An investigation of venom secretion by the venom gland cells of the carpet viper (Echis carinatus)

The indirect immunofluorescent antibody technique was applied to the study of Echis carinatus pyramidum venom antigens in venom gland tissue using semi-thin frozen sections. A total of four rabbit antisera, two monoclonal antibodies active against E. carinatus venom, two monoclonal antibodies active against the rodent malaria parasite, Plasmodium chabaudi, and two monoclonal antibodies active against the human malaria parasite, Plasmodium falciparum, were investigated. The results of this study suggest that each secretory cell within the main part of the gland produces all the venom constituents. The resultant venom is therefore considered to be produced as a single package by each individual secretory cell. The different constituents of the venom studied are not produced at the same time or at the same rate throughout the secretory cycle, some being produced at the beginning and others at a later stage.     

Characterisation of a monoclonal antibody capable of neutralising the haemorrhagic activity of West African Echis carinatus (carpet viper) venom

A murine monoclonal antibody (SV-1/F10) was highly specific immunologically for West African E.carinatus venom both by ELISA and immunoblotting. In cross-protection tests in vivo, it possessed strong antihaemorrhagic activity. This IgGl monoclonal antibody recognised an epitope present in a protein band of 124,000 mol. wt using immunoblotting of non-reduced Nigerian and Ghanaian E.carinatus venoms, as well as in a second protein band of 105,000 mol. wt in Ghanaian E.carinatus venom. The SV-1/F10 monoclonal antibody is of potential use for the isolation of West African E.carinatus venom haemorrhagin from whole venom, allowing the possibility of elucidation of the mechanism of its actions as well as its interactions with antibody.     

Determination of factor X activator in the venom of the saw-scaled viper (Echis carinatus)

Factor X activator in Echis carinatus venom was determined by incubating the zymogen 'factor X' with venom, interrupting the activation process by ethylenediaminetetraacetic acid and measuring the generated proteinase 'factor Xa' by means of a synthetic chromogenic substrate. A comparison of factor X- and prothrombin-activating potencies in E. carinatus venoms of five different geographic origins revealed no correlation between these two procoagulant activities.     

Rapid isolation and partial characterization of two phospholipases from Kenyan Echis carinatus leakeyi (Leakey's saw-scaled viper) venom.

The purification and partial sequencing of two phospholipase A2 toxins from the venom of Kenyan E. carinatus leakeyi is described. The two proteins exhibit sequence homology with other toxic phospholipases. Both have a molecular weight in the region of 16,000 and are purified to homogeneity from crude venom by a single high performance liquid chromatography. The role of these proteins in the toxicity of the venom remains to be established.     

Chromogenic assay for the prothrombin activator ecarin from the venom of the saw-scaled viper (Echis carinatus)

Ecarin, by limited proteolysis and subsequent autocatalytic reactions, causes the conversion of prothrombin into three products with amidolytic activity: meizothrombin, meizothrombin 1 and lpha-thrombin. Ecarin action may be abolished by ethylenediaminetetraacetic acid and the activity of alpha-thrombin can, with a high degree of selectivity, be inhibited by heparin. Thus, ecarin potency may be assayed by measuring the meizothrombin activity generated by ecarin action on human plasma in the presence of heparin. The chromogenic substrate Tosyl-glycyl-L-prolyl-L-arginine-p-nitroanilide (Chromozym TH) is used in this assay.     

The purification and partial characterisation of two novel metalloproteinases from the venom of the West African carpet viper, Echis ocellatus.

Separation of previously uncharacterised Echis ocellatus venom by phenyl-Superose FPLC (Fast Liquid Protein Chromatography) yielded eight protein fractions. Three of these displayed high proteolytic activity when assayed by in vivo and in vitro assays (including enzyme linked immunosorbant assay), and were further separated using Superdex 75 and Mono-Q FPLC. This resulted in the purification of a non-haemorrhagic 24 kDa metalloproteinase (EoVMP1, pI 7.0), and a haemorrhagic 56 kDa metalloproteinase (EoVMP2, pI 5.5). Following tryptic digest, short amino acid sequences of EoVMP1 and EoVMP2 were obtained using Edman degradation. Both sequences displayed homology when aligned with existing snake venom metalloproteinases (SVMPs). The strong homology observed among previously well-characterised SVMPs suggests that principles governing the interaction of substrates and inhibitors are likely to be similar for EoVMP1, EoVMP2 and all members of the reprolysin family.     

Formulation of a liquid ovine Fab-based antivenom for the treatment of envenomation by the Nigerian carpet viper (Echis ocellatus).

Most antivenoms are required for use in tropical or sub-tropical countries where temperatures may be high and refrigerated storage unavailable or unreliable. Although freeze-dried products can be expected to have maximal storage stability, many antivenoms are manufactured in liquid form to lower their cost and ease their use. We developed a liquid formulation of an existing freeze-dried antivenom against the carpet viper (Echis ocellatus) for use in Nigeria. When Fab fragments, prepared from antisera raised in sheep, were exposed to pH between 3.0 and 8.0, time and temperature dependent precipitation of some populations of the Fab product occurred over the range 4.5-7.0. Formulation of the Fab fragments in acetate buffer (20 mM) at pH 4.0 provided a clear, colourless, particle-free and stable product which retained neutralising potency for at least one year at 4 degrees C and room temperature. An accelerated study indicated stability for at least 4 weeks at 37 degrees C which may be considered equivalent to four years at 4 degrees C. The presence of sugars (sorbitol at 20 g/l or mannitol at 50 g/l) or sodium chloride (153 mM) had no effect in stabilising Fab at high temperature (37 degrees C) and, in agreement with the prediction of these accelerated studies, no beneficial effect of mannitol was found in real-time studies after one year.     

Relationship of administered dose to blood venom levels in mice following experimental envenomation by Russell's viper (Vipera russelli) venom

I125 labelled Russell's viper venom was administered i.m. into mice at various doses. Radioactivity in the blood at different intervals was determined and related to the amount injected. A mathematical equation was formulated to represent the relationship of administered dose to blood venom levels. This study suggests that the derivation of such an equation is also possible in humans in order to predict the amount envenomated.     

Pharmacokinetics of Cryptelytrops purpureomaculatus (mangrove pit viper) venom following intravenous and intramuscular injections in rabbits

The pharmacokinetic profiles of Cryptelytrops purpureomaculatus (mangrove pit viper) venom following intravenous and intramuscular injections were investigated in rabbits. The serum levels of the venom were estimated using double-sandwich enzyme-linked immunosorbent assay (ELISA). After intravenous injection (0.2 mg/kg), the serum venom concentration–time course declined in a biexponential manner, consistent with a two-compartment model, with an α-phase half-life of 0.25 h and a β-phase half-life of 27.7 h. The volume of distribution by area was 2.19 L/kg and systemic clearance was 54.7 mL/h/kg. When the venom was injected intramuscularly (0.5 mg/kg), the serum level increased rapidly to reach a peak (500 ng/mL) at about 1 h, which then declined rapidly to a plateau (104–142 ng/mL) at 3–10 h before further gradual decline until the end of the 72-hour study. The terminal half-life (27.0 h), clearance (54.7 mL/h/kg) and volume of distribution (2.13 L/kg) of the venom for intramuscular route were not significantly different from the corresponding values for intravenous route, and the intramuscular bioavailability of the venom was estimated to be 41.6%.     

Coagulant component in Cerastes cerastes (Egyptian sand viper) venom

A coagulant component has been purified from Cerastes cerastes venom, using gel filtration on a Sephadex G 100 (fine) column followed by chromatography on a Bio-Rex 70 column. This compound had a proteolytic effect and could coagulate human plasma deficient in factor VIII or (VIII + IX) with the formation of a firm clot. It could also clot plasma deficient in factor X, but the clot formed was soft and not complete. The compound had no effect on platelet aggregation and was nontoxic. This compound is believed to be primarily a factor X activator, as it could replace factor VIII in hemophilic plasmas.     

Amount of venom injected by Russell's viper (Vipera russelli)

The total yield of venom (desiccated) in 25 adult V. russelli (mean length 111 +/- 1.8 cm (S.E.) ranges from 21-268 mg (127 +/- 13 mg, mean +/- S.E.) and that of 13 juvenile snakes (mean length 79 +/- 2.8 cm) is 8-79 mg (45 +/- 7 mg). The venom yield per milking correlated well with the length of the snake. The average amount of venom injected in the first bite of 31 adults (mean length 107 +/- 1.4 cm) is 63 +/- 7 mg (range 6-147 mg) and by 17 juvenile snakes (mean length 83 +/- 1.1 cm) is 41 +/- 8 mg (range 3-138 mg). Adults inject 45% of the glands' content in the first bite. More than 75 mg of venom (desiccated) was injected by 11 out of 31 adults and 2 out of 17 juvenile snakes. This indicates that in a substantial proportion of cases of envenomation, 40 ml of Burma Pharmaceutical Industry monovalent antivenom, which is commonly used, may not be adequate.     

Albizia lebbeck seed methanolic extract as a complementary therapy to manage local toxicity of Echis carinatus venom in a murine model

Context and objective: Viperid venom-induced chronic local-toxicity continues even after anti-snake venom treatment. Therefore, traditional antidote Albizia lebbeck L. (Fabaceae) seed extract was tested against Echis carinatus S. (Viperidae) venom (ECV)-induced local toxicity to evaluate its complementary remedy.

Materials and methods: Soxhlet extraction of A. lebbeck seeds was performed with the increasing polarity of solvents (n-hexane to water); the extract was screened for phytochemicals (alkaloids, anthraquinones, flavonoids, glycosides, phenolics, saponins, steroids and tannins). In preliminary in vitro analysis, A. lebbeck methanolic extract (ALME) demonstrated significant inhibition of ECV proteases, the major enzyme–toxin responsible for local- toxicity. Therefore, in vitro neutralizing potential of ALME was further evaluated against hyaluronidases and phospholipase A₂ (1:1–1:100 w/w). In addition, alleviation of ECV induced characteristic local- toxicity [haemorrhage (i.d.) and myotoxicity (i.m.)] was determined in mice.

Results: ALME contained high concentrations of phenolics and flavonoids and demonstrated significant in vitro inhibition of ECV protease (IC_50?=_?36.32?μg, p?<?0.0001) and hyaluronidase (IC_50?=_?91.95?μg, p?<?0.0001) at 1:100 w/w. ALME significantly neutralized ECV induced haemorrhage (ED_50?=_?26.37?μg, p?<?0.0001) and myotoxicity by significantly reducing serum creatinine kinase (ED_50?=_?37.5?μg, p?<?0.0001) and lactate dehydrogenase (ED_50?=_?31.44?μg, p?=?0.0021) levels at 1:50 w/w.

Discussion and conclusion: ALME demonstrated significant neutralization of ECV enzymes that contribute in local tissue damage and haemostatic alterations. The study scientifically supports the anecdotal use of A. lebbeck in complementary medicine and identifies ALME as principle fraction responsible for antivenom properties.     

Enzymatic activities of Calloselasma rhodostoma (Malayan pit viper) venom

The enzyme contents of four venom samples of Calloselasma rhodostoma were analyzed. The venoms contained phosphodiesterase, alkaline phosphomonoesterase, 5'-nucleotidase, protease, phospholipase A, L-amino acid oxidase, hyaluronidase, arginine ester hydrolase, arginine amidase, fibrinogenase and coagulant enzyme activities. There is significant variation in the contents of coagulant enzyme, arginine ester hydrolase, hyaluronidase, protease, phosphodiesterase, alkaline phosphomonoesterase and L-amino acid oxidase. DEAE-Sephacel ion exchange chromatography of the venom resolved it into eight major protein fractions. The eight fractions were heterogeneous and exhibited more than one type of enzymatic activity. The 5'-nucleotidase, alkaline phosphomonoesterase, protease, coagulant enzyme, arginine ester hydrolase, arginine amidase and fibrinogenase exist in multiple forms.     

Treatment of envenomation by Echis coloratus (mid-east saw scaled viper): a decision tree

Envenomation by Echis coloratus causes a transient hemostatic failure. Systemic symptoms, hypotension and evident bleeding are rare, with only one reported fatality. In this paper, we examine the decision to treat victims of Echis coloratus by a specific horse antiserum. The decision model considers the mortality of treated and untreated envenomation, and the side effects of antiserum treatment: fatal anaphylaxis, serum sickness and increased risk of death after a possible repeated exposure to horse antiserum in the future. The results of the analysis are not sensitive to variations in the probability of side effects of antiserum treatment. They are sensitive to variations in the risk of bleeding after envenomation, in the degree of reduction of this risk by antiserum treatment and in the risk of dying after an event of bleeding. Prompt administration of antiserum appears to be the treatment of choice if it reduces the risk of bleeding from 23.6% to 20.3% and if 1.6% or more of the bleeding events are fatal. We conclude that presently available data support antiserum treatment of victims of Echis coloratus who present with hemostatic failure, even though the advantage imparted by this treatment appears to be small.     

The haemodynamic effect of Bitis nasicornis (rhinoceros horned viper) venom

1. Intravenous venom (0.0625 mg/kg) in the dog caused an immediate increase in coronary blood flow due to a fall in coronary vascular resistance (CVR). 2. Subsequently, total peripheral resistance (TPR) fell causing a significant reduction in aortic blood pressure (ABP). 3. CVR and TPR returned to normal after 20 min but ABP did not recover completely. 4. The failure of ABP to recover was due to decreased stroke volume and cardiac output (CO). 5. Animals died after four doubling doses of venom following irreversible reductions in CO, ABP and coronary flow.     

Evidence for the presence of histamine in Gaboon viper (Bitis gabonica) venom

Venom from B. gabonica produced a dose-dependent contraction of both isolated guinea-pig ileum and rabbit aortic strip preparations. The venom-induced contraction was not antagonised by pretreatment with atropine, phentolamine, methysergide or indomethacin, however, it was blocked by the prior addition of chlorpheniramine or cyproheptadine. Upon exhaustive dialysis of the venom, the dialysate contracted both preparations, while the dialysed venom had no effect. Paper and thin-layer chromatography of the venom showed a spot with an Rf value corresponding to authentic histamine. Biological assay of the crude venom on the guinea-pig ileum showed that it contained the equivalent of 2-10 micrograms of histamine per mg dry weight of crude venom. pA2 values for chlorpheniramine using either the venom or histamine as agonists were not significantly different.