The protective effects of Mucuna pruriens seed extract (MPE) against the cardio-respiratory depressant and neuromuscular paralytic effects induced by injection of Calloselasma rhodostoma (Malayan pit viper) venom in anaesthetized rats were investigated. While MPE pretreatment did not reverse the inhibitory effect of the venom on the gastrocnemius muscle excitability, it significantly attenuated the venom-induced cardio-respiratory depressant effects (p < 0.05). The protection effects may have an immunological mechanism, as indicated by the presence of several proteins in the venom that are immunoreactive against anti-MPE. However, we cannot rule out the possibility that the pretreatment may exert a direct, non-immunological protective action against the venom.
Seed of Mucuna pruriens (Velvet beans) has been prescribed by traditional medicine practitioners in Nigeria as a prophylactic oral antisnake remedy. In the present studies, we investigated the protective effects of M. pruriens seed extract (MPE) against histopathological changes induced by intravenous injection of Naja sputatrix (Malayan cobra) venom in rats pretreated with the seed extract. Examination by light microscope revealed that the venom induced histopathological changes in heart and blood vessels in liver, but no effect on brain, lung, kidney and spleen. The induced changes were prevented by pretreatment of the rats with MPE. Our results suggest that MPE pretreatment protects rat heart and liver blood vessels against cobra venom-induced damages.
Cross neutralisation of venoms by antivenom raised against closely-related species has been well documented. The spectrum of paraspecific protection of antivenom raised against Asiatic Naja and Bungarus (krait) venoms, however, has not been fully investigated. In this study, we examined the cross neutralisation of venoms from common Southeast Asian cobras and kraits by two widely used polyvalent antivenoms produced in India: Vins Polyvalent Antivenom (VPAV) and Bharat Polyvalent Antivenom (BPAV), using both in vitro and in vivo mouse protection assays. BPAV was only moderately effective against venoms of N. kaouthia (Thailand) and N. sumatrana, and either very weakly effective or totally ineffective against the other cobra and krait venoms. VPAV, on the other hand, neutralised effectively all the Southeast Asian Naja venoms tested, as well as N. naja, B. candidus and Ophiophagus hannah venoms, but the potency ranges from effective to weakly effective. In an in vivo rodent model, VPAV also neutralised the lethality of venoms from Asiatic Naja and B. candidus. In anesthetised rat studies, both antivenoms effectively protected against the N. kaouthia venom-induced cardio-respiratory depressant and neuromuscular blocking effects. Overall, our results suggest that VPAV could be used as alternative antivenom for the treatment of elapid envenomation in Southeast Asian regions including Malaysia, Thailand and certain regions of Indonesia.
Sea snake envenomation is a serious occupational hazard in tropical waters. In Malaysia, the beaked sea snake (Hydrophis schistosus, formerly known as Enhydrina schistosa) and the spine-bellied sea snake (Hydrophis curtus, formerly known as Lapemis curtus or Lapemis hardwickii) are two commonly encountered species. Australian CSL sea snake antivenom is the definitive treatment for sea snake envenomation; it is unfortunately extremely costly locally and is not widely available or adequately stocked in local hospitals. This study investigated the cross-neutralizing potential of three regionally produced anti-cobra antivenoms against the venoms of Malaysian H. schistosus and H. curtus. All three antivenoms conferred paraspecific protection from sea snake venom lethality in mice, with potency increasing in the following order: Taiwan bivalent antivenom < Thai monocled cobra monovalent antivenom < Thai neuro polyvalent antivenom (NPAV). NPAV demonstrated cross-neutralizing potencies of 0.4 mg/vial for H. schistosus venom and 0.8 mg/vial for H. curtus, which translates to a dose of less than 20 vials of NPAV to neutralize an average amount of sea snake venom per bite (inferred from venom milking). The cross-neutralization activity was supported by ELISA cross-reactivity between NPAV and the venoms of H. schistosus (58.4%) and H. curtus (70.4%). These findings revealed the potential of NPAV as a second-line treatment for sea snake envenomation in the region. Further profiling of the cross-neutralization activity should address the antivenomic basis using purified toxin-based assays.
The Senegalese cobra, Naja senegalensis, is a non-spitting cobra species newly erected from the Naja haje complex. Naja senegalensis causes neurotoxic envenomation in Western Africa but its venom properties remain underexplored. Applying a protein decomplexation proteomic approach, this study unveiled the unique complexity of the venom composition. Three-finger toxins constituted the major component, accounting for 75.91% of total venom proteins. Of these, cardiotoxin/cytotoxin (~53%) and alpha-neurotoxins (~23%) predominated in the venom proteome. Phospholipase A2, however, was not present in the venom, suggesting a unique snake venom phenotype found in this species. The venom, despite the absence of PLA2, is highly lethal with an intravenous LD50 of 0.39 µg/g in mice, consistent with the high abundance of alpha-neurotoxins (predominating long neurotoxins) in the venom. The hetero-specific VINS African Polyvalent Antivenom (VAPAV) was immunoreactive to the venom, implying conserved protein antigenicity in the venoms of N. senegalensis and N. haje. Furthermore, VAPAV was able to cross-neutralize the lethal effect of N. senegalensis venom but the potency was limited (0.59 mg venom completely neutralized per mL antivenom, or ~82 LD50 per ml of antivenom). The efficacy of antivenom should be further improved to optimize the treatment of cobra bite envenomation in Africa.
Naja nivea (Cape Cobra) is endemic to southern Africa. Envenoming by N. nivea is neurotoxic, resulting in fatal paralysis. Its venom composition, however, has not been studied in depth, and specific antivenoms against it remain limited in supply. Applying a protein decomplexation approach, this study unveiled the venom proteome of N. nivea from South Africa. The major components in the venom are cytotoxins/cardiotoxins (~75.6% of total venom proteins) and alpha-neurotoxins (~7.4%), which belong to the three-finger toxin family. Intriguingly, phospholipase A2 (PLA2) was undetected-this is a unique venom phenotype increasingly recognized in the African cobras of the Uraeus subgenus. The work further showed that VINS African Polyvalent Antivenom (VAPAV) exhibited cross-reactivity toward the venom and immunorecognized its toxin fractions. In mice, VAPAV was moderately efficacious in cross-neutralizing the venom lethality with a potency of 0.51 mg/mL (amount of venom completely neutralized per milliliter of antivenom). In the challenge-rescue model, VAPAV prevented death in 75% of experimentally envenomed mice, with slow recovery from neurotoxicity up to 24 h. The finding suggests the potential para-specific utility of VAPAV for N. nivea envenoming, although a higher dose or repeated administration of the antivenom may be required to fully reverse the neurotoxic effect of the venom.
Venoms of Calliophis bivirgata and Calliophis intestinalis exhibited moderate binding activities toward Neuro Bivalent Antivenom (Taiwan) but not the other six elapid monovalent or bivalent antivenoms available in the region. All antivenoms failed to neutralize C. bivirgata venom lethality in mice. The findings indicate the need to validate antivenom cross-reactivity with in vivo cross-neutralization, and imply that distinct antigens of Calliophis venoms should be incorporated in the production of a pan-regional poly-specific antivenom.
Arboreal pit vipers of the Trimeresurus complex group are medically important species in Indonesia (west of Wallace's line), but there is no specific antivenom produced in the country for treating related envenomation. Instead, the exiting trivalent Indonesian antivenom, Biosave® Serum Anti Bisa Ular (SABU, indicated for envenoming by Malayan pit viper, Javan spitting cobra and banded krait) is often misused to treat Trimeresus envenoming resulting in poor therapeutic outcome. Here, we investigated the cross-reactivity and neutralization capability of Thai Green Pit Viper Antivenom (GPVAV) against the venoms of four Indonesian Trimeresurus species. Consistently, the venoms of Trimeresurus (Trimeresurus) insularis, Trimeresurus (Trimeresurus) purpureomaculatus, Trimeresurus (Parias) hageni and Trimeresurus (Craspedocephalus) puniceus of Indonesia showed stronger immunoreactivity on ELISA to GPVAV than to Biosave®. The findings correlated with in vivo neutralization results, whereby GPVAV was far more effective than Biosave® in cross-neutralizing the lethality of the venoms by a potency of at least 13 to 80 times higher. The efficacy of GPVAV is partly attributable to its cross-neutralization of the procoagulant effect of the venoms, thereby mitigating the progression of venom-induced consumptive coagulopathy. The paraspecific effectiveness of GPVAV against Trimeresurus species envenoming in Indonesia await further clinical investigation.
Gel filtration chromatography and gel electrophoresis revealed minimal protein degradation in lyophilized antivenoms which were 2-year expired (Hemato Polyvalent, Neuro Polyvalent; Thailand) and 18-year expired (Hemato Bivalent, Neuro Bivalent; Taiwan). All expired antivenoms retained immunological binding activity, and were able to neutralize the hemotoxic or neurotoxic as well as lethal effects of the homologous snake venoms. The findings show that antivenoms under proper storage conditions may remain relatively stable beyond the indicated shelf life.
The intraspecific geographical venom variations of Calloselasma rhodostoma from Malaysia (CR-M), Indonesia (CR-I), Thailand (CR-T) and Vietnam (CR-V) were investigated through 1D SDS-PAGE and nano-ESI-LCMS/MS. The venom antigenicity, procoagulant activities and neutralization using Thai C. rhodostoma Monovalent Antivenom (CRMAV) were also investigated. SDS-PAGE patterns of the venoms were relatively similar with minor variations. Proteomic analysis revealed that snake venom metalloproteinases (SVMPs, particularly P-I class), serine proteases (SVSPs) and snaclecs dominated the venom protein composition (68.96-81.80%), followed by L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2) (7.37-11.08% and 5.18-13.81%, respectively), corroborating C. rhodostoma envenoming effects (hemorrhage, consumptive coagulopathy, thrombocytopenia and local tissue necrosis). Other proteins of lower abundances (2.82-9.13%) identified include cysteine-rich secretory proteins (CRISP), phospholipase B, phosphodiesterase, nerve growth factor, 5'-nucleotidase, aminopeptidase and hyaluronidase. All four venoms exhibited strong procoagulant effects which were neutralized by CRMAV to different extents. CRMAV immunoreactivity was high toward venoms of CR-M, CR-I and CR-T but relatively low for CR-V venom. Among the venom samples from different locales, CR-V venom proteome has the smallest SVMP composition while SVSP, PLA2 and phosphodiesterase were more abundant in the venom. These variations in C. rhodostoma venom protein composition could partly explain the differences seen in immunoreactivity. (198 words).
Trimeresurus wagleri (speckled pit viper) venom exhibited the usual set of enzyme activities occurring in pit viper venoms but the content of alkaline phosphomonoesterase was unusually high, whereas the proportions of protease and arginine ester hydrolase were very low. The venom also exhibited weak thrombin-like activity but did not exhibit hemorrhagic or anticoagulant activity. Analysis of the Sephadex G-200 gel filtration fractions of the venom indicated that the lethal fraction was a low mol.wt protein, and that fractions exhibiting phosphodiesterase, phosphomonoesterase, arginine ester hydrolase, thrombin-like enzyme, L-amino acid oxidase and phospholipase A activities were not lethal. Two lethal toxins, designated as wagleri toxins 1 and 2, were isolated from the venom using Sephadex G-50 gel filtration chromatography followed by SP-Sephadex C-25 ion exchange chromatography. The mol.wts of the two toxins were 8900 by gel filtration. The LD50 (i.v.) values in mice for wagleri toxins 1 and 2 are 0.17 microgram/g and 0.19 microgram/g, respectively.
Trimeresurus purpureomaculatus venom acetylcholinesterase has been partially purified by Sephadex G-200 gel filtration chromatography and DEAE Sephacel ion exchange chromatography. The enzyme has a mol. wt of 58,600. It was strongly inhibited by physostigmine salicylate and edrophonium chloride and exhibited substrate inhibition at high substrate concentration. The content of acetylcholinesterase in Trimeresurus purpureomaculatus venom was estimated to be much less than 0.3%.
The enzymatic activities of four samples of Malayan cobra venom were investigated. There was significant variation in the contents of L-amino acid oxidase, alkaline phosphomonoesterase, acetylcholinesterase, phospholipase A, 5'-nucleotidase and hyaluronidase. The phosphodiesterase content was, however, constant. Storage of the lyophilized venom powder at 25 degrees C for 1 month did not affect the enzymatic activities. The venom enzymatic activities were generally also stable at 4 degrees C in 0.85% saline solution. After incubation at 37 degrees C for 39 days in 0.85% saline solution, the venom still retained considerable amounts of enzymatic activities. SP-Sephadex C-25 ion-exchange chromatography of the venom showed that the phospholipase A, L-amino acid oxidase, 5'-nucleotidase, phosphodiesterase and alkaline phosphomonoesterase exist in multiple forms.
Hannahtoxin, the major hemorrhagin purified from king cobra (Ophiophagus hannah) venom, elicits hemorrhages in rabbits but not in mice. Two antisera against hannahtoxin were prepared: one raised against purified hannahtoxin, while the other was raised against glutaraldehyde cross-linked and detoxified hannahtoxin. The antisera were refined by pepsin digestion and ammonium sulfate precipitation. They are of approximately equal potency in their ability to neutralize the hemorrhagic activity of king cobra venom in rabbits. The antisera did not form a precipitin line with venom of snakes of the Viperidae family nor neutralize hemorrhages elicited in mice by any of these venoms. However, when the hemorrhagic activity was assayed in rabbits, both antisera were able to abolish the hemorrhages elicited by all of the venoms tested. These results suggest that hannahtoxin displays few epitopes in common with hemorrhagins of viperid venoms, except those involved in the neutralization of hemorrhagic activity in rabbits. The epitopes of viperid venom hemorrhagins involved in the neutralization reaction in rabbits are different from those in mice.
Bungarus candidus venom exhibited high hyaluronidase, acetylcholinesterase and phospholipase A activities; low proteinase, 5'-nucleotidase, alkaline phosphomonoesterase and phosphodiesterase activities and moderately high L-amino acid oxidase activity. SP-Sephadex C-50 ion exchange chromatographic fractionation of the venom and Sephadex G-50 chromatography of the major lethal venom fractions indicate that the venom contains at least two highly lethal, basic phospholipases A with LD50 (i.v.) values of 0.02 micrograms/g (F6A) and 0.18 micrograms/g (F4A), respectively; as well as two polypeptide toxins with LD50 (i.v.) values of 0.17 micrograms/g and 0.83 micrograms/g, respectively. The major lethal toxin is the basic lethal phospholipase A, F6A, which accounts for approximately 13% of the venom protein and has a mol. wt of 21,000.
Sumatran pit viper (Trimeresurus sumatranus sumatranus) venom was fractionated by DEAE-Sephacel ion exchange chromatography into seven fractions. Fractions 4, 5 and 6 were lethal to mice and exhibited strong hemorrhagic activity, as well as some enzymatic activities. Fraction 6 also exhibited potent anticoagulant and thrombin-like activities. Analysis of the biological and enzymatic properties of the three lethal fractions suggests that the major lethal component of fractions 4 and 5 may be the hemorrhagic principle, and that the lethality of fraction 6 may be due to the hemorrhagic principle and/or the anticoagulant principle.
Some enzymatic activities and toxic properties of four samples of Ophiophagus hannah (king cobra) venom were investigated. There is little intraspecific variation in enzyme contents, protein composition and toxic properties of the venom. The venom does not exhibit hemolytic or edema-inducing activity but is characterized by an exceptionally high alkaline phosphomonoesterase activity. DEAE-Sephacel ion exchange chromatography and Sephadex G-75 gel filtration chromatography of the venom indicate that the major lethal toxins are the low mol.wt, non-enzymatic basic proteins. Venom fractions exhibiting high enzymatic activities apparently do not play an important role in the lethality in mice of Ophiophagus hannah venom.
The in vivo interactions between alpha-neurotoxin, cardiotoxin and two phospholipases A2 (sputa-phospholipase A2-1 and 3) isolated from Malayan cobra venom were assessed by examining the effects of simultaneous injection of sub-LD50 dose of one toxin on (i) i.v. LD50 S of the other toxins in mice; and (ii) mean survival times of mice injected with lethal doses of the other toxins. While LD50 measurements did not reveal any interaction between the toxins in vivo, survival time measurements suggest a synergy between the neurotoxin and sputa-phospholipase A2-1 and between sputa-phospholipase A2-1 and sputa-phospholipase A2-3. Our results also suggest that both sputa-phospholipases A2 interfere with the lethal action of the cardiotoxin, resulting in prolongation of the mean survival time of mice injected with a lethal dose of cardiotoxin. The patterns of in vivo interactions between phospholipase A2 and other venom toxins appear to depend on the nature and mode of pharmacological action of the phospholipase A2.