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Cross-neutralization by tiger snake (Notechis scutatus) antivenene and sea snake (Enhydrina schistosa) antivenene against several sea snake venoms
Abstract
In vitro cross-neutralization of venoms of nine species of sea snake was studied, using mice and immunodiffusion patterns as indicators. Tiger snake antivenene was more effective than sea-snake antivenene against all species. Both antivenenes neutralized all venoms tested—Aipysurus laevis, Astrotia stokesii, Enhydrina schistosa, Hydrophis cyanocinctus, H. elegans, H. major, H. spiralis, Lapemis hardwickii, Laticauda semifasciata and Notechis scutatus. Immunodiffusion patterns did not always reflect the in vivo observations. The significance of the results in indicating the choice of an antivenene for treatment of sea snake envenomation is discussed.
... In the absence of this antivenom it has been suggested that CSL tiger snake antivenom be used as a substitute, although at a higher starting dosage (four ampoules) than that recommended for sea snake antivenom itself (Sutherland and Tibballs, 2001). This recommendation appears to have been based largely on experiments where antivenom was administered to mice after venom injection or experiments where venom and antivenom were mixed prior to injection (Baxter and Gallichio, 1974, 1976). These studies indicated that tiger snake antivenom was equivalent, or in some cases more effective, in neutralising sea snake venom, than sea snake antivenom. ...
... However, whilst products from Vietnam, Japan and India were briefly available in the past, the only remaining commercially available sea snake antivenom is that made since the early 1960s by CSL in Melbourne, Australia using E. schistosa venom (Tu and Ganthavorn, 1969; Reid, 1975). Early studies by Baxter and Gallichio (1974, 1976) indicated that tiger snake antivenom was effective in neutralising the toxicity of the venoms of true sea snake as well as of sea kraits. Since these studies were conducted, tiger snake antivenom has been recommended for sea snake envenomation if sea snake antivenom is unavailable (Sutherland and Tibballs, 2001). ...
... This was particularly notable in regards to the venom of the taxonomically divergent Laticauda—as these snakes represent an independent marine lineage (Fig. 1). These data confirm earlier work using Laticauda semifaciata venom (Baxter and Gallichio, 1974, 1976). This cross-reactivity may be due to the remarkable streamlining of the sea snake venoms that occurred independently within these two lineages (Fry et al., 2003a) and the low level of phylogenetic variation of the marine 3FTx (Fry et al., 2003b). ...
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.
... Although antivenoms commercially produced for the treatment of Australasian snake envenoming are labelled as monovalent, there have been increasing reports of cross-neutralisation (Baxter and Gallichio, 1974). CSL Ltd. produces six monovalent antivenoms for the five major terrestrial elapid groups and one antivenom for sea snakes (White, 2001). ...
... It has been previously observed that some Australian monovalent antivenoms were able to neutralise the effects of venoms other than ones they were made to neutralise, notably TSAV neutralising sea snake venom (Baxter and Gallichio, 1974;Minton, 1967) and TSAV neutralising the effects of United States coral snake (Micrurus fulvius fulvius) venom in mice (Wisniewski et al., 2003). In the first instance, it was thought that this was because the horses used to produce the TSAV had also been immunised with sea snake. ...
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.
... It has been suggested that several components of the venom may act in a synergistic manner to potentiate toxic effects (Ryan and Yong, 1997). Finally, antivenoms raised against tiger snake (Notechis scutatus) or common sea snake (Enhydrina schistosa) venoms have been shown to have some cross-reactivity towards the venom of A. laevis, although the efficacies of these antivenoms are lower than against the venoms of homologous species (Baxter and Gallichio, 1974). Aiming to further develop understanding of sea snake venoms and to expand knowledge of venom intra-species variability, this study presents the proteomic analysis of the venom of A. laevis, together with an assessment of variability in three different specimens, and of toxicity of all its main protein components in mice. ...
Four specimens of the olive sea snake, Aipysurus laevis, were collected off the coast of Western Australia, and the venom proteome was characterized and quantitatively estimated by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses. A. laevis venom is remarkably simple and consists of phospholipases A2 (71.2%), three-finger toxins (3FTx; 25.3%), cysteine-rich secretory proteins (CRISP; 2.5%), and traces of a complement control module protein (CCM; 0.2%). Using a Toxicity Score, the most lethal components were determined to be short neurotoxins. Whole venom had an intravenous LD50 of 0.07 mg/kg in mice and showed a high phospholipase A2 activity, but no proteinase activity in vitro. Preclinical assessment of neutralization and ELISA immunoprofiling showed that BioCSL Sea Snake Antivenom was effective in cross-neutralizing A. laevis venom with an ED50 of 821 μg venom per mL antivenom, with a binding preference towards short neurotoxins, due to the high degree of conservation between short neurotoxins from A. laevis and Enhydrina schistosa venom. Our results point towards the possibility of developing recombinant antibodies or synthetic inhibitors against A. laevis venom due to its simplicity.
Copyright © 2015. Published by Elsevier Ltd.
... It has been suggested that several components of the venom may act in a synergistic manner to potentiate toxic effects (Ryan and Yong, 1997). Finally, antivenoms raised against tiger snake (Notechis scutatus) or common sea snake (Enhydrina schistosa) venoms have been shown to have some cross-reactivity towards the venom of A. laevis, although the efficacies of these antivenoms are lower than against the venoms of homologous species (Baxter and Gallichio, 1974). ...
Hemorrhage is one of the most significant effects in envenomings induced by viperid snakebites. Damage to the microvasculature, induced by snake venom metalloproteinases (SVMPs), is the main event responsible for this effect. The precise mechanism by which SVMPs disrupt the microvasculature has remained elusive, although recent developments provide valuable clues to deciphering the details of this pathological effect. The main targets of hemorrhagic SVMPs are components of basement membrane (BM) and surrounding extracellular matrix (ECM), which provide mechanical stability to capillaries. P-III SVMPs, comprising disintegrin-like and cysteine-rich domains in addition to the catalytic domain, are more potent hemorrhagic toxins than P-I SVMPs, constituted only by the metalloproteinase domain. This is likely due to the presence of exosites in the additional domains, which contribute to the binding of SVMPs to relevant targets in the microvasculature. Recent in vivo studies have shown that P-III SVMPs are preferentially located in microvessels. On the other hand, the structural determinants responsible for the different hemorrhagic potential of P-I SVMPs remain largely unknown, although backbone flexibility in a loop located near the active site is likely to play a role. Moreover, hemorrhagic and non-hemorrhagic SVMPs differ in their capacity to hydrolyze in vivo key BM proteins, such as type IV collagen and perlecan, as well as other ECM proteins, like types VI and XV collagens, which play a critical role by connecting BM components to perivascular fibrillar collagens. The evidence gathered support a two-step model for the pathogenesis of SVMP-induced hemorrhage: initially, hemorrhagic SVMPs bind to and hydrolyze components of the BM and associated extracellular matrix proteins that play a key role in the mechanical stability of BM. In conditions of normal blood flow in the tissues, such cleavage results in the weakening, distension and eventual disruption of capillary wall due to the action of biophysical forces operating in vivo.
... If used correctly antivenom can be effective even though not given until hours after the bite (Reid, 1962). Both tiger snake antivenom and sea snake antivenom (Commonwealth Serum Laboratories, Melbourne) are effective (Baxter and Gallichio, 1974). Antivenom (1000-10,000 u) should always be given by intravenous infusion, which is the most effective and the safest route. ...
As a rule, bites and stings in travellers are merely a nuisance. But it is sensible to be informed about the more serious possibilities which can result. Systemic diseases can be transmitted, the skin lesions from insects can be troublesome and finally, some bites and stings can cause envenoming. Thus, the bather may be harmed by venomous fish stings, sea urchins, jellyfish and in Asian-Pacific waters by sea-snakes. Land hazards include bites or stings by scorpions, spiders, ticks, centipedes, bees, wasps, caterpillars and snakes. The main clinical features of such bites and stings, including treatment and prevention, are outlined.
... Reid's Institute provided the beaked sea snake (Enhydrina schistosa) venom and ultimately recorded the first instance of the successful use of the CSL sea snake antivenom (Reid, 1962;Kaire, 1964). Baxter, Marr and Lane (then Director of CSL) later demonstrated the very broad specific of the CSL sea snake antivenom (Baxter and Gallichio, 1974), underpinning its place as the only commercial sea snake antivenom in the world (Chetty et al., 2004). ...
It was not until the last decade of the 19th century that an experimental approach (led by Bancroft in Queensland and Martin in Sydney and Melbourne) brought a higher plane of scientific objectivity to usher in the modern era of Australian toxinology. This Australia era, 1895-1905, coincided with and in some respects was the result of the new knowledge emerging from Europe and the Americas of the therapeutic effects of antitoxins. The subsequent systematic study of Australian venoms and toxins through to the 1930s and beyond, by Tidswell, Fairley, Ross, Kellaway and Cleland, set the foundation for Australia's leading reputation in venom research. As elsewhere, this development was to revolutionise the medical management of those victims who in the past had died in Australia from our venomous and toxic fauna. Morgan, Graydon, Weiner, Lane and Baxter at the Commonwealth Serum Laboratories emphasised the importance of cooperation between those expert at catching and milking the venomous creatures and those developing the antivenoms. Commercial antivenom manufacture began in Australia in 1930 with the tiger snake antivenom. This was followed by other antivenoms for the other important species (1955: taipan; 1956: brown snake; 1958: death adder; 1959: Papuan black snake; 1961: sea snake; 1962: polyvalent) including the first marine antivenoms in the world (1956: stonefish antivenom; 1970: box jellyfish) culminating, in 1980, with the release of the funnel web spider antivenom. More recent activity has focused on veterinary antivenoms and production of new generation human antivenoms for export (CroFab and ViperaTAB). This paper reviews some of the milestones of Australian toxinology, and antivenom development in particular, during the 20th century.
Aquatic vertebrate animals—in particular, fish—present a series of traumatic and/or toxic defenses to survive in their environment. A large number of fish species produce toxins. Some have specialized structures to inoculate these substances, and this feature characterizes them as venomous animals. Fish of commercial value (such as catfish) can be venomous, and encounters with rays and many other fish capable of envenomations are common. These injuries occur mainly in professional fishermen. In addition to fish, there are venomous sea snakes and even venomous mammals, which are also discussed in this chapter. The chapter also addresses treatment measures and ways to try and prevent traumatic injuries and envenomations by fish and reptiles.
Snakebite, recently declared a neglected tropical disease and global health priority by the World Health Organization (WHO), results in an estimated 2.5 million envenomations, 138,000 deaths and over 500,000 cases of permanent disability worldwide every year. Snake, spider, and scorpion envenomations are a common environmental and occupational hazard for military forces worldwide. The consequences of an envenomation range from mild local effects to permanent disability or death, and the outcome is largely determined by the time to antivenom treatment and the level of training of the medical providers involved.
Once an envenomation has occurred, the provider and patient are racing against the clock to neutralize active venom components before extensive damage has occurred. Necrosis caused by cytotoxic venoms cannot be reversed, but it can be prevented by early antivenom administration or arrested before further damage can occur in cases of late antivenom treatment. Hemotoxic venoms can induce coagulopathies within an hour of the envenomation which is quickly followed by a standard progression of worsening local and systemic external and internal bleeding. Neurotoxic venoms can act rapidly and be fatal. Africa is one of the few places in the world with snakes like the black mamba that are capable of killing a human within one hour due to direct effects of the venom, and most patients with mamba envenomation who are not rapidly treated with antivenom will die within 2 - 6 hours from respiratory arrest. When a neurotoxic bite occurs, rapid antivenom administration prior to the onset of respiratory muscle weakness can arrest the progression of descending paralysis before serious systemic manifestations develop. Every hour wasted between bite and antivenom administration is strongly associated with sharp increases in mortality and the development of chronic or permanent sequelae including amputation, disfigurement, PTSD, blindness, kidney injury, infections, and partial or complete loss of function of the bitten limb.
This CPG will cover the continuum of snakebite care for snake envenomations in all combatant commands.
Wounds and envenomation caused by fish and other aquatic animals are events that can occur in certain populations, and are sometimes characterized as occupational accidents. A typical example occurs with professional fishermen and their families, when cleaning and preparing fish. In these people, the injuries commonly occur in the hands (mainly) and feet of the victims. Individuals who engage in recreational or sport fishing are also at risk. Another segment of the population affected is bathers, as the most common circumstance is discarded fish (especially marine catfish) on the sand or in shallow water, left by amateur fishermen or by professionals who dismiss small specimens. This is because the fish is of little interest for consumption or trade and the venom can remain active for about 24 h after its death [1–9].
The most abundant venomous snakes are those of the Hydrophiidae. Although none are found in the Atlantic Ocean, members of this family range broadly throughout the Pacific Ocean. High concentrations occur in the Indo-Pacific and near the Malay Archipelago. At least 50 species are known, all of which are venomous, a dozen of which have been implicated in human envenomation. Nevertheless, very little is known about the general biology of the majority of these snakes and much less is known regarding specifics for their venom. Therefore, an expedition was arranged under the direction of the Research Vessel Alpha Helix program to study these animals in their natural habitat. Ashmore Reef was a logical choice because of the known abundance and diversity of sea snakes there.
Differences among venoms of snake species geographically and phylogenetically remote from each other were observed by pioneer workers on snake venoms. Fayrer (1872) noted differences in the effects of various Indian snake venoms on animals. Mitchell and Reichert (1886) found significant differences in the concentrations of globulins and peptones in venoms of the rattlesnake (Crotalus adamanteus), water moccasin (Agkistrodon piscivorus), and cobra (Naja naja). They also found these venoms differed in their pharmacologic effects and in the tissue changes produced. Wolfenden (1886) reported on chemical differences between venoms of the cobra and Russell’s viper (Vipera russellii), concluding that they depended on modifications of the protein molecule. It soon became apparent that the composition of snake venoms could not be adequately defined by the biochemical techniques of the day, and the venom components were generally classified by their pharmacologic effects as neurotoxins, hemorrhagins, cytolysins, etc. Soon after Sewall’s (1887) demonstration of the immunogenic properties of snake venom and the development of bacterial antitoxins, therapeutic antisera to snake venoms were developed on a more or less empiric basis with imperfect understanding of the nature and complexity of the venoms.
Sea snakes are the most abundant and widely dispersed of the world’s venomous reptiles. Around the coastal seas of the Indian and western Pacific oceans, fishermen encounter sea snakes every day. Sea snake venoms are highly toxic. One “drop” (about 0.03 ml) contains enough venom to kill three adult men; some sea snake species can eject seven to eight such “drops” in a single bite. Fortunately for fishermen, sea snakes are not aggressive, and even when they do bite man, rarely inject much of their highly toxic venom. If this were not the case, deaths from sea snakebite would probably stop all sea fishing in many parts of Asia—and in these vast areas, fish are a most valuable source of high-quality protein.
The study of humoral immunity has been enriched in recent decades by a considerable number of observations concerning the origin, structure and specifity of antibodies.
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.
Among a series of 101 patients bitten by sea-snakes in Malaya in the years 1957-64, 80% were fishermen. Bathers and divers are occasionally bitten. Before sea-snake antivenom became available the mortality-rate (despite the high toxicity of sea-snake venom) was only 10%; however, of 11 with serious poisoning, 6 died. Subsequently 10 patients with serious poisoning received specific sea-snake antivenom; 2 patients, admitted moribund, temporarily improved but died, and 8 patients recovered dramatically. In serious poisoning the suitable dosage of intravenous sea-snake antivenom is 3000-10,000 units; in mild poisoning 1000-2000 units should suffice.
Subcutaneous and i.v. ld50 were determined on all venoms used in the study. Statistical examination revealed that the lethality by the i.v. route was significantly greater (95% confidence level) in only six of the eleven sea snake species. The neutralizing potency of four antivenenes, Tiger Snake Antivenene (N. scutatus), monovalent Sea Snake Antivenene (E. schistosa), commercial Sea Snake Antivenene (N. scutatus-E. schistosa), and polyvalent Sea Snake Antivenene (L. hardwickii, L. semi-fasciata and H. cyanocinctus) was determined against all eleven venoms by an in vivo passive protection method. Results were compared with those obtained by in vitro neutralization. In only 4 of the 33 venom-antivenene combinations tested was there a statistically significant difference in potency determined by the two methods. Little difference in neutralization potency was seen when results of tests using the 2ld50 and 5ld50 level of envenomation were statistically examined. Antivenene was more effective in protection when given early after envenomation. Tiger Snake Antivenene proved to be the antivenene of choice for protection against sea snake envenomation.
Monovalent antivenoms were raised in mice against the venoms of Causus maculatus, Vipera ammodytes, Echis carinatus, Cerastes cerastes, Bitis arietans, Agkistrodon rhodostoma and Bothrops atrox. These antivenoms as well as four commercially available antivenoms were tested against the venoms of 15 viperid species by means of immunoelectrophoresis and/or ELISA. Cross-reactive protein bands were determined by immunoblot. ELISA cross-reactions of C. maculatus antivenom were low with all heterologous venoms. When investigating the other viperine antivenoms in ELISA stronger cross-reactions were observed with several heterologous venoms. In immunoblot, two heterologous antivenoms cross-reacted with one or two protein bands of C. maculatus venom whereas there were at least four heterologous antivenoms cross-reacting with each of the other venoms. The findings indicate that there is little antigenic affinity between C. maculatus venom and the other venoms investigated. Broad in vitro cross-reactions between viperine antivenoms and Causus venom which were reported in literature seem to be attributable to the use of antivenoms of commercial grade. Specificity of commercially produced, mono- or polyvalent antivenoms may not be strictly limited to those venoms, against which potency is claimed on the label of the product.
Potentially fatal bites and stings by Australian terrestrial and marine animals are relatively common, but with correct management of victims, death should be an infrequent occurrence. The known pharmacopathological actions of some of these venoms are enumerated and specific antidotes are described. In cases of certain venoms against which no ''specific'' treatment has yet been developed, an approach to management based upon clinical and experimental evidence is presented.
Lethal doses in mice are reported for venoms of six species of snakes collected in the Coral Sea. Three have not previously been evaluated. Venom of Aipysurus duboisii has extremely high lethality exceeded by only one snake species. Secretion from Emydocephalus annulatus is essentially non-toxic.
A rare occurrence of serious envenomation by a sea snake in the waters of a popular Sydney beach is reported. A 19-year-old man was bitten while swimming, then quickly developed major proximal neuromuscular complications. Prompt, effective first aid (firm limb bandaging and splinting), transport to hospital, and administration of antivenom led to the rapid, full recovery of the patient who was discharged from hospital on the following day.
A case of a near-fatal sea snake bite, believed to the the first such case in Australia, is presented. The two-year-old girl victim became unconscious and apnoeic soon after envenomation by an Astrotia stokesii, and required artificial ventilation for 22 hours. She regained consciousness 4 1/2 hours after the administration of antivenom. The recovery phase was marked by hallucinations and tonic spasms. The patient made a full recovery before discharge from hospital.
Conscious monkeys were comfortably restrained for several hours whilst the movement and effects of injected snake venom was studied. Routine clinico-pathological studies were carried out on blood samples obtained at regular intervals and a solid phase radioimmunoassay was used to assay plasma levels of snake venoms and an individual neurotoxin.
When snake venom was injected subcutaneously into the lower limb of a monkey and no first aid was applied, venom could be detected in the plasma of the animals within 15 min of the injection. Plasma venom and neurotoxin levels peaked about 60 min after the injection but signs of neurotoxicity were usually not seen until 120 min after injection. Post mortem studies showed that very little Tiger snake venom remained at the injection site, but high concentrations were found in the regional lymph nodes.
Tiger snake venom components including the neurotoxin, notexin, were excreted in both the bile and urine of monkeys. The high levels of venom and neurotoxin found in the urine of monkeys parallel those found in some human snake bite victims.
Recently developed first aid measures were applied to the envenomed limb of a number of monkeys. These measures consisted of the application of a firm crepe bandage to the length of the injected limb which was then immobilized with a splint. The first aid measures were found to delay the movement of all the major Australian land snake venoms and also the venom of the beaked sea snake (Enhydrina schistosa).
A number of envenomed monkeys infused with antivenom immediately neurotoxic signs developed, barely survived even when given 10 times the amount of antivenom required to neutralize in vitro the dose of venom they had received. Surviving monkeys had persistent ptosis and lethargy for up to 5 d after envenoming. Failure to respond to antivenom was more likely if no effective first aid measure had been employed.
The complete amino acid sequence of E. schistosa VI:5b, a strongly myotoxic phospholipase A2 from the venom of the common sea snake, Enhydrina schistosa, has been determined. The sequence determination was done exclusively by automated Edman degradation on constituent peptides. The main fragmentation was done by cyanogen bromide cleavage yielding two fragments of approximately equal size. Peptides derived by cleavage with trypsin and Staphylococcus aureus V8 protease were used to complete and align the sequence. The results indicate that the myotoxin consists of a single chain of 119 amino acids cross-linked by 7 disulfide bridges and is closely homologous to other phospholipases A2 of elapid origin.
Notexin, a well known neurotoxin, derived from tiger snake venom, was shown to have direct nephrotoxic properties in experimental mice. A single subcutaneous dose of 1.38 micrograms/kg body weight of notexin produced renal tubular and glomerular damage within 24 hours. Renal damage increased in severity proportional to the dose of notexin injected. At a high dose of notexin, thrombotic "spherules" were found in glomeruli. These thrombotic spherules have not been previously reported to be associated with notexin or snake venoms.
1. Crude Prairie rattlesnake (Crotalus viridis viridis) venom was fractionated by HPLC DEAE anion exchange chromatography. Both acidic and basic protein fractions were assayed for hemorrhagic activity in vivo. 2. The acidic fractions that showed the highest hemorrhagic activities were pooled and divided into two samples. One sample was treated with EDTA and heat to inactivate and denature the hemorrhagic toxins; the other sample was left in native form. Both denaturated and native samples were used as immunogens for production of polyvalent antibodies in rabbits. 3. Neutralizing ability for hemorrhage of the antiserum raised against the native sample was about 50% greater than that of antiserum raised against the denatured sample. There was no correlation between ELISA reactivity and neutralizing ability of the antisera. 4. Antiserum raised against native hemorrhagic fractions showed extensive ELISA crossreactivities with the nonhemorrhagic basic proteins. The crossreacting antibodies accounted for 61% of the total ELISA reactivity and 54% of the total neutralizing ability of the antiserum. Treatment of the hemorrhagic fractions with EDTA induced a fundamental conformational change of the molecules, which was reflected in significantly increased ELISA reactivities. Antivenom raised in rabbits had high neutralizing potency compared to Wyeth antivenom (0.38 mg IgG was able to completely neutralize the hemorrhagic lesions induced by 10 micrograms crude C. v. viridis venom). However, when it was quantitatively compared with commercial Wyeth antivenom, no significant advantages were found.
A phospholipase A2 (OHV A-PLA2) from the venom of Ophiophagus hannah (King cobra) is an acidic protein exhibiting cardiotoxicity, myotoxicity, and antiplatelet activity. The complete amino acid sequence of OHV A-PLA2 has been determined using a combination of Edman degradation and mass spectrometric techniques. OHV A-PLA2 is composed of a single chain of 124 amino acid residues with 14 cysteines and a calculated molecular weight of 13719 Da. It contains the loop of residues (62-66) found in pancreatic PLA2s and hence belongs to class IB enzymes. This pancreatic loop is between two proline residues (Pro 59 and Pro 68) and contains several hydrophilic amino acids (Ser and Asp). This region has high degree of conformational flexibility and is on the surface of the molecule, and hence it may be a potential protein-protein interaction site. A relatively low sequence homology is found between OHV A-PLA2 and other known cardiotoxic PLA2s, and hence a contiguous segment could not be identified as a site responsible for the cardiotoxic activity.
Alistair Reid was an outstanding clinician, epidemiologist and scientist. At the Penang General Hospital, Malaya, his careful observation of sea snake poisoning revealed that sea snake venoms were myotoxic in man leading to generalized rhabdomyolysis, and were not neurotoxic as observed in animals. In 1961, Reid founded and became the first Honorary Director of the Penang Institute of Snake and Venom Research. Effective treatment of sea snake poisoning required specific antivenom which was produced at the Commonwealth Serum Laboratories in Melbourne from Enhydrina schistosa venom supplied by the Institute. From the low frequency of envenoming following bites, Reid concluded that snakes on the defensive when biting man seldom injected much venom. He provided clinical guidelines to assess the degree of envenoming, and the correct dose of specific antivenom to be used in the treatment of snake bite in Malaya. Reid demonstrated that the non-clotting blood of patients bitten by the pit viper, Calloselasma rhodostoma [Ancistrodon rhodostoma] was due to venom-induced defibrination. From his clinical experience of these patients, Reid suggested that a defibrinating derivative of C. rhodostoma venom might have a useful role in the treatment of deep vein thrombosis. This led to Arvin (ancrod) being used clinically from 1968. After leaving Malaya in 1964, Alistair Reid joined the staff of the Liverpool School of Tropical Medicine, as Senior Lecturer. Enzyme-linked immunosorbent assay (ELISA) for detecting and quantifying snake venom and venom-antibody was developed at the Liverpool Venom Research Unit: this proved useful in the diagnosis of snake bite, in epidemiological studies of envenoming patterns, and in screening of antivenom potency. In 1977, Dr H. Alistair Reid became Head of the WHO Collaborative Centre for the Control of Antivenoms based at Liverpool.
Neurotoxic venoms are common among tropical marine creatures, which have specialized apparatuses for delivery of the venoms. These include jellyfish and anemones, venomous cone snails, venomous fish, stingrays, sea snakes, and venomous octopuses. Numerous toxic neuropeptides are found within these venoms, and some can discriminate between closely related intracellular targets, a characteristic that makes them useful to define cation channels and attractive for drug development. A synthetic derivative of an omega-conotoxin is now available, representing a new class of analgesics. In general, toxic marine venoms contain proteins that are heat labile, providing opportunity for therapeutic intervention following envenomation, while ingestible seafood toxins are thermostable toxins. Ingestible toxins found in the tropics include those associated with reef fish, pufferfish, and some shellfish, which serve as food-chain vectors for toxins produced by marine microorganisms.
• (1)A survey of north-west Malayan fishing villages suggests that sea-snake bite is much more common than has hitherto been realized.
• (2)Biology of sea-snakes is described with a brief account of them in captivity.
• (3)Venom yields of several sea-snake species are reported, and available information on toxicity quoted.
• (4)The pathological changes in subjects of sea-snake bite are described, and other aspects of basic toxicology considered in the light of relevant work.
• (5)The treatment of neurotoxic snake poisoning is critically reviewed. The prevention of sea-snake bite is briefly discussed.
• (6)A new theory is postulated to account for the apparently large number of sea-snake bites which do not cause serious signs of poisoning.
• (7)The need for further clinical and laboratory research is mentioned.
The immunologic properties of the venom of four species of sea snake from different parts of Southeast Asia were investigated by means of double diffusion and immunoelectrophoretic methods in agar. The effectiveness of antivenin for Enhydrina schistosa venom, manufactured at Commonwealth Serum Laboratories, Melbourne, Australia, was tested against different sea-snake venoms. This antivenin was not only effective for homologous venom, but it also effectively neutralized venom of Hydrophis cyanocinctus from the Strait of Malacca, venom of Pelamis platurus from the East China Sea near Formosa, and venom of Lapemis hardwickii from the Gulf of Thailand.
Seventeen elapid antivenins and one sea snake antivenin were tested for neutralizing activity against a panel of fifteen elapid venom samples (eight Naja, three Bungarus, one each of Ophiophagus, Hemachatus, Walterinnesia and Micrurus) and one sea snake (Enhydrina) sample. Assays were made by injecting mice subcutaneously with 2–10 ld50 of venom and injecting antivenin intravenously immediately thereafter.Antivenins against venoms of certain Australian snakes (tiger snake, death adder, taipan) protected animals against at least fifteen of the venoms used. Tiger Snake Antivenene neutralized ten of the venoms at a dose of 10 ld50. Sea Snake Antivenene showed at least some protective effect against fourteen of the fifteen elapid venoms used, but most elapid antivenins failed to neutralize sea snake venom.Cobra antivenins proved largely genus specific and showed considerable variation in neutralizing capacity. Venoms of Naja n. atra and N. nigricollis were less well neutralized than other cobra venoms. Krait antivenins were relatively ineffective even in homologous systems. Coral snake (Micrurus fulvius) venom proved very difficult to neutralize.Certain implications of these findings are discussed and suggestions made for utilizing the paraspecific effect of elapid antivenins in clinical situations.
• 1)Toxicity tests were made on a number of snake venoms but special attention has been paid to Enhydrina schistosa venom.
• 2)The toxic fraction of E. schistosa venom is rapidly destroyed by heat and readily passes through a cellophane dialysis sac.
• 3)The LD50 dose for a variety of laboratory mammals and one species of fish is 40 to 120 μg./kg. For the frog it is 20 μg./kg.
• 4)Electrophoretic analysis at pH 6.8 showed that the toxic fraction of the venom was more electropositive than the other components.
• 5)Various methods of producing an antivenom were attempted. Using Freund's incomplete adjuvant, an antiserum was prepared of which 1.0 ml. was required to neutralize 100 mouse LD50 of venom, using an in vitro testing method.
• 6)Immunoelectrophoretic analysis suggested that the toxic fraction of the venom is less antigenic than the other protein components.
• 7)By in vitro testing, E. schistosa antivenom afforded significant cross protection against Hydrophis cyanocinctus and Hydrophis spiralis venoms. Antivenoms to krait, cobra and a polyvalent antivenom to Indian cobra, krait, Echis and Russell's viper afforded negligible neutralization of E. schistosa venom.
Preparation d'un serum antivenin d'hydrophüdes. Premiers essais therapeutiques The toxicity and immunological properties of corns nea~nake venoms witb particular reference to that of Erhydrina schlstosa
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1%7) Paraspecific protection by elapid and sea snake antivenins Immunological study of sea snake venom
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Preparation d'un serum antivenin d'hydrophiides
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Immunological study of sea snake venom
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