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.
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.
1. The hemorrhagic, procoagulant, anticoagulant, protease, phosphodiesterase, alkaline phosphomonoesterase, L-amino acid oxidase, acetylcholinesterase, arginine ester hydrolase, phospholipase A, 5'-nucleotidase and hyaluronidase activities of 39 samples of venoms from 13 species (15 taxa) of Australian elapids were determined and the Sephadex G-75 gel filtration patterns for some of the venoms were also examined. 2. The results indicate that Australian elapid venoms can be divided into two groups: procoagulant Australian venoms (including N. scutatus, N. ater, O. scutellatus, O. microlepidotus, P. porphyriacus, T. carinatus, H. stephensii and P. textilis) and non-procoagulant Australian venoms (including A. superbus, P. colletti, P. australis, P. guttatus and A. antarcticus). 3. The non-procoagulant Australian venoms exhibited biological properties similar to other elapid venoms, while the procoagulant Australian venoms exhibited some properties characteristic of viperid venoms. 4. The data show that information on venom biological properties can be used for differentiation of many species of Australian elapids. 5. Particularly useful for this purpose are the hyaluronidase, alkaline phosphomonoesterase, acetylcholinesterase, and the procoagulant activities and the Sephadex G-75 gel filtration patterns of the venoms.
1. The edema-inducing activity of 24 venoms from snakes of the subfamilies of Elapinae, Hydrophiini, Crotalinae and Viperinae was determined. 2. All snake venoms tested are very potent edema inducers. The minimum edema doses of the venoms ranged from 0.16 to 3.41 micrograms per mouse paw. 3. The venoms induced a rapid onset edema which peaked within 1 h of injection and declined thereafter; at low dose, however, some venoms induced a rapid onset edema that sustained over a longer duration.
1. The biological properties of twelve samples of venoms from all four species of Dendroaspis (mamba) were investigated. 2. Dendroaspis venoms generally exhibited very low levels of protease, phosphodiesterase and alkaline phosphomonoesterase; low to moderately low level of 5'-nucleotidase and very high hyaluronidase activities, but were devoid of L-amino acid oxidase, phospholipase A, acetylcholinesterase and arginine ester hydrolase activities. The unusual feature in venom enzyme content can be used to distinguish Dendroaspis venoms from other snake venoms. 3. All Dendroaspis venoms did not exhibit hemorrhagic or procoagulant activity. Some Dendroaspis venoms, however, exhibited strong anticoagulant activity. The intravenous median lethal dose of the venoms ranged from 0.5 microgram/g mouse to 4.2 micrograms/g mouse. 4. Venom biological activities are not very useful for the differentiation of the Dendroaspis species. The four Dendroaspis venoms, however, can be differentiated by their venom SDS-polyacrylamide gel electrophoretic patterns.
1. The biological properties of nine venom samples from six taxa of Micrurus were investigated. The venoms exhibited low protease, phosphodiesterase and 5'-nucleotidase activities, moderate to strong phospholipase A and hyaluronidase activities, variable L-amino acid oxidase activity and were devoid of arginine ester hydrolase and thrombin-like activities. Some venom samples exhibited strong acetylcholinesterase activity. Venoms of M. c. dumerili and M. frontalis exhibited exceptionally high alkaline phosphomonoesterase activity while two of the M. f. fulvius venom samples tested exhibited strong hemorrhagic activity in mice. 2. The polyacrylamide gel electrophoretic patterns of the venoms indicate that most of the Micrurus venom proteins are basic proteins. All Micrurus venoms tested exhibited similar SDS-polyacrylamide gel electrophoretic patterns, with an intense low mol. wt protein band. 3. The Micrurus venoms appear to exhibit biological properties similar to other elapid venoms found in Asia and Africa. There are, however, no common characteristics in the biological properties of the venoms examined at the generic level.
1. The biological properties of four venom pooled samples from adult taipan (Oxyuranus scutellatus) snakes and one pooled venom sample from six juvenile taipan snakes (11 months old) were compared. 2. The intravenous LD50 (median lethal dose), procoagulant activity and enzymatic activities of the juvenile venom were not significantly different from those of the adult venoms. 3. The juvenile and adult venoms exhibited similar polyacrylamide gel electrophoretic (PAGE) and SDS-PAGE patterns, indicating that they possessed a similar protein composition. 4. The results suggest that there is no significant age-dependency in the biological properties of taipan venom.
The biological properties of adult and juvenile inland taipan (Oxyuranus microlepidotus) snake venoms were examined. The enzymatic activities, intravenous median lethal dose and procoagulant activity of the juvenile venom samples were not significantly different from those of the adult venom samples. Also, the juvenile and adult venoms exhibited similar electrophoretic patterns, indicating that they possessed similar protein composition.
Immunization with Bungarus candidus venom was performed in four rabbits at high dose (initial dose, 75 microg/kg) and low dose (initial dose, 50 microg/kg). Each dose group consisted of two rabbits; one rabbit received the venom subcutaneously (s.c.) and the other intradermally (i.d.). The venom was injected as emulsified solutions with the same volume of Freund's complete adjuvant until the 4th immunization, thereafter as plain solutions. By stepwise increments of the immunizing dose, the higher dose group received a dose of 200 microg/kg and the lower dose group 150 microg/kg after the 5th immunization, respectively. Thereafter, seven additional immunizations were performed within six months. All rabbits were sacrificed two weeks after the last immunization (12th). Antilethal activity of the immunized antisera thus obtained was determined not only with the homologous venom but also with two heterologous venoms from Bungarus fasciatus and Bungarus flaviceps. Immunodiffusion analysis was also performed with these venoms. The results obtained in this pilot trial provided useful information for production of Malayan krait antivenom at Queen Saovabha Memorial Institute.
We examined the neurotoxicity of the following sea snake venoms: Enhydrina schistosa (geographical variants from Weipa and Malaysia), Lapemis curtus (Weipa and Malaysia), Laticauda colubrina, Aipysurus laevis, Aipysurus fuscus and Aipysurus foliosquamatus. Venom from a terrestrial snake, Notechis scutatus (tiger snake), was used as a reference. All venoms (1 and 3 microg/ml) abolished indirect twitches of the chick biventer cervicis muscle and significantly inhibited responses to ACh (1 mM) and CCh (20 microM), but not KCl (40 mM), indicating the presence of post-synaptic toxins. Prior administration (10 min) of CSL sea snake antivenom (1 unit/ml) attenuated the twitch blockade produced by N. scutatus venom and all sea snake venoms (1 microg/ml). Prior administration (10 min) of CSL tiger snake antivenom (1 unit/ml) attenuated the twitch blockade of all venoms except those produced by E. schistosa (Malaysia and Weipa) and A. foliosquamatus. Administration of CSL sea snake antivenom (1 unit/ml) at t90 (i.e. time at which 90% inhibition of initial twitch height occurred) reversed the inhibition of twitches (20-50%) produced by the sea snake venoms (1 microg/ml) but not by N. scutatus venom (1 microg/ml). CSL tiger snake antivenom (1 unit/ml) administered at t90 produced only minor reversal (i.e. 15-25%) of the twitch blockade caused by L. curtus (Weipa), A. foliosquamatus, L. colubrina and A. laevis venoms (1 microg/ml). Differences in the rate of reversal of the neurotoxicity produced by the two geographical variants of E. schistosa venom, after addition of CSL sea snake antivenom, indicate possible differences in venom components. This study shows that sea snake venoms contain potent post-synaptic activity that, despite the significant genetic distances between the lineages, can be neutralised with CSL sea snake antivenom. However, the effects of CSL tiger snake antivenom are more variable.
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.
Bungarus candidus and Bungarus fasciatus are two species of krait found in Southeast Asia. Envenoming by these snakes is often characterized by neurotoxicity and, without treatment, causes considerable morbidity and mortality. In this study, the in vitro neurotoxicity of each species, and the effectiveness of two monovalent antivenoms and a polyvalent antivenom, against the neurotoxic effects of the venoms, were examined in a skeletal muscle preparation. Both venoms caused concentration-dependent inhibition of indirect twitches, and attenuated responses to exogenous nicotinic receptor agonists, in the chick biventer preparation, with B. candidus venom being more potent than B. fasciatus venom. SDS-PAGE and western blot analysis indicated different profiles between the venoms. Despite these differences, most proteins bands were recognized by all three antivenoms. Antivenom, added prior to the venoms, attenuated the neurotoxic effect of the venoms. Interestingly, the respective monovalent antivenoms did not neutralize the effects of the venom from the other Bungarus species indicating a relative absence of cross-neutralization. Addition of a high concentration of polyvalent antivenom, at the t90 time point after addition of venom, partially reversed the neurotoxicity of B. fasciatus venom but not B. candidus venom. The monovalent antivenoms had no significant effect when added at the t90 time point. This study showed that B. candidus and B. fasciatus venoms display marked in vitro neurotoxicity in the chick biventer preparation and administration of antivenoms at high dose is necessary to prevent or reverse neurotoxicity.
Bungarus fasciatus is one of three species of krait found in Malaysia. Envenoming by B. fasciatus results in neurotoxicity due to the presence of presynaptic and postsynaptic neurotoxins. Antivenom, either monovalent or polyvalent, is the treatment of choice in systemically envenomed patients. In this study, we have isolated a postsynaptic neurotoxin which we named α-elapitoxin-Bf1b. This toxin has an approximate molecular weight of 6.9 kDa, with LCMS/MS data showing that it is highly homologous with Neurotoxin 3FTx-RI, a toxin identified in the Bungarus fasciatus venom gland transcriptome. α-Elapitoxin-Bf1b also shared similarity with short-chain neurotoxins from Laticauda colubrina and Pseudechis australis. α-Elapitoxin-Bf1b produced concentration- and time-dependent neurotoxicity in the indirectly-stimulated chick biventer cervicis muscle preparation, an effect partially reversible by repetitive washing of the preparation. The pA2 value for α-elapitoxin-Bf1b of 9.17 ± 0.64, determined by examining the effects of the toxin on cumulative carbacol concentration-response curves, indicated that the toxin is more potent than tubocurarine and α-bungarotoxin. Pre-incubation of Bungarus fasciatus monovalent and neuro polyvalent antivenom failed to prevent the neurotoxic effects of α-elapitoxin-Bf1b in the chick biventer cervicis muscle preparation. In conclusion, the isolation of a postsynaptic neurotoxin that cannot be neutralized by either monovalent and polyvalent antivenoms may indicate the presence of isoforms of postsynaptic neurotoxins in Malaysian B. fasciatus venom.
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 venom proteome of Hydrophis schistosus (syn: Enhydrina schistosa) captured in Malaysian waters was investigated using reverse-phase HPLC, SDS-PAGE and high-resolution liquid chromatography-tandem mass spectrometry. The findings revealed a minimalist profile with only 18 venom proteins. These proteins belong to 5 toxin families: three-finger toxin (3FTx), phospholipase A2 (PLA2), cysteine-rich secretory protein (CRISP), snake venom metalloprotease (SVMP) and L-amino acid oxidase (LAAO). The 3FTxs (3 short neurotoxins and 4 long neurotoxins) constitute 70.5% of total venom protein, 55.8% being short neurotoxins and 14.7% long neurotoxins. The PLA2 family consists of four basic (21.4%) and three acidic (6.1%) isoforms. The minor proteins include one CRISP (1.3%), two SVMPs (0.5%) and one LAAO (0.2%). This is the first report of the presence of long neurotoxins, CRISP and LAAO in H. schistosus venom. The neurotoxins and the basic PLA2 are highly lethal in mice with an intravenous median lethal dose of <0.2 μg/g. Cross-neutralization by heterologous elapid antivenoms (Naja kaouthia monovalent antivenom and Neuro polyvalent antivenom) was moderate against the long neurotoxin and basic PLA2, but weak against the short neurotoxin, indicating that the latter is the limiting factor to be overcome for improving the antivenom cross-neutralization efficacy.
Geographical variations of snake venoms can result in suboptimal effectiveness of Indian antivenoms that are currently used in most South Asian countries. This study investigated the toxicity and neutralization profile of the venom and toxins from Pakistani spectacled cobra, Naja naja, using VINS polyvalent antivenom (VPAV, India), Naja kaouthia monovalent antivenom (NKMAV, Thailand), and neuro bivalent antivenom (NBAV, Taiwan). Cation-exchange and reverse-phase high-performance liquid chromatography fractionations followed by toxin identification through liquid chromatography-mass spectrometry (MS)/MS indicated that the venom comprised mainly of postsynaptic neurotoxins (NTXs) (long neurotoxins [LNTXs], 28.3%; short neurotoxins [SNTXs], 8%), cytotoxins (CTXs) (31.2%), and acidic phospholipases A2 (12.3%). NKMAV is the most effective in neutralizing the lethal effect of the venom (potency = 1.1 mg venom/mL) and its LNTX (potency = 0.5 mg toxin/mL), consistent with the high content of LNTX in N. kaouthia venom. VPAV was effective in neutralizing the CTX (potency = 0.4 mg toxin/mL), in agreement with the higher CTX abundance in Indian cobra venom. All the three antivenoms were weak in neutralizing the SNTX (potency = 0.03-0.04 mg toxin/mL), including NBAV that was raised from the SNTX-rich Taiwanese cobra venom. In a challenge-rescue experiment, envenomed mice were prevented from death by a maximal dose of VPAV (intravenous 200 μL) but the recovery from paralysis was slow, indicating the need for higher or repeated doses of VPAV. Our results suggest that optimal neutralization for Pakistani N. naja venom may be achieved by improving the formulation of antivenom production to enhance antivenom immunoreactivity against long and SNTXs.
The Southeast Asian monocled cobras (Naja kaouthia) exhibit geographical variations in their venom proteomes, especially on the composition of neurotoxins. This study compared the neuromuscular depressant activity of the venoms of N. kaouthia from Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V), and the neutralization of neurotoxicity by a monospecific antivenom. On chick biventer cervicis nerve-muscle preparation, all venoms abolished the indirect twitches, with NK-T venom being the most potent (shortest t90, time to 90% twitch inhibition), followed by NK-V and NK-M. Acetylcholine and carbachol failed to reverse the blockade, indicating irreversible/pseudo-irreversible post-synaptic neuromuscular blockade. KCl restored the twitches variably (NK-M preparation being the least responsive), consistent with different degree of muscle damage. The findings support that NK-T venom has the most abundant curarimimetic alpha-neurotoxins, while NK-M venom contains more tissue-damaging cytotoxins. Pre-incubation of tissue with N. kaouthia monovalent antivenom (NKMAV) prevented venom-induced twitch depression, with the NK-T preparation needing the largest antivenom dose. NKMAV added after the onset of neuromuscular depression could only halt the inhibitory progression but failed to restore full contraction. The findings highlight the urgency of early antivenom administration to sequester as much circulating neurotoxins as possible, thereby hastening toxin elimination from the circulation. In envenomed mice, NKMAV administered upon the first neurological sign neutralized the neurotoxic effect, with the slowest full recovery noticed in the NK-T group. This is consistent with the high abundance of neurotoxins in the NK-T venom, implying that a larger amount or repeated dosing of NKMAV may be required in NK-T envenomation.
Cardiovascular effects (e.g., tachycardia, hypo- and/or hypertension) are often clinical outcomes of snake envenoming. Malayan krait (Bungarus candidus) envenoming has been reported to cause cardiovascular effects that may be related to abnormalities in parasympathetic activity. However, the exact mechanism for this effect has yet to be determined. In the present study, we investigated thein vivoandin vitrocardiovascular effects ofB. candidusvenoms from Southern (BC-S) and Northeastern (BC-NE) Thailand. SDS-PAGE analysis of venoms showed some differences in the protein profile of the venoms.B. candidusvenoms (50 µg/kg-100 µg/kg, i.v.) caused dose-dependent hypotension in anaesthetised rats. The highest dose caused sudden hypotension (phase I) followed by a return of mean arterial pressure to baseline levels and a decrease in heart rate with transient hypertension (phase II) prior to a small decrease in blood pressure (phase III). Prior administration of monovalent antivenom significantly attenuated the hypotension induced by venoms (100 µg/kg, i.v.). The sudden hypotensive effect of BC-NE venom was abolished by prior administration of hexamethonium (10 mg/kg, i.v.) or atropine (5 mg/kg, i.v.). BC-S and BC-NE venoms (0.1 µg/kg-100 µg/ml) induced concentration-dependent relaxation (EC50= 8 ± 1 and 13 ± 3 µg/mL, respectively) in endothelium-intact aorta. The concentration-response curves were markedly shifted to the right by pre-incubation with L-NAME (0.2 mM), or removal of the endothelium, suggesting that endothelium-derived nitric oxide (NO) is likely to be responsible for venom-induced aortic relaxation. Our data indicate that the cardiovascular effects caused byB. candidusvenoms may be due to a combination of vascular mediators (i.e., NO) and autonomic adaptation via nicotinic and muscarinic acetylcholine receptors.
The wide distribution of king cobra (Ophiophagus hannah), a medically important venomous snake in Asia could be associated with geographical variation in the toxicity and antigenicity of the venom. This study investigated the lethality of king cobra venoms (KCV) from four geographical locales (Malaysia, Thailand, Indonesia, China), and the immunological binding as well as in vivo neutralization activities of three antivenom products (Thai Ophiophagus hannah monovalent antivenom, OHMAV; Indonesian Serum Anti Bisa Ular, SABU; Chinese Naja atra monovalent antivenom, NAMAV) toward the venoms. The Indonesian and Chinese KCV were more lethal (median lethal dose, LD50 ~0.5 μg/g) than those from Malaysia and Thailand (LD50 ~1.0 μg/g). The antivenoms, composed of F(ab)'2, were variably immunoreactive toward the KCV from all locales, with OHMAV exhibited the highest immunological binding activity. In mice, OHMAV neutralized the neurotoxic lethality of Thai KCV most effectively (normalized potency = 118 mg venom neutralized per g antivenom) followed by Malaysian, Indonesian and Chinese KCV. In comparison, the hetero-specific SABU was remarkably less potent by at least 6 to10 folds, whereas NAMAV appeared to be non-effective. The finding supports that a specific king cobra antivenom is needed for the effective treatment of king cobra envenomation in each region.
The genera Ophiophagus and Naja comprise part of a clade of snakes referred to as cobras, dangerously venomous front-fanged snakes in the family Elapidae responsible for significant human mortality and morbidity throughout Asia and Africa. We evaluated venom enzyme variation for eleven cobra species and three N. kaouthia populations using SDS-PAGE venom fingerprinting and numerous enzyme assays. Acetylcholinesterase and PLA2 activities were the most variable between species, and PLA2 activity was significantly different between Malaysian and Thailand N. kaouthia populations. Venom metalloproteinase activity was low and significantly different among most species, but levels were identical for N. kaouthia populations; minor variation in venom L-amino acid oxidase and phosphodiesterase activities were seen between cobra species. Naja siamensis venom lacked the α-fibrinogenolytic activity common to other cobra venoms. In addition, venom from N. siamensis had no detectable metalloproteinase activity and exhibited an SDS-PAGE profile with reduced abundance of higher mass proteins. Venom profiles from spitting cobras (N. siamensis, N. pallida, and N. mossambica) exhibited similar reductions in higher mass proteins, suggesting the evolution of venoms of reduced complexity and decreased enzymatic activity among spitting cobras. Generally, the venom proteomes of cobras show highly abundant three-finger toxin diversity, followed by large quantities of PLA2s. However, PLA2 bands and activity were very reduced for N. haje, N. annulifera and N. nivea. Venom compositionalenzy analysis provides insight into the evolution, diversification and distribution of different venom phenotypes that complements venomic data, and this information is critical for the development of effective antivenoms and snakebite treatment.