end point of permanently restoring blood coagulability by 6 or 24 hours after the start of treatment in > 40% of patients. Colombian antivenom, however, was the most effective after initial doses of 20 ml (two vials), < 70 ml, and any initial dose at both 6 and 24 hours. An initial dose of 20 ml of Colombian antivenom permanently restored blood coagulability in 64% (46/72) of patients after 6 hours (P = 0.054 compared with the other two antivenoms) and an initial dose of < 70 ml was effective at 6 hours (65%, P = 0.045) and 24 hours (99%, P = 0.06). Early anaphylactoid reactions were common (53%, 73%, and 19%, respectively, for Brazilian, Colombian, and Ecuadorian antivenoms, P < 0.0001) but only three reactions were severe and none was fatal., Conclusions: All three antivenoms can be recommended for the treatment of snakebites in this region, though the reactogenicity of Brazilian and Colombian antivenoms is a cause for concern.

Background: The desert horned vipers (Cerastes cerastes and C. gasperettii) are the most familiar snakes of the great deserts of North Africa and the Middle East, including the plains of Iraq. They are responsible for many human snake bites. In Western countries, they are popular among exotic-snake keepers., Aim: To investigate mechanisms of life-threatening envenoming and treatment., Design: Clinical investigation., Methods: Clinical and laboratory studies with measurement of serum venom antigen concentrations by enzyme immunoassay., Results: Two men bitten while handling captive Saharan horned vipers (Cerastes cerastes) in Europe developed extensive local swelling and life-threatening systemic envenoming, characterized by coagulopathy, increased fibrinolysis, thrombocytopenia, micro-angiopathic haemolytic anaemia and acute renal failure. The clinical picture is explicable by the presence in C. cerastes venom of several thrombin-like, Factor-X-activating, platelet-aggregating, haemorrhagic and nephrotoxic components. In one case, prophylactic use of subcutaneous epinephrine may have contributed to intracranial haemorrhage. The roles in treatment of heparin (rejected) and specific antivenom (recommended) are discussed., Discussion: Cerastes cerastes is capable of life-threatening envenoming in humans. Optimal treatment of envenoming is by early administration of specific antivenom, and avoidance of ineffective and potentially-dangerous ancillary methods.

Bites by many species of venomous snake may result in local necrosis at, or extending from, the site of the bite. The use of prophylactic antibiotics to prevent infection as a complication of local necrotic envenoming is controversial. A double-blind randomized controlled trial was carried out to assess whether antibiotic therapy is effective in this situation. Two hundred and fifty-one patients, with proven envenoming by snakes of the genus Bothrops, admitted to two hospitals in Brazil, between 1990 and 1996, were randomized to receive either oral chloramphenicol (500 mg every six hours for five days) or placebo. One hundred and twenty-two of these patients received chloramphenicol (group 1) and 129 were given placebo (group 2). There were no significant differences between the groups at the time of admission. Necrosis developed in seven (5.7%) patients in group 1 and in five (3.9%) patients in group 2 (P>0.05) while abscesses occurred in six patients (4.9%) in group 1 and in six (4.7%) patients in group 2 (P>0.05). In conclusion, the use of orally-administered chloramphenicol for victims of Bothrops snake bite with signs of local envenoming on admission, is not effective for the prevention of local infections.

A common problem in the development of antibody-based therapeutics is the selection, usually from a large population, of specific antibodies with the desired function. One of our research objectives is to identify antibodies capable of neutralising the most important haemorrhagic and haemostasis-disruptive proteases from viper venom. Here, we describe a modification of conventional gelatin-zymography that permits the identification of antibodies capable of neutralising gelatinolytic proteases. We demonstrate that the gelatinolytic activity of viper venom proteases is neutralised by addition of viper antivenom to the matrix of conventional gelatin-zymograms. Venom protein gelatinolytic activity was unaffected by inclusion of antibody from control, non-immunised animals or immunoglobulin-depleted serum. The application of this antibody zymogram technique for future research on snake venoms is evaluated in the context of identified limitations.

World Health Organization (WHO)-recommended preclinical in vivo and in vitro studies were carried out to compare the efficacy of Brazilian, Peruvian and Colombian antivenoms in neutralizing the venom toxins responsible for the lethal, haemorrhagic, necrotizing, coagulant and defibrinogenating effects of five medically-important Peruvian snake venoms. Overall, the Brazilian antivenom was found to be the most effective followed by the Peruvian and Colombian antivenoms. However, it was concluded that all three antivenoms would be acceptable for use in a randomised clinical trial in envenomed humans in Peru.

Three non-RGD-containing disintegrins, VLO5, EO5, and EC3, belong to the heterodimeric family of these snake venom-derived proteins. They are potent inhibitors of certain leukocyte integrins such as alpha4beta1, alpha4beta7, and alpha9beta1, and act through the MLD motif present in one of their subunits. However, the selectivity of these disintegrins to interact with integrins is related to the amino acid composition of the integrin-binding loop in the MLD-containing subunit. The most important amino acid is that preceding the MLD motif. In vitro experiments in adhesion and ELISA assays revealed that the TMLD-containing disintegrins, VLO5 and EO5, appeared to be very potent inhibitors of human alpha4beta1 and alpha9beta1 and less effective in inhibition of the alpha4beta7 integrin. The reverse effect was observed for the AMLD-containing disintegrin, EC3. The data with native disintegrins were confirmed by experiments with synthetic peptides displaying TMLD and AMLD motifs. The MLD-containing disintegrins showed differential activities to inhibit human and murine alpha4beta1 integrin. EC3 was a weaker inhibitor of human integrin, whereas VLO5 and EO5 less actively inhibited murine alpha4beta1. These data describe a useful set of potent and selective integrin antagonists and suggest conformational requirements of human and mouse integrins for interaction with ligands.

The ability of the P-I metalloproteinase BaP1, isolated from the venom of the snake Bothrops asper, to induce systemic bleeding, thrombocytopenia and defibrinogenation was assessed in an experimental mouse model. Intravenous administration of BaP1 caused neither systemic bleeding nor any evidence of pathology in lungs, kidneys, liver, heart and brain. Moreover, there were no alterations in the whole blood clotting time or in platelet numbers. In addition, BaP1 did not inhibit collagen-induced platelet aggregation in vitro. Proteolytic and hemorrhagic activities of BaP1 were readily inhibited by the plasma proteinase inhibitor, alpha(2)-macroglobulin, and normal mouse serum also inhibited hemorrhage. Such inhibition may explain why BaP1 induces multiple local tissue-damaging effects, but is largely devoid of systemic toxicity.

The efficacies of specific Bothrops atrox-Lachesis and standard Bothrops-Lachesis antivenoms were compared in the north eastern Amazon region of Brazil. The main aim was to investigate whether a specific antivenom raised against the venom of B. atrox, the most important Amazon snake species from a medical point of view, was necessary for the treatment of patients in this region. Seventy-four patients with local and systemic effects of envenoming by Bothrops or Lachesis snakes were randomly allocated to receive either specific (n = 38) or standard (n = 36) antivenoms. In 46 cases (24 in the standard antivenom group, 22 in the other) the snake was identified either by enzyme immunoassay or by examination of the dead snake, as B. atrox in 45, L. muta in one. Patients were similar in all clinical and epidemiological respects before treatment. Results indicated that both antivenoms were equally effective in reversing all signs of envenoming detected both clinically and in the laboratory. Venom-induced haemostatic abnormalities were resolved within 24 h after the start of antivenom therapy in most patients. The extent of local complications, such as local skin necrosis and secondary infection, was similar in both groups. There were no deaths. The incidence of early anaphylactic reactions was 18% and 19%, respectively for specific and standard antivenoms; none was life-threatening. Measurement of serum venom concentrations by enzyme immunoassay (EIA) confirmed that both antivenoms cleared venom antigenaemia effectively. EIA also revealed that one patient had been bitten by Lachesis muta, although the clinical features in this case were not distinctive.

Local tissue damage, characterized by edema, hemorrhage and necrosis, is a common consequence of envenoming by many vipers. We have investigated the contribution of inflammatory responses induced by the venom metalloproteinase jararhagin (isolated from Bothrops jararaca venom) in the development of these lesions. Local venom effects (edema, hemorrhage and necrosis) were induced experimentally in knockout mice deficient in the TNF receptors TNFR1 or TNFR2, IL-1betaR, IL-6 and iNOS. Jararhagin-induced dermal necrosis was abolished in mice deficient in the TNF receptors TNFR1 and TNFR2, and the same activity was significantly reduced in IL-6(-/-) mice. There was no significant difference in edema and hemorrhage activities following jararhagin insult between knockout and WT strains, indicating that these local venom metalloproteinase-induced effects are independent of these pro-inflammatory mediators. The contribution of both TNF receptors and IL-6 in local tissue necrosis raises important therapeutic issues regarding the treatment of local envenoming.

Jararhagin is the most important hemorrhagic component in the venom of the snake Bothrops jararaca, a species of medical importance in South America. It is a P-III zinc-dependent metalloproteinase comprising catalytic, disintegrin-like, and cysteine-rich domains. Jararhagin injected intravenously into mice induced rapid and prominent bleeding in the lungs, whereas other organs were devoid of overt hemorrhagic manifestations. This action depends on the proteolytic activity of jararhagin, since it was abrogated by the synthetic inhibitor batimastat. There were conspicuous ultrastructural alterations in cells at the alveolo-capillary unit, i.e., capillary endothelial cells and type I pneumocytes, with a characteristic pattern of "regional alveolar damage" associated with extravasation. These pathological effects were observed under conditions in which the whole blood clotting time, bleeding time, and fibrinogen levels were not affected. 125I-labeled jararhagin is concentrated mainly in liver and kidneys after iv injection, with little radioactivity observed in the lungs, thereby indicating that the predominance of pulmonary microvascular damage is not due to a preferential concentration of this enzyme in the lungs. Despite the fact that jararhagin is complexed by plasma proteins after iv injection, its hemorrhagic activity was not inhibited by the plasma proteinase inhibitor alpha(2)-macroglobulin, and was only partially reduced by normal mouse serum, suggesting that resistance to inhibition may contribute to its ability to cause pulmonary hemorrhage.

Systemic envenoming by the saw-scaled viper, Echis ocellatus, is responsible for more deaths than any other snake in West Africa. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here we describe the isolation of E. ocellatus venom gland cDNAs encoding a protein of 514 amino acids that showed 91% sequence similarity to Ecarin, a prothrombin-activating metalloproteinase from the venom of the East African viper, E. pyramidum leakeyi, that induces severe consumption coagulopathy. Structural similarities between the E. ocellatus metalloproteinase and analogues in venoms of related vipers suggest that antibodies raised to phylogenetically conserved E. ocellatus metalloproteinase domains may have potential for cross-specific and cross-generic neutralisation of analogous venom toxins.

Envenoming by Echis saw scaled vipers and Bitis arietans puff adders is the leading cause of death and morbidity in Africa due to snake bite. Despite their medical importance, the composition and constituent functionality of venoms from these vipers remains poorly understood. Here, we report the cloning of cDNA sequences encoding seven clusters or isoforms of the haemostasis-disruptive C-type lectin (CTL) proteins from the venom glands of Echis ocellatus, E. pyramidum leakeyi, E. carinatus sochureki and B. arietans. All these CTL sequences encoded the cysteine scaffold that defines the carbohydrate-recognition domain of mammalian CTLs. All but one of the Echis and Bitis CTL sequences showed greater sequence similarity to the beta than alpha CTL subunits in venoms of related Asian and American vipers. Four of the new CTL clusters showed marked inter-cluster sequence conservation across all four viper species which were significantly different from that of previously published viper CTLs. The other three Echis and Bitis CTL clusters showed varying degrees of sequence similarity to published viper venom CTLs. Because viper venom CTLs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis and Bitis CTLs on the basis of sequence alone. The extraordinary level of inter-specific and inter-generic sequence conservation exhibited by the Echis and Bitis CTLs leads us to speculate that antibodies to representative molecules should neutralise the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East and the Indian subcontinent.

A prothrombin activator, named 'basparin A,' was isolated from the venom of the crotaline snake Bothrops asper, the species responsible for the majority of snakebite cases in Central America. It is an acidic (pI 5.4), 70kDa, single chain P-III metalloproteinase comprising, in addition to the metalloproteinase domain, disintegrin-like, and high-cysteine domains. Basparin A is a glycoprotein displaying immunological cross-reactivity with BaH1, a P-III hemorrhagic metalloproteinase isolated from the same venom. It activates prothrombin through the formation of meizothrombin, without requiring additional cofactors; it is, therefore, a class A snake venom prothrombin activator. In contrast with most venom metalloproteinases, it does not degrade components of the extracellular matrix. Apart from its clotting activity, basparin A inhibits collagen-dependent platelet aggregation in vitro, an effect that does not depend on proteolytic activity. Clotting activity on human plasma is not abrogated by the plasma proteinase inhibitors alpha(2) macroglobulin and murinoglobulin, whereas activity is completely inhibited by Costa Rican polyvalent (Crotalinae) anti-venom. Basparin A does not induce local tissue alterations, such as hemorrhage, myonecrosis, and edema, in mice. Moreover, it does not induce systemic hemorrhage, thrombocytopenia nor prolongation of the bleeding time following intravenous administration. At low doses, the only observed effect induced by basparin A, when injected intravenously or intramuscularly into mice, is defibrin(ogen)ation. At higher doses, intravenous administration resulted in sudden death due to numerous occluding thrombi in pulmonary vessels. Basparin A is likely to play an important role in the coagulopathy associated with B. asper envenoming.

BaP1 is a 22.7-kD P-I-type zinc-dependent metalloproteinase isolated from the venom of the snake Bothrops asper, a medically relevant species in Central America. This enzyme exerts multiple tissue-damaging activities, including hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. BaP1 is a single chain of 202 amino acids that shows highest sequence identity with metalloproteinases isolated from the venoms of snakes of the subfamily Crotalinae. It has six Cys residues involved in three disulfide bridges (Cys 117-Cys 197, Cys 159-Cys 181, Cys 157-Cys 164). It has the consensus sequence H(142)E(143)XXH(146)XXGXXH(152), as well as the sequence C(164)I(165)M(166), which characterize the "metzincin" superfamily of metalloproteinases. The active-site cleft separates a major subdomain (residues 1-152), comprising four alpha-helices and a five-stranded beta-sheet, from the minor subdomain, which is formed by a single alpha-helix and several loops. The catalytic zinc ion is coordinated by the N(epsilon 2) nitrogen atoms of His 142, His 146, and His 152, in addition to a solvent water molecule, which in turn is bound to Glu 143. Several conserved residues contribute to the formation of the hydrophobic pocket, and Met 166 serves as a hydrophobic base for the active-site groups. Sequence and structural comparisons of hemorrhagic and nonhemorrhagic P-I metalloproteinases from snake venoms revealed differences in several regions. In particular, the loop comprising residues 153 to 176 has marked structural differences between metalloproteinases with very different hemorrhagic activities. Because this region lies in close proximity to the active-site microenvironment, it may influence the interaction of these enzymes with physiologically relevant substrates in the extracellular matrix.