Venom Of Australian Tiger Snake Research Articles

Revisiting Notechis scutatus venom: on shotgun proteomics and neutralization by the “bivalent” Sea Snake Antivenom

Recent advances in proteomics enable deep profiling of the compositional details of snake venoms for improved understanding on envenomation pathophysiology and immunological neutralization. In this study, the venom of Australian tiger snake (Notechis scutatus) was trypsin-digested in solution and subjected to nano-ESI-LCMS/MS. Applying a relative quantitative proteomic approach, the findings revealed a proteome comprising 42 toxin subtypes clustered into 12 protein families. Phospholipases A2 constitute the most abundant toxins (74.5% of total venom proteins) followed by Kunitz serine protease inhibitors (6.9%), snake venom serine proteases (5.9%), alpha-neurotoxins (5.6%) and several toxins of lower abundance. The proteome correlates with N. scutatus envenoming effects including pre-synaptic and post-synaptic neurotoxicity and consumptive coagulopathy. The venom is highly lethal in mice (intravenous median lethal dose=0.09μg/g). BioCSL Sea Snake Antivenom, raised against the venoms of beaked sea snake (Hydrophis schistosus) and N. scutatus (added for enhanced immunogenicity), neutralized the lethal effect of N. scutatus venom (potency=2.95mg/ml) much more effectively than the targeted H.schistosus venom (potency=0.48mg/ml). The combined venom immunogen may have improved the neutralization against phospholipases A2 which are abundant in both venoms, but not short-neurotoxins which are predominant only in H. schistosus venom. SignificanceA shotgun proteomic approach adopted in this study revealed the compositional details of the venom of common tiger snake from Australia, Notechis scutatus. The proteomic findings provided additional information on the relative abundances of toxins and the detection of proteins of minor expression unreported previously. The potent lethal effect of the venom was neutralized by bioCSL Sea Snake Antivenom, an anticipated finding due to the fact that the Sea Snake Antivenom is actually bivalent in nature, being raised against a mix of venoms of the beaked sea snake (Hydrophis schistosus) and N. scutatus. However, it is surprising to note that bioCSL Sea Snake Antivenom neutralized N. scutatus venom much more effectively compared to the targeted sea snake venom by a marked difference in potency of approximately 6-fold. This phenomenon may be explained by the main difference in the proteomes of the two venoms, where H. schistosus venom is dominated by short-neurotoxins in high abundance – this is a poorly immunogenic toxin group that has been increasingly recognized in the venoms of a few cobras. Further investigations should be directed toward strategies to improve the neutralization of short-neurotoxins, in line with the envisioned production of an effective pan-regional elapid antivenom.

Histopathological analysis and in situ localisation of Australian tiger snake venom in two clinically envenomed domestic animals

Objective To assess histopathological changes in clinically envenomed tiger snake patients and identify tissue specific localisation of venom toxins using immunohistochemistry. Samples One feline and one canine patient admitted to the Murdoch Pet Emergency Centre (MPEC), Murdoch University with tiger snake ( Notechis sp.) envenoming. Both patients died as a result of envenomation. Non-envenomed tissue was also collected and used for comparison. Methodology Biopsy samples (heart, lung, kidney andskeletal muscle tissue) were retrieved 1–2 h post death and processed for histopathological examination using Haemotoxylin and Eosin, Martius Scarlet Blue and Periodic Acid Schiff staining. Tissues were examined by light microscopy and tissue sections subjected to immunohistochemical staining using in-house generated monoclonal and polyclonal antibodies against Notechis venoms. Results Venom-induced pathological changes were observed in the lungs, kidneys and muscle tissue of both patients. Evidence, not previously noted, of procoagulant venom effects were apparent, with formed thrombi in the heart, lungs (small fibrillar aggregates and larger, discrete thrombi) and kidneys. Immunohistochemical assays revealed venom present in the pulmonary tissue, in and around the glomerular capsule and surrounding tubules in renal tissue and scattered throughout the Gastrocnemius muscle tissue. Conclusion This work has shown pathological evidence of procoagulant venom activity supporting previous suggestions that an initial thrombotic state occurs in envenomed patients. We have shown that venom toxins are able to be localised to specific tissues, in this case, venom was detected in the lung, kidney and muscle tissues of clinically envenomed animals. Future work will examine specific toxin localisation using monoclonal antibodies and identify if antivenom molecules are able to reach their target tissues.

Characterization of the structure and function of three phospholipases A 2 from the venom of Agkistrodon halys pallas

Y.-C. Chen, J. M. Maraganore, I. Reardon and R. L. Heinrikson. Characterization of the structure and function of three phospholipases A 2 from the venom of Agkistrodon halys pallas. Toxicon 25, 401 – 409. 1987.—Three monomeric phospholipases A 2 with isoelectric points 4.5, 6.9 and 9.3 were purified from the venom of Agkistrodon halys pallas. The complete amino acid sequence of the acidic enzyme and partial amino acid sequences of the neutral and basic phospholipases were determined in order to relate differences in enzymatic reactivities, pharmacologic activities and cytotoxicities to aspects of structure. Studies reported here and elsewhere demonstrate that the three phospholipases A 2 exhibit pronounced differences relative to function. The acidic enzyme maintains the highest reactivity toward hydrolysis of monolayers at the air — water interface and may share a feature in common with the acidic enzyme from A. h. blomhoffii, namely the inhibition of platelet aggregation. The neutral phospholipase A 2 designated agkistrotoxin, is characterized by potent activity as a pre-synaptic neurotoxin. Agkistrotoxin is the first single polypeptide chain, neurotoxic phospholipase A 2 to be documented with a Group II disulfide pattern and, in several respects, may be considered functionally and structurally analogous to notexin from the Australian tiger snake venom. Finally, the basic enzyme exhibits the abilities to hemolyze erythrocytes and to hydrolyze phospholipids of E. coli membranes in the presence of a bactericidal-permeability-increasing protein from neutrophil sources.

Further observations on the pathological responses of rat skeletal muscle to toxins isolated from the venom of the Australian tiger snake, Notechis scutatus scutatus.

1. Some aspects of the response of mammalian skeletal muscle following the injection of purified toxins from the venom of the Australian tiger snake, Notechis scutatus scutatus, are described. 2. The toxins used were notexin, notechis II-5, notechis II-1 and a modified form of notexin (PBP-notexin). They were injected into the dorso-lateral aspect of one himd limb so that the soleus muscle would be exposed to the toxins. 3. Within 1 h after the injection of notexin, the soleus muscles were oedematous and by 3--6 h, polymorphonuclear leucocytes had entered the interstitial spaces. The invasion of necrotic muscle fibers was extensive by this time. Muscle spindles appeared relatively unaffected by the toxin. 4. The muscle regenerated via myoblasts at 2--3 days to myotubes at 3--5 days, immature muscle fibers at 7--14 days and fully differentiated muscle fibers by 21--28 days. Even after 6 months, however, the nuclei of many muscle fibres remained in a central position. 5. A second component of Australian tiger snake venom was also found to be myotoxic. It was slightly less potent than notexin, but caused qualitatively similar damage to that caused by notexin. It was identified as notechis II-5. A third fraction, notechis II-1, was found to be inactive. 6. Notexin could be neutralized by incubation with tiger snake antivenene; the simultaneous injection of antivenene with notexin did not afford complete protection against muscle damage.

Amino acid sequences of the two polypeptide chains in beta1-bungarotoxin from the venom of Bungarus multicinctus.

The two dissimilar composite polypeptide chains (A and B) in beta1-bungarotoxin were isolated as their reduced and carboxymethylated derivatives as reported in the preceding paper. The N-terminal sequences were determined with a sequenator up to the 39th residue for the RCM-A chain and up to the 25th residue for the RCM-B chain with repetitive yields of 90-95%. The tryptic and chymotryptic peptides from the two chains were isolated and their structures were determined by manual Edman degradation together with dansyl-Edman and carboxypeptidases A and Y. To complete the primary structures of the two chains, information on the tryptic peptides derived from the maleylated derivatives of the two chains was also used. The completed amino acid sequence of the A chain containing 120 residues (molecular weight, 13,500) is similar to that of notexin, a presynaptic neurotoxin from Australian tiger snake venom, and phospholipases A from other snake venoms. The amino acid sequence of the 60 residues in the B chain (molecular weight, 7,000) bears no resemblance to any basic polypeptides from snake venoms. The B chain probably plays a significant role by interacting with some components in presynaptic membranes of neurosmuscular junctions.

Australian tiger snake venom--an inhibitor of transmitter release.

INVESTIGATION of the function of excitable cells and synapses has been aided to a considerable extent by the use of biotoxins with selective sites of action. For example, neurotoxins from snakes of the elapid family, of which the Australian tiger snake (Notechis scutatus scutatus) is a member, have been used recently to determine the number of acetylcholine receptors on muscle fibres1–4, to isolate cholinergic receptors from electro-plaques of Torpedo marmorata and Electrophorus electricus5,6 and to isolate cholinergic receptors from guinea-pig cerebral cortex7. We have investigated the effects of crude tiger snake venom (TSV) on neuromuscular transmission and have found both pre- and post-synaptic effects.