In vitro toxic effects of puff adder (Bitis arietans) venom, and their neutralization by antivenom.

This study investigated the in vitro toxic effects of Bitis arietans venom and the ability of antivenom produced by the South African Institute of Medical Research (SAIMR) to neutralize these effects. The venom (50 µg/mL) reduced nerve-mediated twitches of the chick biventer muscle to 19% ± 2% of initial magnitude (n = 4) within 2 h. This inhibitory effect of the venom was significantly attenuated by prior incubation of tissues with SAIMR antivenom (0.864 µg/µL; 67% ± 4%; P < 0.05; n = 3-5, unpaired t-test). Addition of antivenom at t50 failed to prevent further inhibition or reverse the inhibition of twitches and responses to agonists. The myotoxic action of the venom (50 µg/mL) was evidenced by a decrease in direct twitches (30% ± 6% of the initial twitch magnitude) and increase in baseline tension (by 0.7 ± 0.3 g within 3 h) of the chick biventer. Antivenom failed to block these effects. Antivenom however prevented the venom induced cytotoxic effects on L6 skeletal muscle cells. Venom induced a marginal but significant reduction in plasma clotting times at concentrations above 7.8 µg/100 µL of plasma, indicating poor procoagulant effects. In addition, the results of western immunoblotting indicate strong immunoreactivity with venom proteins, thus warranting further detailed studies on the neutralization of the effects of individual venom toxins by antivenom.

Cross-neutralisation of the neurotoxic effects of Egyptian cobra venom with commercial tiger snake antivenom.

Cross-neutralisation has been demonstrated for haemorrhagic venoms including Echis spp. and Cerastes spp. and for Australia elapid procoagulant toxins. A previous study showed that commercial tiger snake antivenom (TSAV) was able to neutralise the systemic effects of the Egyptian cobra, Naja haje, in vivo but it is unclear if this was true cross-neutralisation. The aim of the current study was to determine whether TSAV can neutralise the in vitro neurotoxic effects of N. haje venom. Both Notechis scutatus (10 µg/ml) and N. haje (10 µg/ml) venoms caused inhibition of indirect (supramaximal V, 0.1 Hz, 0.2 msec.) twitches of the chick biventer cervicis nerve-muscle preparation with t(90) values (i.e. the time to produce 90% inhibition of the original twitch height) of 26 ± 1 min. (n = 4) and 36 ± 4 min.; (n = 4). This effect at 10 µg/ml was significantly attenuated by the prior addition of TSAV (5 U/ml). A comparison of the reverse-phase HPLC profiles of both venoms showed some similarities with peak elution times, and SDS-PAGE analysis elucidated comparable bands across both venoms. Further analysis using Western immunoblotting indicated TSAV was able to detect N. haje venom, and enzyme immunoassay showed that in-house biotinylated polyclonal monovalent N. scutatus antibodies were able to detect N. haje venom. These findings demonstrate cross-neutralisation between different and geographically separated snakes supporting potential immunological similarities in snake toxin groups for a large range of snakes. This provides more evidence that antivenoms could be developed against specific toxin groups to cover a large range of snakes.

α-Elapitoxin-Aa2a, a long-chain snake α-neurotoxin with potent actions on muscle (α1)(2)ßγδ nicotinic receptors, lacks the classical high affinity for neuronal α7 nicotinic receptors.

In contrast to all classical long-chain α-neurotoxins possessing the critical fifth disulfide bond, α-elapitoxin-Aa2a (α-EPTX-Aa2a), a novel long-chain α-neurotoxin from the common death adder Acanthophis antarcticus, lacks affinity for neuronal α7-type nicotinic acetylcholine receptors (nAChRs). α-EPTX-Aa2a (8850Da; 0.1-1µM) caused a concentration-dependent inhibition of indirect twitches, and blocked contractures to cholinergic agonists in the isolated chick biventer cervicis nerve-muscle preparation, consistent with a postsynaptic curaremimetic mode of action. α-EPTX-Aa2a (1-10nM) produced a potent pseudo-irreversible antagonism of chick muscle nAChRs, with an estimated pA(2) value of 8.311±0.031, which was not reversed by monovalent death adder antivenom. This is only 2.5-fold less potent than the prototypical long-chain α-neurotoxin, α-bungarotoxin. In contrast, α-EPTX-Aa2a produced complete, but weak, inhibition of (125)I-α-bungarotoxin binding to rat hippocampal α7 nAChRs (pK(I)=3.670), despite high sequence homology and similar mass to a wide range of long-chain α-neurotoxins. The mostly likely cause for the loss of α7 binding affinity is a leucine substitution, in loop II of α-EPTX-Aa2a, for the highly conserved Arg(33) in long-chain α-neurotoxins. Arg(33) has been shown to be critical for both neuronal and muscle activity. Despite this substitution, α-EPTX-Aa2a retains high affinity for muscle (α1)(2)ßγδ nAChRs. This is probably as a result of an Arg(29) residue, previously shown to be critical for muscle (α1)(2)ßγδ nAChR affinity, and highly conserved across all short-chain, but not long-chain, α-neurotoxins. α-EPTX-Aa2a therefore represents a novel atypical long-chain α-neurotoxin that includes a fifth disulfide but exhibits differential affinity for nAChR subtypes.

Variations in the pharmacological profile of post-synaptic neurotoxins isolated from the venoms of the Papuan (Oxyuranus scutellatus canni) and coastal (Oxyuranus scutellatus scutellatus) taipans.

Based on murine LD(50) values, the taipans (i.e. Oxyuranus microlepidotus, Oxyuranus scutellatus and Oxyuranus scutellatus canni) are the most venomous snake genus in the world. Despite this, little is known about the toxins contained in their venoms. The aim of the present study was to isolate and characterise post-synaptic neurotoxins from the venoms of the Papuan taipan (O. s. canni) and coastal taipan (O. scutellatus), and to compare their pharmacology. A 6770Da toxin (i.e. alpha-oxytoxin 1) and a 6781Da toxin (i.e. alpha-scutoxin 1) were isolated from the venoms of O. s. canni and O. scutellatus, respectively, using reverse-phase high performance liquid chromatography. Both alpha-oxytoxin 1 (0.3-1 microM) and alpha-scutoxin 1 (0.1-1 microM) caused concentration-dependent inhibition of indirect twitches in the chick biventer cervicis nerve-muscle preparation. Contractile responses to exogenous carbachol (CCh), but not potassium chloride (KCl), were inhibited by both toxins, suggesting a post-synaptic mode of action. The inhibitory effect of alpha-oxytoxin 1 was reversed by washing. Cumulative concentration-response curves to CCh were obtained in the presence and absence of the toxins with the subsequently determined pA(2) of alpha-scutoxin 1 being 44.7-fold higher than alpha-oxytoxin 1 (i.e. 8.38+/-0.59 versus 7.62+/-0.04). The current study shows that Papuan taipan and coastal taipan venom both contain potent post-synaptic neurotoxins which exhibit different pharmacological profiles. The effect of alpha-oxytoxin 1 is atypical of most snake venom post-synaptic neurotoxins displaying a 'competitive' mode of action, whereas alpha-scutoxin 1 possesses pseudo-irreversible or non-competitive activity.

An examination of the activity of expired and mistreated commercial Australian antivenoms.

Expired antivenoms may be useful in countries where snake envenoming is common and supplies are limited. This study examined the activity of expired Australasian antivenoms. Expired CSL snake antivenoms, including taipan, brown snake and polyvalent antivenoms, were used. The most current antivenom was used as the reference to compare expired antivenoms. Binding activity was assessed by enzyme immunoassay. Neutralisation of venom clotting effects was assessed by a modified clotting test using changes in optical density. Neutralisation of the in vitro neurotoxic effects of taipan venom was determined using a chick biventer cervicis nerve-muscle preparation. All antivenom batches remained active, with gradual deterioration in activity and binding over time. All batches of taipan antivenom at concentrations equivalent to the administration of one vial (including one 15 years expired) prevented clotting by taipan venom. Brown snake antivenoms also prevented clotting, except two that were 10 years old. All expired taipan/polyvalent antivenom prevented in vitro neurotoxicity at concentrations consistent with antivenom treatment. Freeze-thawing the antivenom or leaving it at room temperature for 3 days caused only small decreases in activity. CSL antivenoms are more robust than indicated on their label and maintain useful activity long past their nominated expiry dates.