Comparison of the in vitro neuromuscular activity of venom from three Australian snakes (Hoplocephalus stephensi,Austrelaps superbus and Notechis scutatus): efficacy of tiger snake antivenom

1. Tiger snake antivenom, raised against Notechis scutatus venom, is indicated not only for the treatment of envenomation by this snake, but also that of the copperhead (Austrelaps superbus) and Stephen's banded snake (Hoplocephalus stephensi). The present study compared the neuromuscular pharmacology of venom from these snakes and the in vitro efficacy of tiger snake antivenom. 2. In chick biventer cervicis muscle and mouse phrenic nerve diaphragm preparations, all venoms (3-10 microg/mL) produced inhibition of indirect twitches. In the biventer muscle, venoms (10 micro g/mL) inhibited responses to acetylcholine (1 mmol/L) and carbachol (20 micromol/L), but not KCl (40 mmol/L). The prior (10 min) administration of 1 unit/mL antivenom markedly attenuated the neurotoxic effects of A. superbus and N. scutatus venoms (10 microg/mL), but was less effective against H. stephensi venom (10 microg/mL); 5 units/mL antivenom attenuated the neurotoxic activity of all venoms. 3. Administration of 5 units/mL antivenom at t90 partially reversed, over a period of 3 h, the inhibition of twitches produced by N. scutatus (10 microg/mL; 41% recovery), A. superbus (10 microg/mL; 25% recovery) and H. stephensi (10 microg/mL; 50% recovery) venoms. All venoms (10-100 microg/mL) also displayed signs of in vitro myotoxicity. 4. The results of the present study indicate that all three venoms contain neurotoxic activity that is effectively attenuated by tiger snake antivenom.     

The in vitro neuromuscular activity of Indo-Pacific sea-snake venoms: efficacy of two commercially available antivenoms.

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.     

Neurotoxic effects of venoms from seven species of Australasian black snakes (Pseudechis): efficacy of black and tiger snake antivenoms

1. Pseudechis species (black snakes) are among the most widespread venomous snakes in Australia. Despite this, very little is known about the potency of their venoms or the efficacy of the antivenoms used to treat systemic envenomation by these snakes. The present study investigated the in vitro neurotoxicity of venoms from seven Australasian Pseudechis species and determined the efficacy of black and tiger snake antivenoms against this activity. 2. All venoms (10 microg/mL) significantly inhibited indirect twitches of the chick biventer cervicis nerve-muscle preparation and responses to exogenous acetylcholine (ACh; 1 mmol/L), but not to KCl (40 mmol/L), indicating activity at post-synaptic nicotinic receptors on the skeletal muscle. 3. Prior administration of either black or tiger snake antivenom (5 U/mL) prevented the inhibitory effects of all Pseudechis venoms. 4. Black snake antivenom (5 U/mL) added at t90 (i.e. the time-point at which the original twitch height was reduced by 90%) significantly reversed the effects of P. butleri (28+/-5%), P. guttatus (25+/-8%) and P. porphyriacus (28+/-10%) venoms. Tiger snake antivenom (5 U/mL) added at the t90 time-point significantly reversed the neurotoxic effects of P. guttatus (51+/-4%), P. papuanus (47+/-5%) and P. porphyriacus (20+/-7%) venoms. 5. We show, for the first time, the presence of neurotoxins in the venom of these related snake species and that this activity is differentially affected by either black snake or tiger snake antivenoms.     

Species-dependent variations in the in vitro myotoxicity of death adder (Acanthophis) venoms.

Based on early studies on Acanthophis antarcticus (common death adder) venom, it has long been thought that death adder snake venoms are devoid of myotoxicity. However, a recent clinical study reported rhabdomyolysis in patients following death adder envenomations, in Papua New Guinea, by a species thought to be different to A. antarcticus. Subsequently, a myotoxic phospholipase A2 component was isolated from A. rugosus (Irian Jayan death adder) venom. The present study examined the venoms of A. praelongus (northern), A. pyrrhus (desert), A. hawkei (Barkly Tableland), A. wellsi (black head), A. rugosus, A. sp. Seram and the regional variants of A. antarcticus for in vitro myotoxicity. Venoms (10-50 microg/ml) were examined for myotoxicity using the chick directly (0.1 Hz, 2 ms, supramaximal V) stimulated biventer cervicis nerve-muscle preparation. A significant contracture of skeletal muscle and/or inhibition of direct twitches were considered signs of myotoxicity. This was confirmed by histological examination. All venoms displayed high phospholipase A2 activity. The venoms (10-50 microg/ml) of A. sp. Seram, A. praelongus, A. rugosus,and A. wellsi caused a significant inhibition of direct twitches and an increase in baseline tension compared to the vehicle (n=4-6; two-way ANOVA, p<0.05). Furthermore, these venoms caused dose-dependent morphological changes in skeletal muscle. In contrast, the venoms (10-50 microg/ml; n=3-6) of A. hawkei, A. pyrrhus, and regional variants of A. antarcticus were devoid of myotoxicity. Prior incubation (10 min) of CSL death adder antivenom (5 U/ml) prevented the myotoxicity caused by A. sp. Seram, A. praelongus, A. rugosus, and A. wellsi venoms (50 microg/ml; n=4-7). In conclusion, clinicians may need to be mindful of possible myotoxicity following envenomations by A. praelongus, A. rugosus, A. sp. Seram, and A. wellsi species.     

Characterisation of the biochemical and biological variations from the venom of the death adder species (Acanthophis antarcticus, A. praelongus and A. pyrrhus)

We report on species variation in the venoms of the three species of death adder; the Common death adder (Acanthophis antarcticus), the Northern death adder (Acanthophis praelongus) and the Desert death adder (Acanthophis pyrrhus). The venoms were found to vary in their biochemical (chromatography) and biological (PLA₂ activity, anticoagulant activity and reactivity with commercial death adder antivenom) properties. Each species produced significant differences in the profile and distribution of PLA₂ activity, when whole venom was applied to a cation-exchange Mono-S column. PLA₂ enzymes were purified from each venom and termed acanthoxin B (from A. praelongus), acanthoxin C (from A. pyrrhus) and the previously characterised acanthoxin A (from A. antarcticus). Acanthoxin B and C showed lower enzymatic activities than acanthoxin A (4.0, 13.7 and 23.9 μmol of phospholipid hydrolyzed/min/mg protein, respectively). N-terminal sequencing revealed acanthoxin B to share highest homology with the numerous PLA₂ isozymes (Pa-12C, Pa-1G, Pa-12A) from the King brown snake (Pseudechis australis) and Acanthin I from the Common death adder. Similar to acanthoxin A, acanthoxin C showed highest homology with Acanthin I/II, and pseudexin A-chain from the Red-bellied black snake (Pseudechis porphyriacus). Whole venom from A. antarcticus, A. praelongus and A. pyrrhus each showed weak anticoagulant activity (being able to prolong coagulation of the plasma for 107, 220 and 195 s, respectively). By immunodiffusion, each venom produced precipitation bands against commercial death adder antivenom.     

In Vitro Neutralization of the Myotoxicity of Australian Mulga Snake (Pseudechis australis) and Sri Lankan Russell’s Viper (Daboia russelii) Venoms by Australian and Indian Polyvalent Antivenoms

We studied the neutralisation of Sri Lankan Russell’s viper (Daboia russelii) and Australian mulga snake (Pseudechis australis) venom-induced myotoxicity by Indian (Vins and Bharat) and Australian (Seqirus) polyvalent antivenoms, using the in vitro chick biventer skeletal muscle preparation. Prior addition of Bharat or Vins antivenoms abolished D. russelii venom (30 µg/mL)-mediated inhibition of direct twitches, while Australian polyvalent antivenom was not protective. Bharat antivenom prevented, while Vins and Australian polyvalent antivenoms partially prevented, the inhibition of responses to exogenous KCl. Myotoxicity of Mulga venom (10 µg/mL) was fully neutralised by the prior addition of Australian polyvalent antivenom, partially neutralised by Vins antivenom but not by Bharat antivenom. Although the myotoxicity of both venoms was partially prevented by homologous antivenoms when added 5 min after the venom, with an increasing time delay between venom and antivenom, the reversal of myotoxicity gradually decreased. However, antivenoms partially prevented myotoxicity even 60 min after venom. The effect of antivenoms on already initiated myotoxicity was comparable to physical removal of the toxins by washing the bath at similar time points, indicating that the action of the antivenoms on myotoxicity is likely to be due to trapping the toxins or steric hindrance within the circulation, not allowing the toxins to reach target sites in muscles.     

Development of a sensitive enzyme immunoassay for measuring taipan venom in serum

The detection and measurement of snake venom in blood is important for confirming snake identification, determining when sufficient antivenom has been given, detecting recurrence of envenoming, and in forensic investigation. Venom enzyme immunoassays (EIA) have had persistent problems with poor sensitivity and high background absorbance leading to false positive results. This is particularly problematic with Australasian snakes where small amounts of highly potent venom are injected, resulting in low concentrations being associated with severe clinical effects. We aimed to develop a venom EIA with a limit of detection (LoD) sufficient to accurately distinguish mild envenoming from background absorbance at picogram concentrations of venom in blood. Serum samples were obtained from patients with taipan bites (Oxyuranus spp.) before and after antivenom, and from rats given known venom doses. A sandwich EIA was developed using biotinylated rabbit anti-snake venom antibodies for detection. For low venom concentrations (i.e. <1 ng/mL) the assay was done before and after addition of antivenom to the sample (antivenom difference method). The LoD was 0.15 ng/mL for the standard assay and 0.1 ng/mL for the antivenom difference method. In 11 pre-antivenom samples the median venom concentration was 10 ng/mL (Range: 0.3–3212 ng/mL). In four patients with incomplete venom-induced consumption coagulopathy the median venom concentration was 2.4 ng/mL compared to 30 ng/mL in seven patients with complete venom-induced consumption coagulopathy. No venom was detected in any post-antivenom sample and the median antivenom dose prior to this first post-antivenom sample was 1.5 vials (1–3 vials), including 7 patients administered only 1 vial. In rats the assay distinguished a 3-fold difference in venom dose administered and there was small inter-individual variability. There was small but measurable cross-reactivity with black snake (Pseudechis), tiger snake (Notechis) and rough-scale snake (Tropidechis carinatus) venoms with the assay for low venom concentrations (<1 ng/mL). The use of biotinylation and the antivenom difference method in venom EIA produces a highly sensitive assay that will be useful for determining antivenom dose, forensic and clinical diagnosis.     

The in vitro neurotoxic and myotoxic effects of the venom from the Suta genus (curl snakes) of elapid snakes

Australia has a tremendous diversity of elapid snakes, including many unique smaller sized species of this venomous snake family. However, little if anything is known about the majority of the venoms of these lesser studied snakes. In the current study, the venoms of Suta suta (curl snake) and Suta punctata (spotted-curl snake) were examined for in vitro activity using a skeletal muscle preparation (i.e. chick biventer cervicis nerve-muscle preparation). Both venoms caused concentration-dependent (3-10 microg/ml) inhibition of nerve-mediated twitches, and inhibited responses to exogenous acetylcholine and carbachol, indicating the presence of postsynaptic neurotoxins. These effects were prevented by prior addition of CSL Ltd. polyvalent snake antivenom (5 units/ml) but only partially reversed by the addition of antivenom (5 units/ml) at the t(90) time-point (i.e. time at which twitches were inhibited by 90%). Suta punctata venom (10 microg/ml) was also myotoxic as indicated by the inhibition of direct twitches of the chick biventer cervicis nerve-muscle preparation. This effect was not reversed by antivenom (5 units/ml). This study highlights the danger of underestimating the potential severe clinical effects posed by these small but highly venomous snakes.     

Cross-neutralisation of Australian brown and tiger snake venoms with commercial antivenoms: Cross-reactivity or antivenom mixtures?

Recently it has been suggested that the Australian snake antivenoms made by CSL Ltd. are in fact not truly monovalent and may contain antibodies to other snake venoms because the horses are injected with multiple snake venoms. It is unclear to what extent various monovalent antivenoms can neutralise the effect of other venoms, whether this is due to a mixture of antibodies or true cross-reactivity, and whether this has any clinical significance. We aimed to study the immunological and functional properties of brown snake (Pseudonaja spp.) antivenom (BSAV) and tiger snake (Notechis spp.) antivenom (TSAV) against their respective venoms using enzyme immunoassays (EIA) and in vitro clotting studies. There was significant overlap between the two antivenoms with both TSAV and BSAV being detected by EIA on brown snake venom (BSV)-coated and tiger snake venom (TSV)-coated wells, respectively. In a competition EIA, increasing amounts of immunoaffinity-purified hen anti-brown antibodies (IgYp) mixed with TSAV reduced TSAV measured on TSV-coated wells. Both BSAV and TSAV prevented the clotting activity of both venoms. IgYp also prevented the clotting activity of TSV, suggesting true cross-reactivity. The cross-reactivity of TSAV and BSAV with BSV and TSV, respectively, was likely due to each being a mixture of anti-brown and anti-tiger antibodies, but there was partial cross-reactivity demonstrated by the effect of IgYp. Single-polyvalent antivenom for brown snake and tiger snake may be feasible in the future.     

The in vitro effects of two chirodropid (Chironex fleckeri and Chiropsalmus sp.) venoms: efficacy of box jellyfish antivenom.

The pharmacological and biochemical isolation of cnidarian venoms has been hindered by difficulties with both extracting pure venom from nematocysts and venom stability. The development of a new technique to extract active, pure venom of Chironex fleckeri and Chiropsalmus sp. has enabled identify both neurotoxic and myotoxic activity in their venoms. These activities are similar, but not identical in each species. Venom (50 micro g/ml) from both species significantly inhibited indirect and direct twitches of the chick biventer nerve-muscle preparation. Pre-incubation with 1U/ml box jellyfish antivenom did not have any significant effect on venom-induced reductions of indirect twitches. However, this activity was markedly attenuated by prior addition of 5U/ml antivenom, albeit to a lesser degree for Chiropsalmus sp. In contrast, prior addition of 5U/ml box jellyfish antivenom did not neutralise the myotoxic activity of C. fleckeri venom (50 micro g/ml), although it did inhibit the myotoxicity produced by Chiropsalmus sp. venom (50 micro g/ml). Antivenom (5U/ml) added 1h after the addition of C. fleckeri venom (50 micro g/ml) had no effect on the indirect or direct twitches of the skeletal muscle preparation. However, it partially restored the reduction in indirect twitch height caused by Chiropsalmus sp. venom (50 micro g/ml). Myotoxicity was confirmed in muscle preparations stained with hematoxylin and eosin.Therefore, although antivenom was able to neutralize the neurotoxic effects of both species, and the myotoxic effects of Chiropsalmus sp., when added prior to venom, it was unable to reverse the effects after venom addition. This suggests that antivenom is unlikely to be useful in the treatment of neurotoxic or myotoxic effects in patients, although these effects are rarely seen clinically.     

In Vitro Neurotoxicity of Chinese Krait (Bungarus multicinctus) Venom and Neutralization by Antivenoms

Bungarus multicinctus, the Chinese krait, is a highly venomous elapid snake which causes considerable morbidity and mortality in southern China. B. multicinctus venom contains pre-synaptic PLA₂ neurotoxins (i.e., β-bungarotoxins) and post-synaptic neurotoxins (i.e., α-bungarotoxins). We examined the in vitro neurotoxicity of B. multicinctus venom, and the efficacy of specific monovalent Chinese B. multicinctus antivenom, and Australian polyvalent elapid snake antivenom, against venom-induced neurotoxicity. B. multicinctus venom (1–10 μg/mL) abolished indirect twitches in the chick biventer cervicis nerve-muscle preparation as well as attenuating contractile responses to exogenous ACh and CCh, but not KCl. This indicates a post-synaptic neurotoxic action but myotoxicity was not evident. Given that post-synaptic α-neurotoxins have a more rapid onset than pre-synaptic neurotoxins, the activity of the latter in the whole venom will be masked. The prior addition of Chinese B. multicinctus antivenom (12 U/mL) or Australian polyvalent snake antivenom (15 U/mL), markedly attenuated the neurotoxic actions of B. multicinctus venom (3 μg/mL) and prevented the inhibition of contractile responses to ACh and CCh. The addition of B. multicinctus antivenom (60 U/mL), or Australian polyvalent snake antivenom (50 U/mL), at the t₉₀ time point after the addition of B. multicinctus venom (3 μg/mL), did not restore the twitch height over 180 min. The earlier addition of B. multicinctus antivenom (60 U/mL), at the t₂₀ or t₅₀ time points, also failed to prevent the neurotoxic effects of the venom but did delay the time to abolish twitches based on a comparison of t₉₀ values. Repeated washing of the preparation with physiological salt solution, commencing at the t₂₀ time point, failed to reverse the neurotoxic effects of venom or delay the time to abolish twitches. This study showed that B. multicinctus venom displays marked in vitro neurotoxicity in a skeletal muscle preparation which is not reversed by antivenom. This does not appear to be related to antivenom efficacy, but due to the irreversible/pseudo-irreversible nature of the neurotoxins.     

In vitro neurotoxic and myotoxic effects of the venom from the black whip snake (Demansia papuensis)

1. Black whip snakes belong to the family elapidae and are found throughout the northern coastal region of Australia. The black whip snake (Demansia papuensis) is considered to be potentially dangerous due to its size and phylogenetic distinctiveness. Previous liquid chromatography-mass spectrometry analysis of D. papuensis venom indicated a number of components within the molecular mass ranges compatible with neurotoxins. For the first time, this study examines the in vitro neurotoxic and myotoxic effects of the venom from D. papuensis. 2. Venom (10 microg/mL) caused significant inhibition of twitches elicited by stimulation (0.2 ms, 0.1 Hz, supramaximal V) of motor nerves in the chick biventer cervicis nerve-muscle preparation. This neurotoxic effect, which was postsynaptic in origin, was weak in comparison to that of most other Australian elapids. Prior addition (10 min) of polyvalent (PSAV) or tiger snake (TSAV) antivenom (5 units/mL) prevented venom-induced twitch inhibition. Addition of PSAV (5 units/mL) at t(50) failed to reverse the inhibitory effect but prevented further inhibition of nerve-mediated twitches. 3. The venom (20-50 microg/mL) is also myotoxic as indicated by a slowly developing contracture and inhibition of twitches elicited by direct stimulation (2 ms, 0.1 Hz, supramaximal V, in the presence of tubocurarine 10 micromol/L) of the chick biventer muscle. This activity was confirmed by histological examination of the muscle. 4. Fractionation and characterization of venom components is required to further investigate the reasons for the weak neurotoxic activity of D. papuensis venom.     

The molecular basis of cross-reactivity in the Australian Snake Venom Detection Kit (SVDK)

The Snake Venom Detection Kit (SVDK) is of major medical importance in Australia, yet it has never been rigorously characterised in terms of its sensitivity and specificity, especially when it comes to reports of false-negative and false-positive results. This study investigates reactions and cross-reactions of five venoms the SVDK is directed against and a number of purified toxins. Snakes showing the closest evolutionary relationships demonstrated the lowest level of cross-reactivity between groups. This was, instead, far more evident between snakes that are extraordinarily evolutionary separated. These snakes: Pseudechis australis, Acanthophis antarcticus and Notechis scutatus, in fact displayed more false-positive results. Examination of individual toxin groups showed that phospholipase A₂s (PLA₂s) tends to react strongly and display considerable cross-reactivity across groups while the three-finger toxins (3FTx) reacted poorly in all but the Acanthophis well. The hook effect was evident for all venoms, particularly Oxyuranus scutellatus. The results of this study show considerable variation in toxin detection, with implications in further development of venom detection, both in Australia and other countries.     

Preliminary characteristics of the prothrombin converting enzyme from venom of Stephen''s banded snake (Hoplocephalus stephensii)

Stephen's banded snake (Hoplocephalus stephensii) is an infrequently encountered Australian elapid species. The crude venom contains coagulant activity and the component responsible is a prothrombin activator requiring factor V for activity. SDS-PAGE of the isolated native protein revealed two bands at 23 and 36 kDa. These findings indicate that the procoagulant is similar to that found in the Australian tiger snake (Notechis scutatus) and thus resembles factor Xa.     

The effects of antivenom on the in vitro neurotoxicity of venoms from the taipans Oxyuranus scutellatus, Oxyuranus microlepidotus and Oxyuranus scutellatus canni.

The venoms of the inland (Oxyuranus microlepidotus), coastal (O. scutellatus) and Papuan (O. s. canni) taipans are among the most potent in the world. The present study compared the in vitro neurotoxic effects of these venoms and the protective effects of taipan antivenom. Venom (10 μg/ml) from all three snakes abolished nerve-mediated twitches of the chick biventer cervicis muscle preparation with the following rank order of potency (based on the time taken to inhibit 90% of the twitch response; t₉₀): O. microlepidotus (27±3 min)>O. scutellatus (42±3 min)=O. S. canni (48±5 min). This inhibitory effect of all three venoms was primarily postsynaptic in origin as evidenced by the inhibition of responses to exogenous acetylcholine (ACh; 1 mM) and carbachol (CCh; 20 μM), but not potassium chloride (40 mM). In contrast, the presynaptic neurotoxins taipoxin (3 μg/ml) and paradoxin (3 μg/ml) abolished nerve-mediated twitches without producing a significant effect on contractile responses to exogenous agonists. Prior incubation of the tissue with taipan antivenom (1 unit/ml for 10 min) markedly attenuated the inhibitory effects of taipoxin (3 μg/ml) and paradoxin (3 μg/ml), as well as O. scutellatus (10 μg/ml) and O. s. canni (10 μg/ml) venom. However, in the presence of antivenom, O. microlepidotus venom (10 μg/ml) still abolished nerve-mediated twitches and responses to ACh and CCh. The results of the current study indicate that taipan antivenom, raised against O. scutellatus venom, is effective, in vitro, against the neurotoxic effects of venom from the Papuan and coastal taipans, as well as the presynaptic effects of venom from the inland taipan. However, the antivenom appears less effective against the postsynaptic effects of the latter. It is possible that inland taipan venom contains a component not neutralised by the antivenom which may contribute to the extreme potency of this venom.     

Isolation and characterisation of acanmyotoxin-2 and acanmyotoxin-3, myotoxins from the venom of the death adder Acanthophis sp. Seram

Death adder (genus Acanthophis) venoms display neurotoxic activity but were thought to be devoid of myotoxic components. Studies from our laboratory have shown that some species (i.e. Acanthophis rugosus and Acanthophis sp. Seram) posses venom with myotoxic activity [Wickramaratna JC, Fry BG, Aguilar M, Kini RM, Hodgson WC. Isolation and pharmacological characterisation of a phospholipase A₂ myotoxin from the venom of the Irian Jayan death adder (A. rugosus). Br J Pharmacol 2003;138:333–342; Wickramaratna JC, Fry BG, Hodgson WC. Species-dependent variations in the in vitro myotoxicity of death adder (Acanthophis) venoms. Toxicol Sci 2003;74:352–360]. The present study describes the isolation and characterisation of two myotoxins (acanmyotoxin-2 and acanmyotoxin-3) from A. sp. Seram venom. Venom was fractionated into approximately 12 major peaks using reverse phase high performance liquid chromatography. Two components caused concentration (0.1–1 μM) dependent inhibition of direct (2 ms, 0.1 Hz, supramaximal V) twitches and an increase in baseline tension in the chick biventer cervicis nerve-muscle. Histological examination of the muscle confirmed damage. PLA₂ activity was detected in both acanmyotoxin-2 (390.2 ± 19.7 μmol/(min mg); n = 4) and acanmyotoxin-3 (14.2 ± 7.7 μmol/(min mg); n = 4). In comparison, A. sp. Seram whole venom had a specific activity of 461.3 ± 90.4 μmol/(min mg) (n = 3). Mass spectrometry analysis indicated acanmyotoxin-2 had a mass of 13,082 Da and acanmyotoxin-2 13,896 Da. Acanmyotoxin-2 and acanmyotoxin-3 accounted for approximately 7 and 4% of total venom composition, respectively. N-terminal sequencing of the first 30 amino acids of each toxin indicated they shared some sequence homology with known myotoxins. In conclusion, clinicians should be aware that symptoms of envenoming by some species of death adder may include signs of myotoxicity as well as neurotoxicity. Future studies will investigate the efficacy of the current antivenom treatment against the myotoxic components of A. sp. Seram venom.     

Exploring the Utility of Recombinant Snake Venom Serine Protease Toxins as Immunogens for Generating Experimental Snakebite Antivenoms

Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies in order to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms, were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed that they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.     

Coagulant effects of black snake (Pseudechis spp.) venoms and in vitro efficacy of commercial antivenom

The coagulant effects of Australasian black snakes (Pseudechis spp.) are poorly understood and differ to the procoagulant venoms of most dangerous snakes in Australia. This study aimed to investigate in vitro coagulant effects of Pseudechis venoms and the efficacy of commercial black snake antivenom (BlSAV), tiger snake antivenom (TSAV) and specific rabbit anti-snake IgG to neutralise these effects. Using a turbidimetric assay, all six Pseudechis venoms had anticoagulant activity, as well as phospholipase A₂ (PLA₂) activity. Inhibition of PLA₂ activity removed anticoagulant effects of the venoms. Pseudechis porphyriacus was unique and had procoagulant activity independent of PLA2 activity. Both BlSAV and TSAV completely inhibited the coagulant and PLA2 activity of all Pseudechis venoms. PLA2 activity was also inhibited completely by p-Bromophenacyl bromide (pBPB) and partially by specific anti-N. scutatus IgG antibodies. Anti-N. scutatus IgG also completely inhibited anticoagulant activity of Pseudechis venom. All Pseudechis venoms showed immunological cross reactivity with specific anti-snake IgG antibodies to P. porphyriacus, Pseudechis australis and Notechis scutatus. Pseudechis venoms have in vitro anticoagulant activity that appears to be attributable to PLA₂ activity. Both antivenoms inhibited anticoagulant and PLA₂ activity at concentrations below those occurring in patients treated with one vial of antivenom. There was cross-neutralisation of Pseudechis venoms and N. scutatus antibodies that might be attributable to immunological similarities between the venoms.     

Isolation and pharmacological characterisation of papuantoxin-1, a postsynaptic neurotoxin from the venom of the Papuan black snake (Pseudechis papuanus)

The Papuan black snake (Pseudechis papuanus) is found throughout the southern coastal regions of Papua New Guinea and is thought to occur in the adjacent region of Iriyan Jaya. Neurotoxicity is a major symptom of envenomation by this species. This study describes the isolation of the first neurotoxin papuantoxin-1 from the venom of P. papuanus. Papuantoxin-1 (6738Da), which accounts for approximately 5% of the whole venom, was purified to homogeneity using successive steps of RP-HPLC. The toxin (0.3-1.0 microM) caused concentration dependent inhibition of indirect twitches (0.1 Hz, 0.2 ms and supramaximal V) and inhibited the responses to nicotinic agonists in the chick biventer cervicis nerve-muscle preparation, indicating a postsynaptic mode of action. However, papuantoxin-1 displayed no signs of myotoxicity. Papuantoxin-1 displayed pseudo-irreversible antagonism of cumulative concentration-response curves to carbachol at the skeletal muscle nicotinic receptors with an estimated pA2 value of 6.9+/-0.3. CSL black snake antivenom, which is raised against the venom of the Australian black snake Pseudechis australis, appears to be effective in reversing the effects of papuantoxin-1. Thus, black snake antivenom should be considered for the treatment of the neurotoxic effects following envenomation by the Papaun black snake.