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Geographical and intraspecies variation in the clinical manifestations of envenoming by snakes
Abstract
This is the first book to present a multidisciplinary approach to venomous snake research. As well as focusing on the medical aspects of snake venoms and the effects of snakebites, the book examines the evolution and ecology of venomous snakes, which are so crucial in the search for snakebite antidotes. The book has been written to be accessible to an audience of varied scientific backgrounds. Due to the wide range of information, it will be of great interest to scientists and students involved in any aspect of venomous snake research, whilst also appealing to the growing band of amateur enthusiasts.
... Treatment of sn-ake-bite(2, 13) General measures: The patient should be reassured, kept lying quietly, and the affected Availability and efficacy of snake antivenoms: Table 1 lists polyvalent snake antivenoms currently (or previously) available in Oman, and their efficacy. The recently introduced Saudi National Guard polyvalent antivenom is manufactured using venom from Arabian snakes, intended to offer protection for bites by regional snakes, and would appear to be suitable for use in Oman. ...
... Presence of haemostatic abnormalities (including an abnormal 20 minute clotting test), neurotoxicity, hypotension, shock, impaired consciousness, or genera.lized rhabdomyolysis (13). ...
... Local swelling or less than half a limb Nonna! clotting test. lgE-mediated hypersensitivity of cutaneous mast cells to equine antigen, and does not presage the onset of serious early reactions i.e. anaphylaxis, which is caused by complement activation by lgG aggregates or Fe fragments or serum sickness which occurs 5 days 3 weeks after adm inistration of antivenom, and results from the deposition in tissues of circulating immune complexes (13). ...
Nine of the 22 terrestrial snake of Oman and all of the 9 sea-snakes are venomous. Snakebite is an uncommon but significant cause of admission to hospital. In most cases of serious envenomation from bites by terrestrial snakes, vipers have been incriminated, especially the saw-scaled or carpet vipers, and less frequently Gasperetti's horned viper. Bites from cobras and puff adders (which occur in Dhofar) have not been reported (apart from one incident involving a caged cobra), and bites by sea snakes have not been reported and appear to be rare. In most cases the snake escapes and is not identified. Coagulopathy and renal failure are the principal complications of viper bites, but fatalities are rare. The current polyvalent antivenoms in use in Oman are imported, and some do not offer protection against bites by some dangerous snakes: their use in Oman has not been evaluated. A project has been started in association with the Venom Research Unit at Liverpool School of Tropical Medicine to develop a bedside, terrestrial snake antigen detection test for use in Oman, and a venom research programme with the ultimate goal of developing polyvalent and monovalent terrestrial snake antivenoms suitable for use in Oman.
... Thirdly, the efficacy of available antivenoms and geographic variation thereof is poorly characterized. Because of limited quality control and case studies, it is often unclear which species or populations were used to create each antivenom, how much of the antivenom is required to effectively treat envenomation by each species, and sometimes even if the antivenom effectively neutralizes venom of a certain species at all (Chippaux et al., 1991;Fry et al., 2003;Gutiérrez et al., 2010Gutiérrez et al., , 2011Saravia et al., 2002;Warrell, 1997;Williams et al., 2011). These issues are exacerbated by substantial intraspecific venom variation (Casewell et al., 2014(Casewell et al., , 2020Currier et al., 2010;Daltry et al., 1996;Pla et al., 2019;Senji Laxme et al., 2021a, 2021b, and limited knowledge on the geographic distribution of different intraspecific 'venom lineages'. ...
... with greener areas shown as darker shades of grey. Such geographic variation, along with ontogenetic, intra-population, and other forms of venom variation, has important consequences for snakebite management: antivenom efficacy can vary amongst localities depending on which populations were originally used for antivenom production and differences in enzyme activity can cause different clinical manifestations of envenomation (Casewell et al., 2014;Chippaux et al., 1991;Warrell, 1997). ...
Venomous snakebite is a neglected tropical disease that annually leads to hundreds of thousands of deaths or long-term physical and mental ailments across the developing world. Insufficient data on spatial variation in snakebite risk, incidence, human vulnerability, and accessibility of medical treatment contribute substantially to ineffective on-ground management. There is an urgent need to collect data, fill knowledge gaps and address on-ground management problems. The use of novel, and transdisciplinary approaches that take advantage of recent advances in spatio-temporal models, ‘big data’, high performance computing, and fine-scale spatial information can add value to snakebite management by strategically improving our understanding and mitigation capacity of snakebite. We review the background and recent advances on the topic of snakebite related geospatial analyses and suggest avenues for priority research that will have practical on-ground applications for snakebite management and mitigation. These include streamlined, targeted data collection on snake distributions, snakebites, envenomings, venom composition, health infrastructure, and antivenom accessibility along with fine-scale models of spatio-temporal variation in snakebite risk and incidence, intraspecific venom variation, and environmental change modifying human exposure. These measures could improve and ‘future-proof’ antivenom production methods, antivenom distribution and stockpiling systems, and human-wildlife conflict management practices, while simultaneously feeding into research on venom evolution, snake taxonomy, ecology, biogeography, and conservation.
... The proteomic characterization of venoms contributes to a better understanding on biology, ecology and physiopathology of envenomations caused by these animals. Moreover, the knowledge of different families of toxins may be relevant for the creation of immunization protocols based on production of antibodies with greater specificity and effectiveness that conventional ones, produced in horses (69). ...
... Nowadays, the understanding of molecular mechanisms that participate in venom variations, including ontogenetic and geographic characteristics, may lead to a great impact on the treatment of snakebite victims, as well as in the selection of specimens for the generation of improved antivenoms (69). ...
Accidents caused by snakes, especially in tropical and subtropical countries, still constitute a serious public health problem due to the lack of knowledge of health professionals and the precariousness of health systems in the regions where most accidents occur. Snake venoms contain a range of molecules that may provoke local swelling, pain, renal and respiratory insufficiencies. The study of the effects of each molecule on humans can help the development of complementary therapy. Similarly, the knowledge of clinical aspects of envenomations provides a better identification and implementation of appropriate treatment. In addition, to understand Bothrops envenomations and improve the therapeutic strategy, it is necessary to understand and study the role of important inflammatory mediators, particularly nitric oxide (NO), cytokines and the complement system.
... The proteomic characterization of venoms contributes to a better understanding on biology, ecology and physiopathology of envenomations caused by these animals. Moreover, the knowledge of different families of toxins may be relevant for the creation of immunization protocols based on production of antibodies with greater specificity and effectiveness that conventional ones, produced in horses (69). ...
... Nowadays, the understanding of molecular mechanisms that participate in venom variations, including ontogenetic and geographic characteristics, may lead to a great impact on the treatment of snakebite victims, as well as in the selection of specimens for the generation of improved antivenoms (69). ...
Accidents caused by snakes, especially in tropical and subtropical countries, still constitute a serious public health problem due to the lack of knowledge of health professionals and the precariousness of health systems in the regions where most accidents occur. Snake venoms contain a range of molecules that may provoke local swelling, pain, renal and respiratory insufficiencies. The study of the effects of each molecule on humans can help the development of complementary therapy. Similarly, the knowledge of clinical aspects of envenomations provides a better identification and implementation of appropriate treatment. In addition, to understand Bothrops envenomations and improve the therapeutic strategy, it is necessary to understand and study the role of important inflammatory mediators, particularly nitric oxide (NO), cytokines and the complement system.
... 1,3,5,6 Variation in snake venom composition is notably influenced by adaptation to available prey species, [7][8][9][10] and can lead to regionally distinct clinical patterns observed in envenomed patients. 7,[11][12][13][14] This venom composition diversity-also occurring within speciescan lead to regionally distinct clinical patterns on envenomed patients. 7,14 Intraspecific venom variations have been shown also in some European viperids, eg, in the long-nosed viper (Vipera ammodytes) in Croatia 15 and the asp vipers (Vipera aspis) in France. ...
... 16,[18][19][20]30,31 Similar diversity of symptoms was reported in studies on the Russell's viper (Daboia russelii) envenomings in Asia. 13,14,32 These clinical reports demonstrate the great medical relevance of the regional venom variability of a given snake species. 7 A large number of reports have been issued about the clinical aspects of V. berus envenoming since Reid's pioneering work. ...
We report a recent case of common adder (Vipera berus) envenoming causing paralytic signs and symptoms. A 12-year-old girl was bitten by the nominate subspecies of the common adder (V. berus berus) in eastern Hungary on May 2, 2012, 22 km away from where the first neurotoxic V. berus berus envenoming was reported in 2008. The patient developed unambiguous cranial nerve disturbances, manifested in bilateral impairment characterized by oculomotor paralysis with partial ptosis, gaze paresis, and diplopia. Drowsiness and photophobia were her additional symptoms; both occurred only during the first day of envenoming. Until now among viper envenomings in Europe, photophobia has only been documented by victims of Vipera aspis. Supportive and symptomatic treatments were administered during 3 days of hospitalization. Although case reports of V. berus berus envenomings are often published, clinical experience with neurotoxicity by this subspecies still remains rare. Population-based and geographic variation of venom composition in V. berus berus seems to include neurotoxic envenomings in certain populations. This second authenticated case provides new clinical evidence for the existence of a possible neurotoxic V. berus berus population in a restricted geographical area in eastern Hungary.
... 1,3,5,6 Variation in snake venom composition is notably influenced by adaptation to available prey species, [7][8][9][10] and can lead to regionally distinct clinical patterns observed in envenomed patients. 7,[11][12][13][14] This venom composition diversity-also occurring within speciescan lead to regionally distinct clinical patterns on envenomed patients. 7,14 Intraspecific venom variations have been shown also in some European viperids, eg, in the long-nosed viper (Vipera ammodytes) in Croatia 15 and the asp vipers (Vipera aspis) in France. ...
... 16,[18][19][20]30,31 Similar diversity of symptoms was reported in studies on the Russell's viper (Daboia russelii) envenomings in Asia. 13,14,32 These clinical reports demonstrate the great medical relevance of the regional venom variability of a given snake species. 7 A large number of reports have been issued about the clinical aspects of V. berus envenoming since Reid's pioneering work. ...
Consequences of bites by the Common adder (Vipera berus) were reviewed in this study. Patients bitten by snakes from different populations may develop variable symptoms due to geographical venom variation. The correct diagnosis of snake bites and the knowledge of the distribution of venomous snake taxa have a crucial impact on snake bite therapy. The characteristic symptoms of patients bitten by V. berus in Hungary are highlighted. The habitat characteristics, seasonal activity and the Hungarian distribution of the adder are described based on literature data, museum specimens and field observations. However, envenomings are uncommon in Hungary, the annual 3 to 4 incidents have to be taken seriously, regardless of the age and actual health condition of the patients. Contrary to beliefs persisting both among laymen and professionals, the venom of V. berus is powerful. Medical observation of the patients is necessary in the first 5 to 6 hours. Any systemic symptom or progression of the edema requires hospital admission.
... The venom's phospholipases A 2 , hemorrhagins, and procoagulant enzymes, including Factors X and V protease activators , are presumed to be responsible for these clinical manifestations [10] [12]. However, striking variations in the clinical symptoms of envenomed victims [12] [13] appeared to reflect some geographical differences in venom components [13]. Relative to other regions, Daboia snakebites in Myanmar and eastern India are known to cause severe internal bleeding and higher mortality [14] [15]. ...
... The venom's phospholipases A 2 , hemorrhagins, and procoagulant enzymes, including Factors X and V protease activators , are presumed to be responsible for these clinical manifestations [10] [12]. However, striking variations in the clinical symptoms of envenomed victims [12] [13] appeared to reflect some geographical differences in venom components [13]. Relative to other regions, Daboia snakebites in Myanmar and eastern India are known to cause severe internal bleeding and higher mortality [14] [15]. ...
Two homologous P-III hemorrhagic metalloproteinases were purified from Russell's viper venoms from Myanmar and Kolkata (eastern India), and designated as daborhagin-M and daborhagin-K, respectively. They induced severe dermal hemorrhage in mice at a minimum hemorrhagic dose of 0.8–0.9 μg. Daborhagin-M specifically hydrolyzed an Aα-chain of fibrinogen, fibronectin, and type IV collagen in vitro. Analyses of its cleavage sites on insulin chain B and kinetic specificities toward oligopeptides suggested that daborhagin-M prefers hydrophobic residues at the P1, P1′, and P2′ positions on the substrates. Of the eight Daboia geographic venom samples analyzed by Western blotting, only those from Myanmar and eastern India showed a strong positive band at 65 kDa, which correlated with the high risk of systemic hemorrhagic symptoms elicited by Daboia envenoming in both regions. The full sequence of daborhagin-K was determined by cDNA cloning and sequencing, and then confirmed by peptide mass fingerprinting. Furthermore, molecular phylogenetic analyses based on 27 P-IIIs revealed the co-evolution of two major P-III classes with distinct hemorrhagic potencies, and daborhagin-K belongs to the most hemorrhagic subclass. By comparing the absolute complexity profiles between these two classes, we identified four structural motifs probably responsible for the phylogenetic subtyping and hemorrhagic potencies of P-III SVMPs.
... By contrast, the immunization mixtures used for antivenom production are specific for every country or region, due to the intraspecific venom variability and to the fact that different snake species are responsible for the majority of envenomings in different countries. The inter-and intraspecies heterogeneity in venom composition may account for differences in the clinical symptoms observed in human victims of envenoming by the same snake species in different geographical regions [17,18]. Understanding the variation in antigenic constituents of venoms from snakes of distinct geographic origin represents thus a key challenge towards the design of novel, toxin-specific approaches for the immunotherapy of snake bite envenoming. ...
... Geographic and ontogenetic variation in venom composition: implications for the preparation of venom pools Intraspecies variation in snake venom composition has been extensively reported [17,18]. This phenomenon is particularly notorious among species that have a wide distribution range. ...
Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact, particularly in Asia, Africa and Latin America. A global initiative, aimed at increasing antivenom production and accessibility, is being promoted by the World Health Organization and others. This work discusses several aspects of antivenom manufacture and control in which the proteomic analysis of snake venoms, for which the term 'snake venomics' has been coined, might play a relevant supporting role. Snake venomics has already shown its usefulness for generating knowledge at different levels (ontogenetic, individual, and geographic) on inter- and intraspecies venom variability. This information has applications for the quality control of venom preparations used in antivenom manufacture. Moreover, the design of the best venom mixtures for immunization, aimed at increasing the effectiveness of antivenoms, may also be guided by venom proteome analysis, including molecular studies of the cross-reactivity of antivenoms and heterologous venoms through a recently developed methodological approach termed 'antivenomics'. Results generated by proteomic protocols should be complemented by preclinical testing of antivenom efficacy using functional neutralization assays. Snake venomics might be also helpful in designing alternative in vitro tests for the assessment of antivenom efficacy that would eventually substitute current in vivo tests.
... These snakes possess a highly specialized system of venom delivery, formed by tubular frontal fangs attached to mobile maxillas (solenoglyph dentition), highly kinetic cranial elements and muscles that compress venom glands connected to the fangs by ducts (Cundall, 2002;Jackson, 2003;Deufel and Cundall, 2006). Viperids are animals of great biomedical importance given the potential lethality of their bites, and because of the beneficial pharmacological properties of some components of their venoms (Chippaux et al., 1991;Warrell, 1997;Lipps, 1999;Fry et al., 2003;Koh and Kini, 2012). ...
Crotalines (pitvipers) in the Americas are distributed from southern Canada to southern Argentina, and are represented by 13 genera and 163 species that constitute a monophyletic group. Their phylogenetic relationships have been assessed mostly based on DNA sequences, while morphological data have scarcely been used for phylogenetic inquiry. We present a total‐evidence phylogeny of New World pitvipers, the most taxon/character comprehensive phylogeny to date. Our analysis includes all genera, morphological data from external morphology, cranial osteology and hemipenial morphology, and DNA sequences from mitochondrial and nuclear genes. We performed analyses with parsimony as an optimality criterion, using different schemes for character weighting. We evaluated the contribution of the different sources of characters to the phylogeny through analyses of reduced datasets and calculation of weighted homoplasy and retention indexes. We performed a morphological character analysis to identify synapomorphies for the main clades. In terms of biogeography, our results support a single colonization event of the Americas by pitvipers, and a cladogenetic event into a Neotropical clade and a North American/Neotropical clade. The results also shed light on the previously unstable position of some taxa, although they could not sufficiently resolve the position of Bothrops lojanus, which may lead to the paraphyly of either Bothrops or Bothrocophias. The morphological character analyses demonstrated that an important phylogenetic signal is contained in characters related to head scalation, the jaws and the dorsum of the skull, and allowed us to detect morphological convergences in external morphology associated with arboreality.
... Clinical and venom-based data suggest a geographical variation in the envenoming species and also in the nature of the venom. 13,14 A review of geographical variation of snake bite syndromes showed that in southern India snake bites were predominantly haemotoxic with renal failure and in northern India they were predominantly neurotoxic. Russell's viper bites were scarce in northern India. ...
Background.
Acute kidney injury (AKI) is a common complication of snake envenomation. However, the long-term renal outcomes of such patients are not well defined. We aimed to determine the proportion of patients who developed AKI, characterize the presenting syndromes and ascertain the long-term resolution of AKI.
Methods.
We did a cohort study with prospective follow- up from two centres in southern India. All admitted patients >15 years of age with snake envenomation and serum creatinine ≥1.5 mg/dl over the past 10 years were identified through their discharge summaries. These patients were prospectively contacted, interviewed telephonically and requested to come for a hospital review.
Results.
Of the 866 patients screened, 1 84 developed AKI (21.2%). Among these, 53% had combined renal, haematological and neurological manifestations; 33.6% required admission to the intensive care unit and 38% were dialysed. On follow-up of hospital records the creatinine of 49% of patients had normalized. Of those admitted, 36% were contacted and none had a known renal disease or were on dialysis. Among these, 16 patients came to the hospital for review and only 2 had an elevated creatinine. The total mortality was 1 4.
Conclusion.
AKI is an important cause of morbidity with snake envenomation and a proportion will require dialysis. The mortality in our study was low and long-term renal outcomes were relatively good.
... Da) that constitutes 19.2% of the crude venom [32]. Although damage to the central nervous system, particularly ischemic complications, after D. russelii bite are rare, the risk of anterior pituitary infarction resulting in fatal acute pituitary adrenal failure or chronic panhypopituitarism has been reported in southern India [20,29,30,33,34]. Moreover, rare cases of ischemic stroke following Russell's viper snake bites in India have been documented [35][36][37]. ...
Russell's viper (Daboia russelii) is, together with Naja naja, Bungarus caeruleus and Echis carinatus, a member of the medically important 'Big Four' species responsible for causing a large number of morbidity and mortality cases across the Indian subcontinent. Despite the wide distribution of Russell's viper and the well-documented ubiquity of the phenomenon of geographic variability of intraspecific snake venom composition, Indian polyvalent antivenoms against the "Big Four" venoms are raised against venoms sourced mainly from Chennai in the southeastern Indian state of Tamil Nadu. Biochemical and venomics investigations have consistently revealed notable compositional, functional, and immunological differences among geographic variants of Russell's viper venoms across the Indian subcontinent. However, these studies, carried out by different laboratories using different protocols and involving venoms from a single geographical region, make the comparison of the different venoms difficult. To bridge this gap, we have conducted bioactivities and proteomic analyses of D. russelii venoms from the three corners of the Indian subcontinent, Pakistan, Bangladesh, and Tamil Nandu (India) and Sri Lanka, along with comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms used in India, Bangladesh and Sri Lanka. These analyses let us to propose two alternative routes of radiation for Russell's viper in the Indian subcontinent. Both radiations, towards the northeast of India and Bangladesh and towards south India and Sri Lanka, have a common origin in Pakistan, and provide a phylovenomics ground for rationalizing the geographic variability in venom composition and their distinct immunoreactivity against available antivenoms. BIOLOGICAL SIGNIFICANCE: Russell's viper (Daboia russelii), the Indian cobra (Naja naja), the common krait (Bungarus caeruleus), and the saw-scaled viper (Echis carinatus) constitute the 'Big Four' snake species responsible for most snakebite envenomings and deaths in the Indian subcontinent. Despite the medical relevance of Daboia russelii, and the well documented variations in the clinical manifestations of envenomings by this wide distributed species, which are doubtless functionally related to differences in venom composition of its geographic variants, antivenoms for the clinical treatment of envenomings by D. russelii across the Indian subcontinent are invariably raised using venom sourced mainly from the southeastern Indian state of Tamil Nadu. We have applied a phylovenomics approach to compare the venom proteomes of Russell's vipers from the three corners of the Indian subcontinent, Pakistan, Bangladesh, and South India/Sri Lanka, and have assessed the in vitro (third-generation antivenomics) and in vivo preclinical efficacy of a panel of homologous antivenoms. The identification of two dispersal routes of ancestral D. russelii into the Indian subcontinent provides the ground for rationalizing the variability in composition and immunoreactivity of the venoms of extant geographic variants of Russell's viper. Such knowledge is relevant for envisioning strategies to improve the clinical coverage of anti- D. russelii antivenoms.
... Kidney damage, with hematuria and proteinuria, eventually triggering renal failure (attributed to poor availability or delayed administration of antivenom), have been noted frequently in Russell's viper envenoming [11][12][13][14][15][16][17][18][19][20]. Variation in venom effects of D. siamensis from different geographical areas in Myanmar and Thailand have been also reported [12][13][14][15][16][17][18][19][20][21][22]. Particularities in clinical manifestations are undoubtedly functionally related to differences in venom composition across the wide geographic range of Siamese Russell's viper. ...
The venom proteome of Siamese Russell’s viper from Taiwan, alongside complementary in vivo lethality neutralization assay and in vitro third-generation antivenomics assessment of the preclinical efficacy of the homologous antivenom manufactured in Taiwan CDC’s Vaccine Center, are here reported. Taiwanese Russell’s viper venom proteome comprised 25 distinct gene products, with the heterodimeric PLA2 viperotoxin-F representing the most abundant toxin (47.5% of total venom proteome). Coagulation FV-activating serine proteinase (RVV-V, 14%), the PIV-SVMP activator of FX (RVV-FX, 8.5%), and less abundant toxins from nine protein families, make up its venom proteome. Venom composition-pathology correlations of D. siamensis envenomings in Taiwan are discussed. The lethal effect of Taiwanese D. siamensis venom was 0.47 mg/g mouse. Antivenomics-guided assessment of the toxin recognition landscape of the Taiwanese Russell’s viper antivenom, in conjunction with complementary in vivo neutralization analysis, informed the antivenom’s maximal toxin immunorecognition ability (14 mg total venom proteins/vial), neutralization capacity (6.5 mg venom/vial), and relative content of lethality neutralizing antibodies (46.5% of the toxin-binding F(ab’)2 antibodies). The antigenomic analysis also revealed suboptimal aspects of the CDC-Taiwan antivenom. Strategies to improve them are suggested.
... Snake venom composition varies by species, but also within each species with variation due to geographical and seasonal factors, and snake maturity. 16 Venom contains around a hundred toxic and non-toxic proteins, along with carbohydrates, lipids, amines and other small molecules. 17 The most important venom components are pro-coagulant enzymes leading to consumption coagulopathy that contributes to bleeding; cytolytic toxins, digestive enzymes that destroy cell membranes; haemolytic and myolytic phospholipases, causing widespread effects including neurotoxicity and rhabdomyolysis; neurotoxins that act both presynaptically and postsynaptically at neuromuscular junctions leading to paralysis; and haemorrhagings which damage vascular endothelium and cause spontaneous bleeding. ...
Snakebite envenoming is rare among military patients, with few cases reported in recent years. Increasingly, however, military operations are taking place in remote parts of Africa, which are inhabited by numerous species of venomous snake, and in Europe, where dangerous species exist but are less common. Bites from a venomous snake may prove fatal, and therefore military medics must be adequately prepared to manage them. This paper reviews the most medically significant species of venomous snake present in Africa and Europe, before suggesting an evidence-based approach to snakebite prevention and management, including possible changes to the UK’s Clinical Guidelines for Operations.
... Differences in venom composition have resulted in variations in the clinical manifestations of envenoming throughout the range of distribution [23][24][25][26][27][28]. The clinical presentation of envenomed patients may vary dramatically among regions. ...
Asia, the largest landscape on Earth, provides habitat to the majority of Earth’s human beings as well as to a colossal diversity of snakes. More than 150 venomous species, representing several snake families: the Viperidae (e.g., vipers, pit vipers, and Fea’s viper), the Elapidae (e.g., kraits, cobras, king cobras, and Asian coral snakes), and the broad assemblage previously contained within Family Colubridae (e.g., non-front-fanged-colubrids (NFFC snakes), including “rear-fanged” snakes) are known to exist.
... Differences in venom composition have resulted in variations in the clinical manifestations of envenoming throughout the range of distribution [23][24][25][26][27][28]. The clinical presentation of envenomed patients may vary dramatically among regions. ...
Asia, the largest landscape on Earth, provides habitat to the majority of Earth’s human beings as well as to a colossal diversity of snakes. More than 150 venomous species, representing several snake families: the Viperidae (e.g., vipers, pit vipers, and Fea’s viper), the Elapidae (e.g., kraits, cobras, king cobras, and Asian coral snakes), and the broad assemblage previously contained within Family Colubridae (e.g., non-front-fanged-colubrids (NFFC snakes), including “rear-fanged” snakes) are known to exist.
... [1,2]. The often extensive compositional variation between conspecific populations or between closely related species has been of particular interest, partly due to its medical consequences [3][4][5] and partly due to its potential as a model system for understanding adaptive evolution at the molecular level (e.g., [6][7][8]): many species display extreme intraspecific geographic variation in venom composition, and this variation may bear little relationship to population genetic structure or organismal phylogeny [9,10]. Natural selection for optimization of venom to the diet of snakes has been identified as a likely key driver of venom evolution in several groups [9,[11][12][13]. ...
Venomous snakes often display extensive variation in venom composition both between and within species. However, the mechanisms underlying the distribution of different toxins and venom types among populations and taxa remain insufficiently known. Rattlesnakes (Crotalus, Sistrurus) display extreme inter- and intraspecific variation in venom composition, centered particularly on the presence or absence of presynaptically neurotoxic phospholipases A2 such as Mojave toxin (MTX). Interspecific hybridization has been invoked as a mechanism to explain the distribution of these toxins across rattlesnakes, with the implicit assumption that they are adaptively advantageous. Here, we test the potential of adaptive hybridization as a mechanism for venom evolution by assessing the distribution of genes encoding the acidic and basic subunits of Mojave toxin across a hybrid zone between MTX-positive Crotalus scutulatus and MTX-negative C. viridis in southwestern New Mexico, USA. Analyses of morphology, mitochondrial and single copy-nuclear genes document extensive admixture within a narrow hybrid zone. The genes encoding the two MTX subunits are strictly linked, and found in most hybrids and backcrossed individuals, but not in C. viridis away from the hybrid zone. Presence of the genes is invariably associated with presence of the corresponding toxin in the venom. We conclude that introgression of highly lethal neurotoxins through hybridization is not necessarily favored by natural selection in rattlesnakes, and that even extensive hybridization may not lead to introgression of these genes into another species.
... Inadequate attention to these long-understood geographic variations in venoms is one of the reasons for the increasingly common reports from clinicians about the ineffectiveness of commercially available antivenoms [31][32][33] . Other issues include the sourcing of venom from unlicensed producers, misleading/outdated medical information in the instruction leaflets, noncompliance with WHO standards and protocols for venom and antivenom production. ...
Snakebite is a medically and socially significant issue in India, but the quality of treatment and reporting protocols need to be upgraded to international standards. There are currently seven pharmaceutical laboratories in India which produce antivenom against four medically important Indian snake species (cobra (Naja sp.), krait (Bungarus sp.), Russell's viper (Daboia russelii) and sawscaled viper (Echis carinatus sp.), the 'big four'. Most venom for antivenom production is sourced from Chennai, South India. While the 'big four' are responsible for a majority of serious and fatal bites, the situation is actually much more complex. In this article, we review the production of venom and antivenom in India and suggest areas of improvement. We show that several factors complicate the treatment of snakebite in India. The first is geographic, intra-species variation in venoms of cobras and Russell's vipers. Secondly, there are four species of cobra, eight species of kraits, two distinct sub-species of saw-scaled viper. In view of these observations, it is felt that identifying, evaluating and implementing changes to venom and antivenom production protocols, public education, snakebite treatment and policy in India should be an immediate priority.
... In order to guarantee an appropriate geographical spectrum of effi cacy of an antivenom, it is essential to know in which parts of the country or region the specifi c envenoming is predominant. The control of the neutralizing ability of an antivenom preparation should be performed using pools of well-characterized venoms, taking into account the known causes of intra-specifi c variation in venom composition and antigenicity (35). Currently, there are many lacunae in the preparation and use of venom pools for antivenom standardization and control. ...
... Ontogenic variations in snake venoms may be important. 40 In the genus Bothrops, the venoms of the young snakes of the majority of species contain larger amounts of both factor II and factor X activators than those of adult snakes. 41 This results in relatively more severe haemostatic disorders following envenoming by young snakes. ...
The South American tropical rattlesnake (Crotalus durissus subspp) is responsible for ;10% of bites from venomous snakes in Brazil. We studied 24 victims of bites by this species over 3 years, in southeastern Brazil, particularly investigating haemostatic alterations. Thirteen patients were defined as moderately envenomed and 11 as severe. There were two deaths, which were not attributed to venom-induced haemostatic disturbances. However, envenoming by C. durissus is frequently associated with haemostatic disorders, which are probably attributable mainly to the action of the thrombin-like enzyme, with possible additional effects secondary to the powerful myotoxic activity of the venom.
... In order to guarantee an appropriate geographical spectrum of effi cacy of an antivenom, it is essential to know in which parts of the country or region the specifi c envenoming is predominant. The control of the neutralizing ability of an antivenom preparation should be performed using pools of well-characterized venoms, taking into account the known causes of intra-specifi c variation in venom composition and antigenicity (35). Currently, there are many lacunae in the preparation and use of venom pools for antivenom standardization and control. ...
The people most affected by rabid dog bites, snake bites and scorpion stings usually live in poor rural communities where medical resources are often sparse. Because they lack a strong political voice, their problems tend to be overlooked by politicians and health authorities who are based in capital cities and are poorly informed about major public health issues affecting rural areas. Consequently, the impact of these health issues, although dramatic and economically significant, does not appear as a priority in the design of national public health programmes. These are therefore the most neglected among today’s neglected global health problems. The gravity of this situation should be discussed explicitly in national, regional and global health fora, in order to give these neglected diseases and their abandoned victims the attention they deserve. The situation is particularly poignant because, in contrast to some other diseases, a highly effective treatment already exists: the timely administration of specific antiserum. Rabies, for instance, is entirely preventable even after severe exposure, provided post-exposure prophylaxis, completed with rabies immunoglobulin, can be given. Similarly, the mortality and morbidity of snake bites and scorpion stings can be reduced to very low levels by timely administration of appropriate antivenoms. The state of antisera production worldwide varies greatly. Public access to production technology following GMP standards, together with a concerted exchange process through workshops, direct technical assistance and innovation in specific aspects of manufacture and training, should allow the less developed manufacturing laboratories to strengthen their technical and production capacities. The possibility of partnerships should be promoted, including, in the case of antivenoms, the creation of groups developing the skills needed for the preparation of high quality venom pools and the subsequent preparation of antivenoms. In summary, the current situation of the management of potentially rabid mammal bites and envenomings by snake bites or scorpion stings worldwide is a global public health emergency. There is a lack of awareness of the magnitude of the problem by health authorities and politicians alike, due to both the scarcity of adequate statistics on the real impact of these diseases, and the lack of advocacy by and on behalf of the affected groups, mostly children and rural agricultural workers. Worldwide production of these antisera has declined, due to economic constraints that have forced the withdrawal of some private producers, and to the weakening of public-sector manufacturers in the public sector in many countries. Moreover, the poor quality of some antisera and the resulting deficiency in their efficacy and safety, together with deficient distribution policies and inadequate training of medical and nursing staff requires an urgent international action. The gravity of this problem, and the complexity of its causes, demands from the public health community, and especially from the WHO and humanitarian international agencies, a concerted, rapid and effective global response to reduce the burden of human suffering incurred by rabies, and snake and scorpion envenomings.
... Moreover, some species present a conspicuous ontogenetic variability in the composition of their venoms, such as the Central American rattlesnake Crotalus simus (Calvete et al., 2010a) and the lance-head viper Bothrops asper (Alape-Girón et al., 2008). This high variability in venom composition has evident implications for the clinical manifestations of envenoming (Warrell, 1997) and for the preparation of antivenoms (Gutiérrez et al., 2009a). ...
... The occurrence of within-species geographic, individual, and ontogenetic variability in the venom composition, and in the clinical manifestation of envenomings, has long been appreciated by herpetologists, toxinologists, and clinicians [34,35]. This phenomenon is particularly notorious for highly adaptable species, such as B. atrox, that have a wide distribution range. ...
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Parenteral administration of animal-derived antivenoms constitutes the mainstay in the treatment of snakebite envenomings. Despite the fact that this therapy has been available for over a century, the detailed understanding of the neutralizing and immunoreactivity profiles of the majority of antivenoms is pending. Currently, a combination of preclinical neutralization tests and 'antivenomics', i.e. a proteomic-based assessment of antivenom immunoreactivity, provides a powerful analytical platform to investigate the preclinical efficacy of antivenoms. In this review, the studies performed on the polyvalent antivenom manufactured by Instituto Clodomiro Picado, Costa Rica, are summarized. This antivenom is prepared by immunizing horses with a mixture of the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys, and is used in Central America for the treatment of envenomings by viperid species. Overall, the antivenom shows a widespread pattern of immunological reactivity against homologous and heterologous venoms, which correlates with its ability to neutralize lethal, hemorrhagic, myotoxic, coagulant, defibrinogenating, phospholipase A2 and proteinase activities of viperid venoms. At the same time, antivenomics detected several venom components against which the antivenom shows only partial or negligible immunorecognition, such as low molecular mass vasoactive peptides, disintegrins, and some phospholipases A2, P-I metalloproteinases and serine proteinases. Such information can be used to design strategies for enhancing the antibody response of horses against poorly immunogenic, toxicologically-relevant venom components in order to further improve the efficacy of this antivenom.
Biological significance:
The timely parenteral administration of an appropriate antivenom remains, more than a century after the development of the first serum antivenimeux by Calmette and Phisalix and Bertrand, the only currently effective treatment for snakebite envenomings. A key technical issue in the generation of novel antivenoms is the design of optimized immunization venom mixtures that ensure that the resulting antidotes will be effective against the highest number of venoms from snakes of medical concern across the geographical range where they will be used. Antivenomics is a proteomics-based protocol developed to complement in vitro and in vivo standard preclinical tests in the qualitative and quantitative characterization of the immunological profile and the extent of cross-reactivity of antivenoms against homologous and heterologous venoms. Antivenomics is translational venomics. The combination of antivenomics and neutralization assays represents a powerful analytical platform to investigate the efficacy of antivenoms at the molecular and preclinical levels. This article is part of a Special Issue entitled: Proteomics of non-model organisms.
... Other studies show the existence of inter-and intraspecies heterogeneity in venom composition, which produces different clinical features for various envenomings. [39][40][41][42] In this study, in the first and second phases of nociception response monitoring, the VAVL group showed statistically significant reduction in nociceptive activity. These data suggest that the effects of the combined treatments can help in reducing the pain of snakebite envenomations, a result that corroborates those of other studies. ...
Antivenom therapy has been ineffective in neutralizing the tissue damage caused by snakebites. Among therapeutic strategies to minimize effects after envenoming, it was hypothesized that a low level laser would reduce complications and reduce the severity of local snake venom effects. In the current study, the effect of a low-level semiconductor gallium arsenide (GaAs) laser on the local pathological alterations induced by B. moojeni snake venom was investigated. The experimental groups consisted of five male mice, each administered either B. moojeni venom (VB), B. moojeni venom + antivenom (VAV), B. moojeni venom + laser (VL), B. moojeni venom + antivenom + laser (VAVL), or sterile saline solution (SSS) alone. Paw oedema was induced by intradermal administration of 0.05 mg kg(-1) of B. moojeni venom and was expressed in mm of directly induced oedema. Mice received by subcutaneous route 0.20 mg kg(-1) of venom for evaluating nociceptive activity and the time (in seconds) spent in licking and biting the injected paw was taken as an indicator of pain response. Inflammatory infiltration was determined by counting the number of leukocytes present in the gastrocnemius muscle after venom injection (0.10 mg kg(-1)). For histological examination of myonecrosis, venom (0.10 mg kg(-1)) was administered intramuscularly. The site of venom injection was irradiated by the GaAs laser and some animals received antivenom intraperitoneally. The results indicated that GaAs laser irradiation can help in reducing some local effects produced by the B. moojeni venom in mice, stimulating phagocytosis, proliferation of myoblasts and the regeneration of muscle fibers.
... Among these, hemorrhage on the site of bite and unhealed wounds of the victim were severe and major manifestation of envenomation, though renal failure is considered as the primary cause of death (Kularatne et al., 2011) (Warrell, 1989 ). Recent studies and clinical observations have indicated higher case fatalities and more severe hemorrhagic manifestations in D. russelii envenoming in Eastern India, Myanmar and in other regions (Kumar and Gowda, 2006; Mukherje et al., 2000; Senthilkumaran et al., 2012) (Warrell, 1997). Although hemorrhage is a well-known phenomenon in the Daboia russelii bites, its mechanism of action needs to be elucidated. ...
Snake venoms are complex mixture of enzymatic and non-enzymatic proteins. Non-covalent protein-protein interaction leads to protein complexes, which bring about enhanced pharmacological injuries by their synergistic action. Here we report identification and characterization of a new Daboia russelii hemorrhagic complex I (DR-HC-I) containing phospholipase A2 (PLA2) and non-enzymatic peptide. DR-HC-I was isolated from the venom of Daboia russelii by CM-Shepadex-C25 and gel permeation chromatography. Individual components were purified and identified by RP-HPL chromatography, mass spectrometry and N-terminal amino acid sequencing. DR-HC-I complex was lethal to mice with the LD50 dose of 0.7mg/kg body weight with hemorrhagic and neurotoxic properties. DR-HC-I complex consists of non-hemorrhagic PLA2 and neurotoxic non-enzymatic peptide. The non-enzymatic peptide quenched the intrinsic fluorescence of PLA2 in a dose dependent manner, signifying the synergistic interaction between two proteins. PLA2 and peptide toxin in a 5:2 molar ratio induced skin hemorrhage in mice with MHD 20μg. However, addition of ANS (1-Anilino-8-naphthalene sulfonate) to DR-HC-I complex inhibited skin hemorrhagic effect and also synergic interaction. But there was no impact on PLA2 due to this synergistic interaction, and indirect hemolytic or plasma re-calcification activity. However, the synergistic interaction of PLA2 and non-enzymatic peptide contributes to the enhanced venom-induced hemorrhage and toxicity of Daboia russellii venom.
... Although the present review is concerned entirely with systematics at or above the species and subspecies level, we emphasise that venom variation in snakes happens at all systematic levels, including between individuals and populations of a single subspecies (Warrell, 1997). Obtaining a correct identification to species or subspecies level will not necessarily predict the qualities of the venom to be found. ...
... However, understanding the molecular mechanisms of the evolutionary trends and the ecological constraints that underlie inter-and intra-specific venom variation may provide insights into snakebite pathology Gutierrez et al., 2008Gutierrez et al., , 2010Alape-Girón et al., 2008;Valente et al., 2009;Boldrini-França et al., 2010;Öhler et al., 2010;Rey-Suárez et al., 2011;Massey et al., 2012;Lomonte et al., 2012aLomonte et al., , 2012bHerrera et al., 2012;Madrigal et al., 2012). The occurrence of variability in the biochemical composition of venoms and in the symptomatology after envenomation by snakes from different geographical locations and age has long been appreciated by herpetologist and toxinologists (Chippaux et al., 1991;Warrell, 1997), though detailed comparative analyses are scarce in the literature prior to 2008. A specific question that we have assessed in recent years using a Table 2 of venomics approach include the occurrence and onset of geographic and ontogenetic variations in Bothrops asper (Alape-Girón et al., 2008), Bothrops atrox , the Crotalus durissus complex (Boldrini-França et al., 2010;Calvete et al., 2010a), Crotalus scutulatus scutulatus (Massey et al., 2012) (Fig. 4), S. m. barbouri , and across genus Lachesis (Madrigal et al., 2012) (Fig. 5). ...
... Exploring variation in snake venom is important because symptoms caused by snakebite highly correlate with the venom composition and content [12][13][14][15][16][17]. Previous studies have mostly focused on differences in venom composition among species, among populations of the same species, and among individuals of the same population using venoms from adults. ...
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Snake venoms undergo ontogenetic shifts in biochemical and pharmacological activities. This may be related to variation in venom components associated with the ontogenetic shift in diet. We used the short-tailed pit viper Gloydius brevicaudus that displays ontogenetic shifts in diet to examine whether the species displays a neonate-to-adult transition of snake venomics. Venoms from neonates and adults were pooled separately and then analyzed by 2-DE, MALDI-TOF-MS/MS and iTRAQ technologies. The 2-DE profiles showed that the main components in both types of venoms were acidic proteins, and that neonates and adults differed in snake venomics. The proteins with molecular masses/pI of ~12-39kDa/4.0-4.6, ~36-57kDa/5.6-7.0 and ~65-92kDa/4.5-5.8 were more abundant in the neonate venom, while the proteins with molecular masses/pI of ~12-19kDa/4.6-6.4, ~23-30kDa/5.4-6.3 and ~35-62kDa/4.6-5.4 were more abundant in the adult venom. The iTRAQ analysis showed quantitative changes in various toxin families, including mainly metalloproteinases, serine proteinases, phospholipase A2s and C-type lectins. The N-deglycosylation analysis demonstrated that glycosylation was an important post-translational modification of snake venom. Our results show a neonate-to-adult transition of snake venomics in G. brevicaudus. Such a transition might be driven by the divergence in dietary habits between neonates and adults.
Biological significance:
This study is first to demonstrate a neonate-to-adult transition of snake venomics in G. brevicaudus, and the results will be helpful in predicting and treating clinical pathologic symptoms caused by the snake at different developmental stages.
... Generally, each allopatric patch has a "conventional" subspecies (i.e. based on superficial morphological criteria) with five to seven being recognizedsee Wüster et al. (1992) and Wüster (1998) for reviews. These subspecies do not relate to the considerable geographic variation in the clinical manifestations of envenoming of humans reviewed in Warrell (1989Warrell ( , 1997 and Belt et al. (1997). While the clinical symptoms may be functionally related to the differences in venom composition and enzyme activity (Jayanthi & Gowda, 1988;Woodhams et al., 1990), the pattern of variation does not relate either to the conventional subspecies, or very obviously to the efficacy of anti-venoms (Wüster, 1998). ...
The Russell's viper complex has a patchy (relict) distribution over large areas of Asia from Pakistan to Taiwan and the Lesser Sunda islands. In many areas it is the primary cause of snakebite mortality, and hence a serious medical problem. A multigene mitochondrial gene tree, supported by multivariate morphometry and basic colour pattern, suggests a primary split in the organismal phylogeny giving distinct, diagnosable, eastern and western forms that we recognize as full species: Daboia russelii (west of the Bay of Bengal) and Daboia siamensis (east of the Bay of Bengal). The clinical symptoms of human envenoming show marked geographic variations that are broadly unrelated to the phylogeny. The molecular phylogeny, together with current distribution and fossil record, suggests cycles of extreme expansion and contraction for this complex. Further studies on venom variation, diet and local population phylogeny are required, but the local and regional variation in symptoms may be the result of fixation of venom genes during cyclical bottlenecks which could explain the haphazard, non-phylogenetic pattern of symptoms of envenomation in this species complex.
... It also has been shown that the functional differences between members of a venom protein family usually are related to substitutions at the surface sites while the protein folds are conserved (Kini and Chan, 1999). However, the genetic relationships may also be complicated by intra-species variations, possibly resulting from prey-ecology (Daltry et al., 1997), geographic separation, ontology (Warrell, 1997), or other factors. Indeed, we found that the three Taiwanese viperid species, D. r. siamensis (Tsai et al., 1996), P. mucrosquamatus venoms (Tsai et al., 2001), and T. stejnegeri (Tsai et al., 2004), show considerable intra-species variations in their venom PLA isoforms, mainly due to differential expression of each isoforms and geographic separation of the snakes. ...
The major viperid snakes present in Taiwan today are: Daboia russelli siamensis, Deinagkistrodon acutus, Protobothrops mucrosquamatus, Trimeresurus stejnegeri, Ovophis monticola and Ovophis gracilis. As suggested by the species trees deduced from mtDNA sequences of the family Viperidae, these species apparently belong to five different genera. Molecular cloning, N-terminal sequencing, and mass spectrometry have facilitated the sequence-determination of venom proteins. Previous examples of using venom proteins as characteristics for the phylogenetic and evolutionary studies are reviewed herein. Two new phylogeny trees using protein sequence datasets for the crotalid venom acidic phospholipases and the disintegrins are shown. Both trees grossly reveal phylogeographic relationships between the species, although some inconsistencies exist. Moreover, distinct protein markers in each of the Taiwanese viperid venoms and their relations to the snakebite symptoms are discussed in terms of the known snake systematics.
... The occurrence of intraspecies variability in the biochemical composition and symptomatology after envenomation by snakes from different geographical location and age has long been appreciated by herpetologists and toxinologists, and appear to be a general trend of snake venoms [25]. The high variability in venom composition has evident implications for the clinical manifestations of envenoming [26] and consequently for the selection of specimens for antivenom production [27]. In this work, we have explored the age-dependent variation of newborn, juvenile and adult Central American bushmaster, L. stenophrys venoms. ...
We report the proteomic analysis of ontogenetic changes in venom composition of the Central American bushmaster, Lachesis stenophrys, and the characterization of the venom proteomes of two congeneric pitvipers, Lachesis melanocephala (black-headed bushmaster) and Lachesis acrochorda (Chochoan bushmaster). Along with the previous characterization of the venom proteome of Lachesis muta muta (from Bolivia), our present outcome enables a comparative overview of the composition and distribution of the toxic proteins across genus Lachesis. Comparative venomics revealed the close kinship of Central American L. stenophrys and L. melanocephala and support the elevation of L. acrochorda to species status. Major ontogenetic changes in the toxin composition of L. stenophrys venom involves quantitative changes in the concentration of vasoactive peptides and serine proteinases, which steadily decrease from birth to adulthood, and age-dependent de novo biosynthesis of Gal-lectin and snake venom metalloproteinases (SVMPs). The net result is a shift from a bradykinin-potentiating and C-type natriuretic peptide (BPP/C-NP)-rich and serine proteinase-rich venom in newborns and 2-years-old juveniles to a (PI>PIII) SVMP-rich venom in adults. Notwithstanding minor qualitative and quantitative differences, the venom arsenals of L. melanocephala and L. acrochorda are broadly similar between themselves and also closely mirror those of adult L. stenophrys and L. muta venoms. The high conservation of the overall composition of Central and South American bushmaster venoms provides the ground for rationalizing the "Lachesis syndrome", characterized by vagal syntomatology, sensorial disorders, hematologic, and cardiovascular manifestations, documented in envenomings by different species of this wide-ranging genus. This finding let us predict that monospecific Lachesic antivenoms may exhibit paraspecificity against all congeneric species.
... Unless otherwise stated, for MS/MS analyses, cysteine residues were carbamidomethylated; molecular masses were determined by electrospray-ionisation mass spectrometry or SDS-PAGE of reduced (▼) samples; n.p., non peptidic material found. Variation in venom composition is one of the key complicating factors in toxinological research and the design and use of antivenoms [174,175]. The relationship between systematic affinities, phylogeny, venom composition and antivenom cross-neutralisation is highly variable: phylogenetic relationships have been able to predict the symptoms of envenoming in some cases, such as the unique thrombotic syndrome of envenoming shared by the closely related Caribbean island pitvipers B. lanceolatus and Bothrops caribbaeus [176]. ...
... Several factors, such as seasonal variation, habitat, age, and sexual dimorphism are known to produce venom variation (Chippaux et al., 1991; Furtado et al., 2006; Menezes et al., 2006; Pimenta et al., 2007), although it is still controversial to which extent the diet influences the protein profile of snake venoms (Daltry et al., 1996; Sasa, 1999). In fact, intraspecies (geographical, seasonal and ontogenetic) variation in venom composition may account for the variability observed in severity and pattern of snakebite envenomation (for review see Warrell (1997)). Differences in the clinical picture and severity of people bitten by adult and young B. jararaca have been reported since the pioneer observations by Monteiro (1610), who noticed that patients bitten by " jararaca soatinga " (jararaca with white tail tip,Fig. 1) have intense bleeding and rarely survive. ...
Different clinical manifestations have been reported to occur in patients bitten by newborn and adult Bothrops jararaca snakes. Herein, we studied the chemical composition and biological activities of B. jararaca venoms and their immunoneutralization by commercial antivenin at these ontogenetic stages. Important differences in protein profiles were noticed both in SDS-PAGE and two-dimensional electrophoresis. Newborn venom showed lower proteolytic activity on collagen and fibrinogen, diminished hemorrhagic activity in mouse skin and hind paws, and lower edematogenic, ADPase and 5'-nucleotidase activities. However, newborn snake venom showed higher l-amino oxidase, hyaluronidase, platelet aggregating, procoagulant and protein C activating activities. The adult venom is more lethal to mice than the newborn venom. In vitro and in vivo immunoneutralization tests showed that commercial Bothrops sp antivenin is less effective at neutralizing newborn venoms. These findings indicate remarkable differences in biological activities of B. jararaca venom over its development. We suggest that not only venom from adult specimens, but also from specimens at other ontogenetic stages should be included in the venom pool used for raising antibodies. Thus, Bothrops antivenin can efficaciously neutralize proteins lacking in the adult venom pool, especially those that promote more intense hemostatic disturbances in victims of newborn snakes.
... Hence, besides ecological implications, knowledge of the natural history and toxin composition of venoms is of fundamental importance for the treatment of bite victims and in the selection of specimens for the preparation of venom pools for antivenom production [35,36]. This is particularly relevant for highly adaptable and widely distributed species, in which intraspecific (ontogenetic, individual, and geographic) venom variability represents a well documented phenomenon [37][38][39] and a source of diversity of the pathological effects of snakebites in different geographical regions [40,41]. Here we sought to characterize the venom phenotypes of adult specimens of subspecies cascavella and collilineatus of Brazilian C. durissus and neonate C. d. terrificus. ...
We report the comparative proteomic and antivenomic characterization of the venoms of subspecies cascavella and collilineatus of the Brazilian tropical rattlesnake Crotalus durissus. The venom proteomes of C. d. collilineatus and C. d. cascavella comprise proteins in the range of 4-115 kDa belonging to 9 and 8 toxin families, respectively. Collilineatus and cascavella venoms contain 20-25 main toxins belonging to the following protein families: disintegrin, PLA(2), serine proteinase, cysteine-rich secretory protein (CRISP), vascular endothelial growth factor-like (VEGF), L-amino acid oxidase, C-type lectin-like, and snake venom metalloproteinase (SVMP). As judged by reverse-phase HPLC and mass spectrometry, cascavella and collilineatus share about 90% of their venom proteome. However, the relative occurrence of the toxin families departs among the two C. durissus subspecies venoms. The most notable difference is the presence of the myotoxin crotamine in some C. d. collilineatus specimens (averaging 20.8% of the total proteins of pooled venom), which is absent in the venom of C. d. cascavella. On the other hand, the neurotoxic PLA(2) crotoxin represents the most abundant protein in both C. durissus venoms, comprising 67.4% of the toxin proteome in C. d. collilineatus and 72.5% in C. d. cascavella. Myotoxic PLA(2)s are also present in the two venoms albeit in different relative concentrations (18.1% in C. d. cascavella vs. 4.6% in C. d. collilineatus). The venom composition accounts for the clinical manifestations caused by C. durissus envenomations: systemic neurotoxicity and myalgic symptoms and coagulation disturbances, frequently accompanied by myoglobinuria and acute renal failure. The overall compositions of C. d. subspecies cascavella and collilineatus venoms closely resemble that of C. d. terrificus, supporting the view that these taxa can be considered geographical variations of the same species. Pooled venom from adult C.d. cascavella and neonate C.d. terrificus lack crotamine, whereas this skeletal muscle cell membrane depolarizing inducing myotoxin accounts for approximately 20% of the total toxins of venom pooled from C.d. collilineatus and C.d. terrificus from Southern Brazil. The possible relevance of the observed venom variability among the tropical rattlesnake subspecies was assessed by antivenomics using anti-crotalic antivenoms produced at Instituto Butantan and Instituto Vital Brazil. The results revealed that both antivenoms exhibit impaired immunoreactivity towards crotamine and display restricted ( approximately 60%) recognition of PLA(2) molecules (crotoxin and D49-myotoxins) from C. d. cascavella and C. d. terrificus venoms. This poor reactivity of the antivenoms may be due to a combination of factors: on the one hand, an inappropriate choice of the mixture of venoms for immunization and, on the other hand, the documented low immunogenicity of PLA(2) molecules. C. durissus causes most of the lethal snakebite accidents in Brazil. The implication of the geographic variation of venom composition for the treatment of bites by different C. durissus subspecies populations is discussed.
... In order to guarantee an appropriate geographical spectrum of effi cacy of an antivenom, it is essential to know in which parts of the country or region the specifi c envenoming is predominant. The control of the neutralizing ability of an antivenom preparation should be performed using pools of well-characterized venoms, taking into account the known causes of intra-specifi c variation in venom composition and antigenicity (35). Currently, there are many lacunae in the preparation and use of venom pools for antivenom standardization and control. ...
... Antivenoms differ from other immunotherapeutics in that the nature of the antigen used for antibody generation varies depending on the species of snake, in contrast to tetanus antitoxin, or diphtheria antitoxin, which use a single specific immunogen. It is increasingly recognized that geographical intraspecies variation in venom composition and immunogenicity may be sufficiently great to affect clinical efficacy of antivenoms (Chippaux et al., 1991;Warrell, 1997;Saravia et al., 2002). The correct composition of venom mixtures to be used in animal immunization for antivenom production is crucial and demands meticulous attention. ...
Snakebite envenoming is a neglected public health challenge of compelling importance in many regions of the world, particularly sub-Saharan Africa, Asia, Latin America and Papua-New Guinea. Addressing the problem of snakebite effectively demands an integrated multifocal approach, targeting complex problems and involving many participants. It must comprise: (a) Acquisition of reliable information on the incidence and mortality attributable to snakebite envenoming, and the number of people left with permanent sequelae. (b) Improvements in production of effective and safe antivenoms, through strategies aimed at strengthening the technological capacity of antivenom manufacturing laboratories. (c) Increasing the capacity of low-income countries to produce specific immunogens(snake venoms) locally, and to perform their own quality control of antivenoms. (d) Commitments from regional producers to manufacture antivenoms for countries where antivenom production is not currently feasible. (e) Implementation of financial initiatives guaranteeing the acquisition of adequate volumes of antivenom at affordable prices in low-income countries. (f) Performance of collaborative studies on the safety and effectiveness of antivenoms assessed preclinically and by properly designed clinical trials. (g) Development of antivenom distribution programmes tailored to the real needs and epidemiological situations of rural areas in each country. (h) Permanent training programmes for health staff, particularly in rural areas where snakebites are frequent.(i) Implementation of programmes to support those people whose snakebites resulted in chronic disabilities. (j) Preventive and educational programmes at the community level, with the active involvement of local organizations and employing modern methods of health promotion. Such an integrated approach, currently being fostered by the Global Snake Bite Initiative of the International Society on Toxinology and by the World Health Organization, will help to alleviate the enormous burden of human suffering inflicted by snakebite envenoming.
... The selection of venoms for antivenom production must therefore be based on epidemiological knowledge on the medical relevance of the various snake species, on the immunological crossneutralization of venom components and, especially, on the meticulous preclinical and clinical assessment of the neutralizing ability of the newly developed antivenoms. Likewise, the preparation of venom pools for immunization must take into consideration the regional intra-and inter-species venom variability that characterizes snake venoms, especially in species showing a wide African distribution (Chippaux et al., 1991;Warrell, 1997;Barlow et al., 2009). ...
A preclinical assessment was performed on the neutralizing efficacy of a whole IgG polyspecific antivenom (EchiTAb-Plus-ICP), designed for the treatment of snakebite envenomings in Nigeria. It was generated by immunizing horses with the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the most medically important species in Nigeria. Antivenom was tested against the venoms of E. ocellatus, Echis leucogaster, Echis pyramidum leakeyi, B. arietans, Bitis gabonica, Bitis rhinoceros and Bitis nasicornis. The neutralization of the venom toxins responsible for the lethal, hemorrhagic, coagulant and local necrotizing activities was assessed, since these are the most significant effects that characterize envenoming by these species. Echis sp venoms exerted lethal, hemorrhagic, coagulant and necrotizing effects, whereas the Bitis sp venoms tested induced lethality, hemorrhage and necrosis, but were devoid of coagulant activity. The antivenom was effective in the neutralization of all effects tested in all venoms. Highest neutralization was achieved against the venoms of E. ocellatus and B. arietans, and the lowest neutralizing potency was against the venom of B. nasicornis, a species that has a low clinical relevance. It is concluded that EchiTAb-Plus-ICP, whilst specifically designed for Nigeria, has a good preclinical neutralizing profile against homologous and heterologous viperid venoms from other sub-Saharan African locations. It therefore constitutes a promising therapeutic option for the treatment of snakebite envenoming in this region.
... Hence, besides ecological implications, knowledge of the natural history and toxin composition of venoms is of fundamental importance for the treatment of bite victims and in the selection of specimens for the preparation of venom pools for antivenom production [35,36]. This is particularly relevant for highly adaptable and widely distributed species, in which intraspecific (ontogenetic, individual, and geographic) venom variability represents a well documented phenomenon [37][38][39] and a source of diversity of the pathological effects of snakebites in different geographical regions [40,41]. Here we sought to characterize the venom phenotypes of adult specimens of subspecies cascavella and collilineatus of Brazilian C. durissus and neonate C. d. terrificus. ...
Crotalus durissus rattlesnakes are responsible for the most lethal cases of snakebites in Brazil. Crotalus durissus collilineatus subspecies is related to a great number of accidents in Southeast and Central West regions, but few studies on its venom composition have been carried out to date. In an attempt to describe the transcriptional profile of the C. durissus collilineatus venom gland, we generated a cDNA library and the sequences obtained could be identified by similarity searches on existing databases. Out of 673 expressed sequence tags (ESTs) 489 produced readable sequences comprising 201 singletons and 47 clusters of two or more ESTs. One hundred and fifty reads (60.5%) produced significant hits to known sequences. The results showed a predominance of toxin-coding ESTs instead of transcripts coding for proteins involved in all cellular functions. The most frequent toxin was crotoxin, comprising 88% of toxin-coding sequences. Crotoxin B, a basic phospholipase A(2) (PLA(2)) subunit of crotoxin, was represented in more variable forms comparing to the non-enzymatic subunit (crotoxin A), and most sequences coding this molecule were identified as CB1 isoform from Crotalus durissus terrificus venom. Four percent of toxin-related sequences in this study were identified as growth factors, comprising five sequences for vascular endothelial growth factor (VEGF) and one for nerve growth factor (NGF) that showed 100% of identity with C. durissus terrificus NGF. We also identified two clusters for metalloprotease from PII class comprising 3% of the toxins, and two for serine proteases, including gyroxin (2.5%). The remaining 2.5% of toxin-coding ESTs represent singletons identified as homologue sequences to cardiotoxin, convulxin, angiotensin-converting enzyme inhibitor and C-type natriuretic peptide, Ohanin, crotamin and PLA(2) inhibitor. These results allowed the identification of the most common classes of toxins in C. durissus collilineatus snake venom, also showing some unknown classes for this subspecies and even for C. durissus species, such as cardiotoxins and VEGF.
... The main function of these toxins is directly related to the immobilization/death of preys and at the same time, to begin digestion of the prey. In human snake bite accidents, these toxins can induce severe or fatal envenoming (Rosenfeld, 1971;Tu, 1996;Warrell, 1996Warrell, , 1997Chippaux and Goyffon, 1998). ...
A comparative study of venoms from juvenile, sub-adult and adult wild Bothrops atrox specimens captured in Manaus region (Brazil) was performed. All venoms tested had acidic pH (5.5) and the human plasma coagulant activity was higher in venoms from juvenile and sub-adult specimens than in adults. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the most intense bands in adult venoms corresponded to polypeptides of 23 and 50kDa. The 23kDa protein was not detected in juvenile venoms. The 23 and 50kDa proteins were purified by two steps of reversed phase-HPLC followed by size exclusion HPLC. Partial amino acid sequence of the 23kDa protein showed homology to metalloproteinases from other snake venoms. Electrospray ionization mass spectrometric analysis (ESI-MS) showed that the 23kDa band contained at least three isoforms of 23030, 23300 and 23645Da. The 50kDa polypeptide was N-terminally blocked for Edman degradation and presented molecular masses ranging from 46.8 to 49.4kDa by ESI-MS. Both proteins were detected by anti-mutalysin II antibodies in immunoblotting assay indicating that they belong to the metalloproteinase family. Immunoblotting analysis also showed that the 23kDa band increased in intensity from juvenile to adult specimens.SDS-PAGE analysis of juvenile and adult venoms following autoproteolysis in pH 7.4 suggested that endogenous venom metalloproteinases can digest the 50kDa metalloproteinase, originating a new protein band of 27kDa. It was also demonstrated in juvenile venoms that the 23kDa band was not the result of proteolytic processing of the 50kDa metalloproteinase.
... Besides their intrinsic biological relevance, studies on the ontogeny of snake venoms have practical clinical and immunological implications (Warrell, 1997). Results obtained with B. atrox and B. asper venoms suggest that, despite the low amount of venom that young specimens may inject in a bite, envenomations by newborns and juveniles may cause prominent vasculotoxic effects, i.e. hemorrhage and edema. ...
The lancehead snakes Bothrops asper and Bothrops atrox inflict 70-90% of the 3000 bites reported every year in Colombia. In this work, the venoms of B. atrox from Meta (Villavicencio, 33 specimens) and B. asper from Antioquia (San Carlos, 45 specimens), all of them born in captivity, were obtained at different ages (0-6 months; 1, 2 and 3-years old) and compared in terms of their pharmacological and immunochemical characteristics. A conspicuous ontogenetic variability was observed in venom samples from both species. Venoms from newborn and juvenile specimens showed higher lethal, hemorrhagic, edema-forming and coagulant activities, whereas venoms from 3-year old specimens showed higher indirect hemolytic, i.e. phospholipase A2 activity, being more significant in the case of B. asper. SDS-polyacrylamide gel electrophoresis of whole venom for both species evidenced a predominance of high mol. mass bands in the venoms from specimens of <1 year of age, with a change towards bands having lower mol. mass as snakes aged. Gel filtration chromatography showed five peaks in the venoms of B. asper of <6 months and in those from 3-year old specimens. Venom of adult specimens showed a higher number of peaks with indirect hemolytic activity than venom of newborn specimens. Polyvalent antivenom produced in Costa Rica recognized all the bands of both venoms from specimens at all ages tested, when assayed by Western blotting.
The unavailability of effective snake antivenom immunoglobulins (antivenoms) to treat the
specific types of snakebite envenomings encountered in various regions of the world has become
a critical health issue at global level. The crisis has reached its greatest intensity in sub-Saharan
Africa, but other regions, such as south-east Asia, are also suffering from a lack of effective and
affordable products.
The complexity of the production of antivenoms, in particular the importance of preparing
appropriate snake venom mixtures for the production of hyperimmune plasma (the source of
antivenom immunoglobulins), the decreasing number of producers and the fragility of the
production systems in developing countries further jeopardize the availability of effective
antivenoms in Africa, Asia, the Middle East and South America. Most of the remaining current
producers are located in countries where the application of quality and safety standards needs to
be improved.
In October 2005, the WHO Expert Committee on Biological Standardization (ECBS) recognized
the extent of the problem and asked the WHO Secretariat to support and strengthen world
capacity to ensure long-term and sufficient supply of safe and efficient antivenoms. In March
2007, snake antivenom immunoglobulins were included in the WHO Model List of Essential
Medicines (1), acknowledging their role in a primary health care system.
Urgent measures are needed to support the design of immunizing snake venom mixtures that can
be used to make the right polyspecific antivenoms for various geographical areas of the world.
Sustainable availability of effective and safe antivenom immunoglobulins should be ensured and
production systems for these effective treatments should be strengthened at global level.
Meaningful preclinical assessment of the neutralizing capacity of snake antivenom
immunoglobulins needs to be done before these products are used in humans and medicines
regulatory authorities should enforce the licensing of these products before they are used in the
population.
The present “WHO Guidelines for the production, control and regulation of snake antivenoms
immunoglobulins” were developed in response to the above-mentioned needs. These Guidelines
cover all the steps involved in the production, control and regulation of venoms and antivenoms,
as well as an Appendix providing detailed information about the distributions of the most
important snake venoms for use in antivenoms preparation in each country, territory or
geographical area. It is hoped that this document, by covering comprehensively the current
existing experience in the manufacture, preclinical and clinical assessment of these products will
serve as a guide to national control authorities and manufacturers in the support of worldwide
production of these essential medicines. The production of snake antivenoms following good
manufacturing practices should be the aim of all countries involved in the manufacture of these
life-saving biological products.
In addition to the need to produce appropriate antivenoms, other issues that need to be addressed
include ensuring that antivenoms are appropriately used and that outcomes for envenomed
patients are improved. This will entail availability of antivenoms and appropriate distribution
policies, affordability of envenoming treatment and training of health workers to allow safe and
effective use of antivenoms and effective management of snakebite envenomings. These
important issues are beyond the scope of this document and will not be further addressed
specifically here, but should be considered as vital components in the care pathway for
envenoming.
The direct estimate of 46,000 snakebite deaths in India in 2005 (1 for every 2 HIV/AIDS deaths), based on verbal autopsies, renders unrealistic the total of only 47,000 snakebite deaths in the whole world in 2010, obtained indirectly as part of the "Global Burden of Disease 2010" study. Persistent underestimation of its true morbidity and mortality has made snakebite the most neglected of all the WHO's "neglected tropical diseases", downgrading its public health importance. Strategies to address this neglect should include the improvement of antivenom, the only specific antidote to envenoming. To accommodate increased understanding of geographical intraspecific variation in venom composition and the range of snake species that are medically important in India, the design of antivenoms (choice of venom sources and species coverage) should be reconsidered. Methods of preclinical and clinical testing should be improved. The relatively new science of venomics involves techniques and strategies for assessing the toxin composition of snake venoms directly through proteomics-centred approaches or indirectly via high-throughput venom gland transcriptomics and bioinformatic analysis. Antivenomics is translational venomics: a proteomics-based protocol to quantify the extent of cross-reactivity of antivenoms against homologous and heterologous venoms. These approaches could revolutionize the preclinical assessment of antivenom efficacy, leading to a new generation of antivenoms that are clinically more effective.
Toxins from the same protein family present in venoms from snakes belonging to different genera often share antigenic determinants. A practical consequence of this circumstance is that it might be possible to formulate on an immunologically sound basis a mixture of venoms for generating antivenoms against a wide range of species. A deep insight into the inter- and intraspecific variation of the antigenic constituents of venoms from snakes of different geographic origin represents the key for designing novel polyvalent pan-generic antivenoms. This review illustrates how proteomic protocols ('venomics' and 'antivenomics') can aid in assessing the crossreactivity of antivenoms against homologous and heterologous venoms, establishing thus the range of clinical application. Recent work showing how the knowledge of evolutionary trends along with venom phenotyping may have an impact in designing a mixture of venoms for immunization aimed to produce a pan-American anti-crotalic antivenom is discussed.
Russell's viper (Daboia russelii russelii) is an important cause of morbidity and mortality in Sri Lanka. In a study in 1985, Haffkine equine polyspecific antivenom in doses up to 20 g proved ineffective in clearing antigenaemia and caused a high incidence of anaphylactoid reactions. A new, monospecific ovine Fabantivenom (Polonga TAb) has been developed against the venom of Sri Lankan Russell's viper and, to assess its safety and efficacy, we carried out (in 1997) an open, randomized comparison of this with the Haffkine antivenom currently in use in the country. Patients with systemic envenoming following Russell's viper bite were randomized to receive an initial intravenous dose of either 1 g of Polonga TAb (n = 23) or 10 g of Haffkine antivenom (n = 20). One dose of Polonga TAb permanently restored blood coagulability in only 9 (41%) of 22 patients and 13 needed repeated doses, whereas the majority (14/20; 70%) had restored coagulability after 1 dose of Haffkine antivenom. There was a tendency towards more rapid resolution of local swelling and systemic manifestations in the Haffkine group. Venom antigenaemia was eliminated more quickly in the Haffkine group and ovine Fab was cleared from the circulation more rapidly than equine F(ab')2. To evaluate safety, patients were closely observed for adverse reactions. Following a severe reaction with Haffkine antivenom all subsequent patients in this group were treated prophylactically with hydrocortisone and chlorpheniramine. Despite this, the incidence of adverse reactions was significantly higher in the Haffkine group compared with the PolongaTAb group (81% compared with 48%) and 4 patients had a severe anaphylactic reaction in the former group. In conclusion, the new antivenom is safer than Haffkine antivenom but, to avoid repeated doses, an initial dose higher than 1 g is needed in the treatment of Sri Lankan Russell's viper envenoming. The safety of this larger dose is the subject of further studies.
A comparative study was performed on the venoms of the crotaline snake Atropoides nummifer from Guatemala and Honduras. SDS-polyacrylamide gel electrophoresis, under reducing conditions, revealed a highly similar pattern of these venoms, and between them and the venom of the same species from Costa Rica. Similar patterns were also observed in ion-exchange chromatography on CM-Shephadex C-25, in which a highly basic myotoxic fraction was present. This fraction was devoid of phospholipase A(2) activity and strongly reacted, by enzyme-immunoassay, with an antiserum against Bothrops asper myotoxin II, a Lys-49 phospholipase A(2) homologue. A basic myotoxin of 16 kDa was isolated to homogeneity from the venom of A. nummifer from Honduras, showing amino acid composition and N-terminal sequence similar to those of Lys-49 phospholipase A(2) variants previously isolated from other crotaline snake venoms. Guatemalan and Honduran A. nummifer venoms have a qualitatively similar toxicological profile, characterized by: lethal; hemorrhagic; myotoxic; edema-forming; coagulant; and defibrinating activities, although there were significant quantitative variations in some of these activities between the two venoms. Neutralization of toxic activities by two commercially-available antivenoms in the region was studied. Polyvalent antivenom produced by Instituto Clodomiro Picado was effective in the neutralization of: lethal; hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A(2) activities, but ineffective against edema-forming activity. On the other hand, MYN polyvalent antivenom neutralized: hemorrhagic; myotoxic; coagulant; defibrinating; and phospholipase A(2) activities, albeit with a lower potency than Instituto Clodomiro Picado antivenom. MYN antivenom failed to neutralize lethal and edema-forming activities of A. nummifer venoms.
The cobras Naja naja and N. kaouthia are abundant in eastern and north-eastern India, accounting for maximum snakebite deaths. Here we report on variation in the composition of Naja kaouthia and N. naja venom from eastern India on corresponding differences in the severity of pathogenesis. These two venoms differ in chromatographic elution profile through Sephadex G-50 and enzyme activity, protein and carbohydrate contents associated with each fraction. The presence of greater amounts of basic phospholipase A2, L-amino acid oxidase and low molecular weight membrane active polypeptides in the N. naja venom makes it more toxic than N. kaouthia venom. A commercial polyvalent antivenom raised against N. naja venom inactivates lethality and variety of toxic effects of homologous venom more effectively than N. kaouthia venom.
Outdated nomenclature and incorrect taxonomic characterisation of snake venoms in the current toxinological literature have serious implications for the replicability of results from snake venom toxin research. The situation has not improved, despite attempts to supply toxinologists with regular updates on snake systematics. Here, we demonstrate the successful extraction of DNA, and subsequent sequencing of the mitochondrial 12S gene, from dried snake venoms. This approach offers a new and potentially straightforward method for accurate species identification. Mitochondrial DNA (mtDNA) sequences isolated from snake venom can be used to clarify or validate snake species identification through comparison against existing sequences in the GenBank database, and through phylogenetic analyses with other sequences. Pooled venoms can also be screened a priori for the presence of multiple species, and the species names on the labels of commercial venoms verified. Moreover, if the species from which the venom sample has been taken is known, and the specimen is available as a voucher, the mtDNA sequence of the haplotype isolated from that species venom sample could serve as a sequence standard (or 'DNA barcode') for that species. Our new method of DNA barcoding venoms ensures the identification of venoms even after future taxonomic changes.
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