Potent Venom Research Articles

Subtranscriptome analysis of phospholipases D in Loxosceles venom glands: Confirmation of predominance, intra-species diversity, and description of novel isoforms

Spiders of Loxosceles genus, or Brown spiders produce a potent venom with minimal volume and protein content. Among its toxins, phospholipases D (PLDs) are notable for causing primary local and systemic manifestations observed following envenomation. They degrade cellular phospholipids, mainly sphingomyelin and lysophosphatidylcholine. We present a robust and detailed analysis of PLD transcripts from venom glands of three major clinically relevant South American species—L. intermedia, L. laeta, and L. gaucho—using next-generation sequencing. Results confirmed that PLDs are the most highly expressed toxins, accounting for 65.4 % of expression in L. intermedia, 71.8 % in L. gaucho, and 50.4 % in L. laeta. These findings further support the idea that these enzymes form a protein family both within and across species. Eighteen contigs for PLDs were found for L. gaucho, 24 for L. intermedia, and 21 for L. laeta. A detailed analysis revealed that, although all contigs display conserved amino acid residues directly involved in catalysis, magnesium coordination, and substrate affinity, they also possess distinct primary sequences with important substitutions. Such data reinforces the hypothesis that these toxins may act synergistically. Furthermore, new PLD sequences were identified within the contigs. For L. intermedia, 14 potential new isoforms were identified; 16 for L gaucho; and 16 novel sequences for L. laeta. This indicates that there is still a wealth of undisclosed information about these toxins. These data will help identify structural and functional differences among these proteins, support future functional studies, and to the comprehensive understanding of the mechanism of action of PLDs.

Prey specificity of predatory venoms.

Venom represents a key adaptation of many venomous predators, allowing them to immobilise prey quickly through chemical rather than physical warfare. Evolutionary arms races between prey and a predator are believed to be the main factor influencing the potency and composition of predatory venoms. Predators with narrowly restricted diets are expected to evolve specifically potent venom towards their focal prey, with lower efficacy on alternative prey. Here, we evaluate hypotheses on the evolution of prey-specific venom, focusing on the effect of restricted diet, prey defences, and prey resistance. Prey specificity as a potential evolutionary dead end is also discussed. We then provide an overview of the current knowledge on venom prey specificity, with emphasis on snakes, cone snails, and spiders. As the current evidence for venom prey specificity is still quite limited, we also overview the best approaches and methods for its investigation and provide a brief summary of potential model groups. Finally, possible applications of prey-specific toxins are discussed.

Potential therapeutic biomolecules of hymenopteran venom against SARS-CoV-2 from Egyptian patients

The therapeutic potential of insect-derived bioactive molecules as anti-SARS-CoV-2 agents has shown promising results. Hymenopteran venoms, notably from Apis mellifera (honeybee) and Vespa orientalis (oriental wasp), were examined for the first time in an in vitro setting for their potential anti-COVID-19 activity. This assessment utilized an immunodiagnostic system to detect the SARS-CoV-2 nucleocapsid antigen titer reduction. Further analyses, including cytotoxicity assays, plaque reduction assays, and in silico docking-based screening, were performed to evaluate the efficacy of the most potent venom. Results indicated that bee and wasp venoms contain bioactive molecules with potential therapeutic effects against SARS-CoV-2.Nevertheless, the wasp venom exhibited superior efficacy compared to bee venom, achieving a 90% maximal (EC90) concentration effect of antigen depletion at 0.184 mg/mL, in contrast to 2.23 mg/mL for bee venom. The cytotoxicity of the wasp venom was assessed on Vero E6 cells 48 h post-treatment using the MTT assay. The CC 50 of the cell growth was 0.16617 mg/mL for Vero E6 cells. The plaque reduction assay of wasp venom revealed 50% inhibition (IC50) at a 0.208 mg/mL concentration. The viral count at 50% inhibition was 2.5 × 104 PFU/mL compared to the initial viral count of 5 × 104 PFU/mL. In silico data for the wasp venom revealed a strong attraction to binding sites on the ACE2 protein, indicating ideal interactions. This substantiates the potential of wasp venom as a promising viral inhibitor against SARS-CoV-2, suggesting its consideration as a prospective natural preventive and curative antiviral drug. In conclusion, hymenopteran venoms, particularly wasp venom, hold promise as a source of potential therapeutic biomolecules against SARS-CoV-2. More research and clinical trials are needed to evaluate these results and investigate their potential for translation into innovative antiviral therapies.

Anticancer potency of Egyptian venom snakes on MCF-7 and HepG2 carcinoma cells.

Breast and hepatic cancers are the leading incidences in the globe occurring of the human sufferings from various cancers. Snake venoms have been reported to provide effective therapeutic agents. The current study investigates the anticancer potency of Egyptian venoms snakes on two cells: breast cancer cells (MCF-7) and hepato-cancer cells (HepG2) (In vitro assay). The examined venoms were more potent on MCF-7 than HepG2 cells. Their inhibition % on MCF-7 ranged from 71.47 to 99.02% with medium inhibition concentrations (IC50s): 3.48, 3.60, 3.70, 4.33, and 4.49 μg/ml for venoms: Echis pyramid (E.H), Cerastes vipera (C.V), Naja haje (N.H), Echis coloratus (E.C), and Cerastes cerastes (C.C), respectively. The values of IC50s on HepG2 were 4.32, 17.77, 59.72, 63.75, and 217.90 μg/ml for toxins: E.C, E.P, C.V, C.C, and N.H, respectively. Some biomarkers were conducted to investigate the apoptotic effects of toxins into the cells. Increasing profiles of lactate dehydrogenase (LDH) activity and levels of glutathione content (GSH) and malodialdhyde (MDA) as well as repairment of DNA indicated such these actions. So, more reliable investigations on these venoms were needed to provide intelligent therapeutic agent for cancer treatment.

Venoms with oral toxicity towards insects

Animal venoms are a promising source of potential bioinsecticides. To find hits with pronounced oral insect toxicity, we screened 82 venoms using Achroia grisella (Lepidoptera) and Tenebrio molitor (Coleoptera) larvae, and adult Drosophila melanogaster (Diptera). We also injected the most potent venoms in adult D. melanogaster to compare their efficiency in different routes of administration. 18 venoms from spiders and snakes show high oral toxicity and can be further exploited to isolate new insecticides.

Congruence between ultraconserved element-based matrices and phylotranscriptomic datasets in the scorpion Tree of Life.

Scorpions are ancient and historically renowned for their potent venom. Traditionally, the systematics of this group of arthropods was supported by morphological characters, until recent phylogenomic analyses (using RNAseq data) revealed most of the higher-level taxa to be non-monophyletic. While these phylogenomic hypotheses are stable for almost all lineages, some nodes have been hard to resolve due to minimal taxonomic sampling (e.g. family Chactidae). In the same line, it has been shown that some nodes in the Arachnid Tree of Life show disagreement between hypotheses generated using transcritptomes and other genomic sources such as the ultraconserved elements (UCEs). Here, we compared the phylogenetic signal of transcriptomes vs. UCEs by retrieving UCEs from new and previously published scorpion transcriptomes and genomes, and reconstructed phylogenies using both datasets independently. We reexamined the monophyly and phylogenetic placement of Chactidae, sampling an additional chactid species using both datasets. Our results showed that both sets of genome-scale datasets recovered highly similar topologies, with Chactidae rendered paraphyletic owing to the placement of Nullibrotheas allenii. As a first step toward redressing the systematics of Chactidae, we establish the family Anuroctonidae (new family) to accommodate the genus Anuroctonus.

A history of accidental widow spider (genus Latrodectus ) introductions on the island of Ireland with a new irish record for Latrodectus hesperus (Chamberlin and Ivie, 1935), and additional records of Latrodectus geometricus (C.L. Koch, 1841).

ABSTRACT: The widow spiders of the genus Latrodectus (Araneae: Theridiidae) have a well-documented global medical significance due to the potent venom many of them possess. This medical importance partially stems from the global range expansion the genus has undergone, facilitated in part by human trade. This expansion has had an impact on human health in countries where Latrodectus spiders establish populations. Ireland has a detailed report history of accidental widow spider introductions, spanning over 30 years, which assists in understanding the potential such species have for establishing. Here we present three additional cases: a second and third record of Latrodectus geometricus (C.L. Koch, 1841) and the first record of Latrodectus hesperus (Chamberlin and Ivie, 1935). We also review all published reports of Latrodectus introductions in Ireland and discuss the potential for populations to establish. While case histories suggest that L. hesperus and L. geometricus can survive Irish winters, it is not certain that they can complete an egg-to-egg life cycle or even reproduce. Nevertheless, it seems possible they could do so in favourable micro-habitats in anthropogenic habitats such as hot houses. Identification of instances of Latrodectus introductions into Ireland is essential to prevent potential establishments that could lead to envenoming cases.

The complete genome sequence of Androctonus mauritanicus, the Moroccan black thick-tailed scorpion.

Androctonus mauritanicus is a large scorpion indigenous to North Africa. Notable for its extremely potent venom, it is responsible for several human deaths a year. We present the whole genome sequence of this species. Illumina sequencing was performed on a genetic sample from a single wild-caught individual. The reads were assembled using a de novo method followed by a finishing step. The raw and assembled data are publicly available via GenBank: Sequence Read Archive (SRR10738938) and Assembly (GCA_011317285).

Spiders feeding on vertebrates is more common and widespread than previously thought, geographically and taxonomically

According to a recent global literature survey, a total of 39 out of the 129 known spider families (∼30%) contain species capable of capturing vertebrate prey. The finding that the percentage of spider families engaged in vertebrate predation is so high is novel. Two groups of vertebrate-eating spiders are distinguished: “habitual vertebrate-eaters” vs. “occasional vertebrate-eaters”. The habitual vertebrate-eaters comprise ten spider families (Araneidae, Atracidae, Ctenidae, Lycosidae, Nephilidae, Pisauridae, Theraphosidae, Theridiidae, Trechaleidae, and Sparassidae) to which can be attributed 91% of all reported vertebrate predation incidents. The habitual vertebrate-eaters have evolved prey-capture adaptations such as (1) sufficient physical strength coupled with large body size, (2) the use of potent venoms, and (3) the use of highly efficient prey-catching webs. By contrast, unexpected feeding on vertebrates by the occasional vertebrate-eaters (i.e., Actinopodidae, Agelenidae, Amaurobiidae, Anyphaenidae, Barychelidae, Clubionidae, Corinnidae, Ctenizidae, Cyrtaucheniidae, Deinopidae, Desidae, Dipluridae, Eresidae, Filistatidae, Gnaphosidae, Haplonoproctidae, Linyphiidae, Liocranidae, Miturgidae, Oxyopidae, Pholcidae, Porrhothelidae, Salticidae, Selenopidae, Sicariidae, Sparassidae, Tetragnathidae, and Thomisidae) might be considered as chance events that took place when a tiny vertebrate crossed the path of an opportunistic spider. For a few families (e.g., Idiopidae) their status as habitual or occasional vertebrate predators is still unclear. In conclusion, our survey unveiled a large number of spider taxa previously not anticipated to feed on vertebrate prey. These findings improve our general understanding of spider feeding ecology and provide a first assessment of the significance of vertebrate prey as a food source for spiders.

Anti-trypanosomal Activity of Bufonidae (Toad) Venom Crude Extract on Trypanosoma brucei brucei in Swiss Mice

Trypanosomiasis afflicts about 6 ~ 7 million people globally and to a large extent impedes livestock production in Africa. Naturally, trypanosomal parasites undergo genetic mutation and have developed resistance over a wide range of therapies. The utilization of animals and plants products has presented therapeutic potential for identifying novel anti-trypanosomal drugs. This study evaluated toad venom for anti-trypanosomal potency invivo in Swiss mice. Toads were collected from July to August 2019. The acute oral toxicity and biochemical characterization of the toad venom were determined. The experimental mice were administered various doses (130 mg/kg, 173 mg/kg and 217 mg/kg) of the toad venom crude extract and 0.75 mg/mL of Diamizan Plus standard drug for the treatment of trypanosomiasis, once daily for 3 days. The in-vivo anti-trypanosomal activity was evaluated by a curative test, after infecting the mice with Trypanosoma brucei brucei. The pre-patent period was 72 hours before treatment commenced. The overall results showed that trypanosomal load was highest in the control group while the group treated with Diamizan drug had the least trypanosomal load. As such, the mean trypanosomal load in relation to treatments showed a very high significant difference (P<0.05). Also, the mean trypanosomal load in Swiss mice in relation to the highest dosage of toad venom versus Diamizan drug showed a very high significant difference (P<0.05). The mean change in relation to the haematological parameters across treatments groups varied significantly (P<0.05) with the exception of Hb which showed no significant difference (P>0.05) across treatment groups. The over 50% reduction in the trypanosomal load in the 130 mg/kg group in comparison with the control group brings to bare the anti-trypanosomal potency of the toad venom. The anti-trypanosomal activity demonstrated by the toad venom has provided basis for development of new therapeutic agents from different toad species. The study recommends further studies (both in-vivo and invitro) followed by the characterization of the active compounds present in the toad venom responsible for the anti-tyrpanosomal activity observed alongside the management and conservation of these species.

Proteomic insight into the venom composition of the largest European rear-fanged snake, Malpolon monspessulanus monspessulanus

Snake envenomations constitute a worldwide neglected tropical disease, with the vast majority of lethal bites inflicted by front-fanged snakes from the viperid and elapid groups. Rear-fanged snakes (colubrids) were often considered harmless and as a result, are much less studied, but several documented deaths have suggested potent venom in this group as well. The largest European snake (Malpolon monspessulanus monspessulanus), known as the “Montpellier snake”, is such a rear-fanged snake that belongs to the Lamprophiidae family. Its venom remains largely unknown but cases of envenomation with neurological symptoms have been reported. Here, we provide the first insights into the composition of its venom using mass spectrometry methods. First, liquid chromatography coupled mass spectrometry analysis of the manually collected venom samples reveals a complex profile, with the majority of masses encompassing the range 500–3000 Da, 4000–8000 Da, and 10 000–30 000 Da. Next, shotgun proteomics allowed the identification of a total of 42 different known families of proteins, including snake venom metalloproteinases, peptidase M1, and cysteine-rich secretory proteins, as the most prominent. Interestingly, three-finger toxins were not detected, suggesting that neurotoxicity may occur via other, yet to be determined, toxin types. Overall, our results provide the basis for a better understanding of the effects of a peculiar snake venom on human symptomatology, but also on the main prey consumed by this species.

A poisonous shield, a potent venom: these worms mean business

A poisonous shield, a potent venom: these worms mean business

Acute Venom Toxicity Determinations for Five Iranian Vipers and a Scorpion

Background: Poisoning due to the bites and stings of venomous snakes and scorpions is a neglected public health problem, particularly in rural areas. Poor health facilities and inadequate knowledge of health care personnel are the major factors that result in envenomated human victims not receiving adequate care and medical attention. There is a great need for up-to-date and effective healthcare knowledge and awareness of the potency and lethality of venomous creatures in Iran. Assessment of the potency, acute toxicity, and lethal effects of venomous creatures come from a variety of specific tests, such as the 50% median lethal dose (LD50) and ample animal experimentations. Methods: In the present study, using modified Reed-Muench method, the LD0, LD50, and LD100 values of the venoms from five Iranian vipers and one scorpion were determined. The studied venomous creatures were: Macrovipera lebetina, Vipera albicornuta, Vipera raddei, Caucasicus intemedius agkistrodon, Montivipera latifii, and one scorpion Hemiscorpius lepturus. The venoms were injected in Albino mice (n=204) intraperitoneally, and their toxicities determined. Results: The results revealed that the LD50 values of the above-mentioned creatures were 3.87, 2.05, 1.63, 1.45, 0.84, and 6.33 mg/kg, respectively. Among the vipers, M. latifii had the most potent venom while M. lebetina’s venom had the lowest toxicity. Conclusion: Theoretically, the determined LD50 values provide for objective comparisons of the toxicity among of the venoms. However, comparison becomes complicated due to variations in the venoms’ LD50. Further, based on the venoms’ toxicity levels, H. lepturus’ venom caused the lowest toxicity in the Albino mice.

Scorpion Species with Smaller Body Sizes and Narrower Chelae Have the Highest Venom Potency.

Scorpionism is a global health concern, with an estimation of over one million annual envenomation cases. Despite this, little is known regarding the drivers of scorpion venom potency. One widely held view is that smaller scorpions with less-developed chelae possess the most potent venoms. While this perception is often used as a guide for medical intervention, it has yet to be tested in a formal comparative framework. Here, we use a phylogenetic comparative analysis of 36 scorpion species to test whether scorpion venom potency, as measured using LD50, is related to scorpion body size and morphology. We found a positive relationship between LD50 and scorpion total length, supporting the perception that smaller scorpions possess more potent venoms. We also found that, independent of body size, scorpion species with long narrow chelae have higher venom potencies compared to species with more robust chelae. These results not only support the general perception of scorpion morphology and potency, but also the presence of an ecology trade-off with scorpions either selected for well-developed chelae or more potent venoms. Testing the patterns of venom variations in scorpions aids both our ecological understanding and our ability to address the global health burden of scorpionism.

Biophysical studies on latarcins: antimicrobial peptides from spider venom

The ant spider Lachesena tarabaevi produces one of the most potent venom that is largely composed of membrane active peptides. A group of seven linear antimicrobial peptides isolated from the venom of this spider which belong to the latarcin family are reported to be cytolytic and highly membrane active[1]. However, there is no single study where the secondary structure, membrane alignment of all peptides have been systematically addressed. Here, using circular dichroism(CD) spectroscopy, oriented CD, solid-state 15N-NMR and fluorescence spectroscopy, we have characterized the secondary structure and helix alignment of all seven peptides in their membrane-bound state [2].

Phylogenetic Relationships of Scorpion Compsobuthus matthiesseni Based on Sequences of Internal Transcribed Spacer 2 Gene from Khuzestan Province, Iran.

Buthidae family includes scorpions with highly potent venom such as Compsobuthus matthiesseni is important due to the prevalence of scorpion stings in Khuzestan Province, Iran. Morphometric comparison of males and females (n=5 each) showed that the body and carapas of the females were longer and wider (32.57±0.23 mm and 3.75±0.22 mm, respectively) than those of males (28.89±0.25 mm and 3.55±0.12 mm, respectively). From the seven specimens of C. matthiesseni scorpion, 410-bp gene fragments of ribosomal internal transcribed spacer 2 were amplified by polymerase chain reaction. The specimens of CM1 and CM2 (isolated from Baghmalek, Khuzestan) were in the same group with bootstrap values of 87%. Nevertheless, CM4 and CM3 (isolated from Shushtar and Bidroobe, Khuzestan) with bootstrap values of 73% and 62% were separated from the two specimens of Baghmalek, respectively. The two specimens CF3 and CM5 (isolated from Masjed Soleiman, Khuzestan) with bootstrap values of 88% were placed next to each other in a separate group. CF2 was separated from the rest of the specimens with a bootstrap value of 54%. Out of the seven scorpions that were examined, six specimens (CM1, CM2, CM3, CM4, CM5, and CF3) showed the greatest similarity between 1.1% and 4%. However, the genetic distance between CF2 and the rest of the specimens was at the range of 10.8-14.2%. It can be concluded that all C. matthiesseni scorpions from Khuzestan Province belonged to one species; nonetheless, differences were observed within the species, especially in the case of CF2, which might be intraspecies.

Diversity of Conidae (Caenogastropoda: Conoidea) from the Kerala Coast, India

ABSTRACT The Conidae are predatory marine gastropods and they produce one of the most potent venoms in nature. They are ecologically vital for regulating trophic positions and act as primary carnivores in oceans. Amateur shell collectors target some species and ecosystems are intensively exploited by collectors of ornamental shells and commercial traders. Moreover, increasing coastal development, habitat modification and fragmentation, and overexploitation of resources threaten cone snail diversity. This study documents 46 species of Conidae from the Kerala coast, India. Fifteen species are newly recorded from the coast, and one species is reported for the first time from the Indian coast. We also revalidate Conus ceylanensis and Conus bizona.

Expression of Brown and Southern Black Widow Spider (Araneae: Theridiidae) Latrotoxins Is Tissue- and Life Stage-Specific for α-Latroinsectotoxins and δ-Latroinsectotoxins and Is Ubiquitous for α-Latrotoxins.

Widow spiders are widely known for their potent venom toxins that make them among the few spiders of medical concern. The latrotoxins are the most well-studied widow toxins and include both the vertebrate-specific latrotoxins and the insect-specific latroinsectotoxins (LITs). Previous studies have shown that toxins are not limited to expression in the venom glands of adult spiders; however, gaps exist in latrotoxin screening across all life stages for brown widows, Latrodectus geometricus and southern black widows, Latrodectus mactans. In this study, we screened male and female venom gland, cephalothorax, and abdomen tissues, spiderling cephalothorax and abdomen tissues, and eggs of both L. geometricus and L. mactans, for the presence of three latrotoxins: α-latrotoxin (α-LTX), and α- and δ-latroinsectotoxins (α/δ-LITs). Widows were locally collected. Extracted RNA was used to prepare cDNA that was analyzed by PCR for the presence or absence of latrotoxin expression. Results show that expression profiles between the two species are very similar but not identical. Expression of α-LTX was found in all life stages in all tissues examined for both species. For both species, no LIT expression was detected in eggs and variable patterns of α-LIT expression were detected in spiderlings and adults. Notably, δ-LIT could only be detected in females for both species. Our results show that latrotoxin expression profiles differ within and between widow species. Data on their expression distribution provide further insight into the specific latrotoxins that contribute to toxicity profiles for each life stage in each species and their specific role in widow biology.

Phylogenetic and Selection Analysis of an Expanded Family of Putatively Pore-Forming Jellyfish Toxins (Cnidaria: Medusozoa).

Many jellyfish species are known to cause a painful sting, but box jellyfish (class Cubozoa) are a well-known danger to humans due to exceptionally potent venoms. Cubozoan toxicity has been attributed to the presence and abundance of cnidarian-specific pore-forming toxins called jellyfish toxins (JFTs), which are highly hemolytic and cardiotoxic. However, JFTs have also been found in other cnidarians outside of Cubozoa, and no comprehensive analysis of their phylogenetic distribution has been conducted to date. Here, we present a thorough annotation of JFTs from 147 cnidarian transcriptomes and document 111 novel putative JFTs from over 20 species within Medusozoa. Phylogenetic analyses show that JFTs form two distinct clades, which we call JFT-1 and JFT-2. JFT-1 includes all known potent cubozoan toxins, as well as hydrozoan and scyphozoan representatives, some of which were derived from medically relevant species. JFT-2 contains primarily uncharacterized JFTs. Although our analyses detected broad purifying selection across JFTs, we found that a subset of cubozoan JFT-1 sequences are influenced by gene-wide episodic positive selection compared with homologous toxins from other taxonomic groups. This suggests that duplication followed by neofunctionalization or subfunctionalization as a potential mechanism for the highly potent venom in cubozoans. Additionally, published RNA-seq data from several medusozoan species indicate that JFTs are differentially expressed, spatially and temporally, between functionally distinct tissues. Overall, our findings suggest a complex evolutionary history of JFTs involving duplication and selection that may have led to functional diversification, including variability in toxin potency and specificity.

Biogeographic venom variation in Russell's viper (Daboia russelii) and the preclinical inefficacy of antivenom therapy in snakebite hotspots.

BackgroundSnakebite in India results in over 58,000 fatalities and a vast number of morbidities annually. The majority of these clinically severe envenomings are attributed to Russell’s viper (Daboia russelii), which has a near pan-India distribution. Unfortunately, despite its medical significance, the influence of biogeography on the composition and potency of venom from disparate D. russelii populations, and the repercussions of venom variation on the neutralisation efficacy of marketed Indian antivenoms, remain elusive.MethodsHere, we employ an integrative approach comprising proteomic characterisation, biochemical analyses, pharmacological assessment, and venom toxicity profiling to elucidate the influence of varying ecology and environment on the pan-Indian populations of D. russelii. We then conducted in vitro venom recognition experiments and in vivo neutralisation assays to evaluate the efficacy of the commercial Indian antivenoms against the geographically disparate D. russelii populations.FindingsWe reveal significant intraspecific variation in the composition, biochemical and pharmacological activities and potencies of D. russelii venoms sourced from five distinct biogeographic zones across India. Contrary to our understanding of the consequences of venom variation on the effectiveness of snakebite therapy, commercial antivenom exhibited surprisingly similar neutralisation potencies against the majority of the investigated populations, with the exception of low preclinical efficacy against the semi-arid population from northern India. However, the ability of Indian antivenoms to counter the severe morbid effects of Daboia envenoming remains to be evaluated.ConclusionThe concerning lack of antivenom efficacy against the north Indian population of D. russelii, as well as against two other ‘big four’ snake species in nearby locations, underscores the pressing need to develop pan-India effective antivenoms with improved efficacy in high snakebite burden locales.