BoaγPLI from Boa constrictor Blood is a Broad-Spectrum Inhibitor of Venom PLA2 Pathophysiological Actions.

The use of venom in predation exerts a corresponding selection pressure for the evolution of venom resistance. One of the mechanisms related to venom resistance in animals (predators or prey of snakes) is the presence of molecules in the blood that can bind venom toxins, and inhibit their pharmacological effects. One such toxin type are venom phospholipase A2s (PLA2s), which have diverse effects including anticoagulant, myotoxic, and neurotoxic activities. BoaγPLI isolated from the blood of Boa constrictor has been previously shown to inhibit venom PLA2s that induced myotoxic and edematogenic activities. Recently, in addition to its previously described and very potent neurotoxic effect, the venoms of American coral snakes (Micrurus species) have been shown to have anticoagulant activity via PLA2 toxins. As coral snakes eat other snakes as a major part of their diet, neonate Boas could be susceptible to predation by this sympatric species. Thus, this work aimed to ascertain if BoaγPLI provided a protective effect against the anticoagulant toxicity of venom from the model species Micrurus laticollaris in addition to its ability shown previously against other toxin types. Using a STA R Max coagulation analyser robot to measure the effect upon clotting time, and TEG5000 thromboelastographers to measure the effect upon clot strength, we evaluated the ability of BoaγPLI to inhibit M. laticollaris venom. Our results indicate that BoaγPLI is efficient at inhibiting the M. laticollaris anticoagulant effect, reducing the time of coagulation (restoring them closer to non-venom control values) and increasing the clot strength (restoring them closer to non-venom control values). These findings demonstrate that endogenous PLA2 inhibitors in the blood of non-venomous snakes are multi-functional and provide broad resistance against a myriad of venom PLA2-driven toxic effects including coagulotoxicity, myotoxicity, and neurotoxicity. This novel form of resistance could be evidence of selective pressures caused by predation from venomous snakes and stresses the need for field-based research aimed to expand our understanding of the evolutionary dynamics of such chemical arms race.

cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom.

A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.

Integrated Venomics and Venom Gland Transcriptome Analysis of Juvenile and Adult Mexican Rattlesnakes Crotalus simus, C. tzabcan, and C. culminatus Revealed miRNA-modulated Ontogenetic Shifts.

Adult rattlesnakes within genus Crotalus express one of two distinct venom phenotypes, type I (hemorrhagic) and type II (neurotoxic). In Costa Rican Central American rattlesnake, ontogenetic changes in the concentration of miRNAs modulate venom type II to type I transition. Venomics and venom gland transcriptome analyses showed that adult C. simus and C. tzabcan expressed intermediate patterns between type II and type I venoms, whereas C. culminatus had a canonical type I venom. Neonate/juvenile and adult Mexican rattlesnakes showed notable inter- and intraspecific variability in the number, type, abundance and ontogenetic shifts of the transcriptional and translational venom gland activities. These results support a role for miRNAs in the ontogenetic venom compositional changes in the three congeneric Mexican rattlesnakes. It is worth noting the finding of dual-action miRNAs, which silence the translation of neurotoxic heterodimeric PLA2 crotoxin and acidic PLA2 mRNAs while simultaneously up-regulating SVMP-targeting mRNAs. Dual transcriptional regulation potentially explains the existence of mutually exclusive crotoxin-rich (type-II) and SVMP-rich (type-I) venom phenotypic dichotomy among rattlesnakes. Our results support the hypothesis that alterations of the distribution of miRNAs, modulating the translational activity of venom gland toxin-encoding mRNAs in response to an external cue, may contribute to the mechanism generating adaptive venom variability.

Variability in antivenom neutralization of Mexican viperid snake venoms.

BACKGROUND: Each year, 3,800 cases of snakebite envenomation are reported in Mexico, resulting in 35 fatalities. The only scientifically validated treatment for snakebites in Mexico is the use of antivenoms. Currently, two antivenoms are available in the market, with one in the developmental phase. These antivenoms, produced in horses, consist of F(ab')2 fragments generated using venoms from various species as immunogens. While previous studies primarily focused on neutralizing the venom of the Crotalus species, our study aims to assess the neutralization capacity of different antivenom batches against pit vipers from various genera in Mexico. METHODOLOGY: We conducted various biological and biochemical tests to characterize the venoms. Additionally, we performed neutralization tests using all three antivenoms to evaluate their effectiveness against lethal activity and their ability to neutralize proteolytic and fibrinogenolytic activities. RESULTS: Our results reveal significant differences in protein content and neutralizing capacity among different antivenoms and even between different batches of the same product. Notably, the venom of Crotalus atrox is poorly neutralized by all evaluated batches despite being the primary cause of envenomation in the country's northern region. Furthermore, even at the highest tested concentrations, no antivenom could neutralize the lethality of Metlapilcoatlus nummifer and Porthidium yucatanicum venoms. These findings highlight crucial areas for improving existing antivenoms and developing new products. CONCLUSION: Our research reveals variations in protein content and neutralizing potency among antivenoms, emphasizing the need for consistency in venom characteristics as immunogens. While Birmex neutralizes more LD50 per vial, Antivipmyn excels in specific neutralization. The inability of antivenoms to neutralize certain venoms, especially M. nummifer and P. yucatanicum, highlights crucial improvement opportunities, given the medical significance of these species.

Venom of the neotropical rattlesnake, Crotalus culminatus: Intraspecific variation, neutralization by antivenoms, and immunogenicity in rabbits.

Crotalus culminatus is a medically significant species of rattlesnake in Mexico [1]. While the proteomic composition of its venom has been previously reported for both juvenile and adult specimens, there has been limited research into its functional properties, with only a few studies, including one focusing on coagulotoxicity mechanisms. In this study, we aimed to compare the biochemical and biological activities of the venom of juvenile and adult snakes. Additionally, we assessed antibody production using the venoms of juveniles and adults as immunogens in rabbits. Our findings reveal lethality and proteolytic activity differences between the venoms of juveniles and adults. Notably, juvenile venoms exhibited high proportions of crotamine, while adult venoms displayed a reduction of this component. A commercially available antivenom demonstrated effective neutralization of lethality of both juvenile and adult venoms in mice. However, it failed to neutralize the paralytic activity induced by crotamine, which, in contrast, was successfully inhibited by antibodies obtained from hyperimmunized rabbits. These results suggest the potential inclusion of C. culminatus venom from juveniles in commercial antivenom immunization schemes to generate antibodies targeting this small myotoxin.

Taking the sting out of scorpions: Electrophysiological investigation of the relative efficacy of three antivenoms against medically significant Centruroides species.

In this study, we report the innovative application of whole-cell patch-clamp electrophysiology in assessing broad-spectrum neutralisation by three different antivenoms, of venoms from the medically significant scorpion genus Centruroides. Envenomations by as many as 21 species from the Centruroides genus result in up to 300,000 envenomations per year in Mexico, which poses significant and potentially life-threatening pathophysiology. We first evaluated the in vitro manifestation of envenomation against two human voltage-gated sodium (hNaV) channel subtypes: hNaV1.4 and hNaV1.5, which are primarily expressed in skeletal muscles and cardiomyocytes, respectively. The neutralisation of venom activity was then characterised for three different antivenoms using a direct competition model against the more potent target, hNaV1.4. While broad-spectrum neutralisation was identified, variation in neutralisation arose for Centruroides elegans, C. limpidus, C. noxius and C. suffusus venoms, despite the presence of a number of these venoms within the immunising mixture. This raises questions regarding the truly "broad" neutralisation capacity of the antivenoms. This study not only extends previous validation of the in vitro investigation of antivenom efficacy utilising the whole-cell patch-clamp technique but also underscores the potential of this animal-free model in exploring cross-reactivity, experimental scalability, and most importantly, informing clinical management practices regarding the administration of antivenom in Mexico.

Quantifying venom production: A study on Micrurus snakes in Mexico.

Our study quantifies venom production in nine Mexican coral snake species (Micrurus), encompassing 76 specimens and 253 extractions. Noteworthy variations were observed, with M. diastema and M. laticollaris displaying diverse yields, ranging from 0.3 mg to 59 mg. For animals for which we have length data, there is a relationship between size and venom quantity. Twenty-eight percent of the observed variability in venom production can be explained by snake size, suggesting that other factors influence the amount of obtained venom. These findings are pivotal for predicting venom effects and guiding antivenom interventions. Our data offer insights into Micrurus venom yields, laying the groundwork for future research and aiding in medical response strategies. This study advances understanding coral snake venom production, facilitating informed medical responses to coral snake bites.

Therapeutic itineraries of snakebite victims and antivenom access in southern Mexico.

Access to antivenoms in cases of snakebite continues to be an important public health issue around the world, especially in rural areas with poorly developed health care systems. This study aims to evaluate therapeutic itineraries and antivenom accessibility following snakebites in the states of Oaxaca and Chiapas in southern Mexico. Employing an intercultural health approach that seeks to understand and bridge allopathic and traditional medical perceptions and practices, we conducted field interviews with 47 snakebite victims, documenting the therapeutic itineraries of 54 separate snakebite incidents that occurred between 1977 and 2023. Most victims used traditional remedies as a first line of treatment, often to withstand the rigors of a long journey to find antivenoms. The main obstacles to antivenom access were distance, poor antivenom availability, and cost. Standard antivenom treatment is highly valued and sought after, even as traditional beliefs and practices persist within a cultural framework known as the "hot-cold" system. The findings are crucial for informing future enhancements to antivenom distribution systems, health education initiatives, and other interventions aimed at mitigating the impact of snakebites in the region.

Exploring venom diversity in Mixcoatlus browni and Mixcoatlus barbouri: A comparative analysis of two rare Mexican snake species with crotoxin-like presence.

The genus Mixcoatlus is composed of three species: Mixcoatlus barbouri, M. browni, and M. melanurus, of which the venom composition of M. melanurus, the most common species of the three, has only recently been described. However, very little is known about the natural history of M. barbouri and M. browni, and the venom composition of these two species has remained thus far unexplored. In this study we characterize the proteomic profiles and the main biochemical and toxic activities of these two venoms. Proteomic data obtained by shotgun analysis of whole venom identified 12 protein families for M. barbouri, and 13 for M. browni. The latter venom was further characterized by using a quantitative 'venomics' protocol, which revealed that it is mainly composed of 51.1 % phospholipases A2 (PLA2), 25.5 % snake venom serine proteases (SVSP), 4.6 % l-amino oxidases (LAO), and 3.6 % snake venom metalloproteases (SVMP), with lower percentages other six protein families. Both venoms contained homologs of the basic and acidic subunits of crotoxin. However, due to limitations in M. barbouri venom availability, we could only characterize the crotoxin-like protein of M. browni venom, which we have named Mixcoatlutoxin. It exhibited a lethal potency in mice like that described for classical rattlesnake crotoxins. These findings expand knowledge on the distribution of crotoxin-like heterodimeric proteins in viper snake species. Further investigation of the bioactivities of the venom of M. barbouri, on the other hand, remains necessary.

Comparative Analysis of Alpha-1 Orthosteric-Site Binding by a Clade of Central American Pit Vipers (Genera Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium).

The distribution and relative potency of post-synaptic neurotoxic activity within Crotalinae venoms has been the subject of less investigation in comparison with Elapidae snake venoms. No previous studies have investigated post-synaptic neurotoxic activity within the Atropoides, Metlapilcoatlus, Cerrophidion, and Porthidium clade. Given the specificity of neurotoxins to relevant prey types, we aimed to uncover any activity present within this clade of snakes that may have been overlooked due to lower potency upon humans and thus not appearing as a clinical feature. Using biolayer interferometry, we assessed the relative binding of crude venoms to amphibian, lizard, bird, rodent and human α-1 nAChR orthosteric sites. We report potent alpha-1 orthosteric site binding in venoms from Atropoides picadoi, Metlapilcoatlus occiduus, M. olmec, M. mexicanus, M. nummifer. Lower levels of binding, but still notable, were evident for Cerrophidion godmani, C. tzotzilorum and C. wilsoni venoms. No activity was observed for Porthidium venoms, which is consistent with significant alpha-1 orthosteric site neurotoxicity being a trait that was amplified in the last common ancestor of Atropoides/Cerrophidion/Metlapilcoatlus subsequent to the split by Porthidium. We also observed potent taxon-selective activity, with strong selection for non-mammalian targets (amphibian, lizard, and bird). As these are poorly studied snakes, much of what is known about them is from clinical reports. The lack of affinity towards mammalian targets may explain the knowledge gap in neurotoxic activity within these species, since symptoms would not appear in bite reports. This study reports novel venom activity, which was previously unreported, indicating toxins that bind to post-synaptic receptors may be more widespread in pit vipers than previously considered. While these effects appear to not be clinically significant due to lineage-specific effects, they are of significant evolutionary novelty and of biodiscovery interest. This work sets the stage for future research directions, such as the use of in vitro and in vivo models to determine whether the alpha-1 orthosteric site binding observed within this study confers neurotoxic venom activity.

Ontogenetic change in the venom composition of one Mexican black-tailed rattlesnake (Crotalus molossus nigrescens) from Durango, Mexico.

To corroborate the ontogenetic shift in the venom composition of the Mexican Black-tailed Rattlesnake (Crotalus molossus nigrescens) previously reported through the census approach, we evaluated the shift in the protein profile, lethality, and proteolytic and phospholipase activities of four venom samples obtained in 2015, 2018, 2019, and 2021 from one C. m. nigrescens individual (CMN06) collected in Durango, Mexico. We demonstrated that the venom of C. m. nigrescens changed from a myotoxin-rich venom to a phospholipase A2 and snake venom metalloproteinase-rich venom. Additionally, the proteolytic and phospholipase activities increased with age, but the lethality decreased approximately three times.

Cloud serpent coagulotoxicity: The biochemical mechanisms underpinning the anticoagulant actions of Mixcoatlus and Ophryacus venoms.

Mexico is home to an extreme diversity of herpetofauna, with venomous snakes imposing a significant burden upon public health. However, little is known about the pathophysiological venom actions of a number of potentially medically important species, including those from the genera Mixcoatlus and Ophryacus. Our study aimed to fill this knowledge gap by ascertaining the effects of Mixcoatlus melanurus, Ophryacus smaragdinus and Ophryacus sphenophrys venoms upon the coagulation cascade utilising a series of well-validated coagulation assays. While M. melanurus venom exhibited no significant coagulotoxic activities, both O. smaragdinus and O. sphenophrys venoms exerted multiple coagulotoxic activities upon the coagulation cascade which would be contributing towards a net anticoagulant venom activity. O. sphenophrys significantly inhibited the spontaneous clotting of plasma but O. smaragdinus did not. They differed in that O. sphenophrys inhibited the clotting enzymes factor IXa and factor XIa. However, O. smaragdinus was able to inhibit factor Xa in isolation-assays. Both O. smaragdinus and O. sphenophrys degraded fibrinogen, with O. smaragdinus venom causing a significantly weaker fibrinogen clot than O. sphenophrys. In vitro antivenom efficacy assays were undertaken to ascertain the efficacy of Antivipmyn-Tri antivenom (which is made using Bothrops, Crotalus, and Lachesis venoms). This antivenom was chosen due to the phylogenetic uncertain position of the Ophryacus, but with some molecular genetics' studies placing it as sister to Lachesis. Despite the complexity of the antivenom immunising mixture, the anticoagulant activity of O. sphenophrys venom was relatively poorly neutralised by the antivenom. This work contributes to the understanding of the functional activity of Mixcoatlus and Ophryacus venoms, laying a foundation for future work investigating the coagulotoxins present within Ophryacus venoms in addition to providing data useful for the evidence-based design of clinical management strategies for the envenomed patient.

Characterization of the venom and external morphology of a natural hybrid between Crotalus atrox and Crotalus mictlantecuhtli.

Here we report, for the first time, a natural hybrid between Crotalus atrox and C. mictlantecuhtli based on intermediate characteristics of the external morphology and venom. Morphologically, the individual had characteristics of both parent species. The hybrid's venom exhibited an intermediate composition including the presence of crotoxin which has never been documented in C. atrox but is well documented in C. mictlantecuhtli. The hybrid's venom was highly toxic and showed an intermediate proteolytic activity between the parental species. The two Mexican antivenoms were able to neutralize the hybrid's venom's lethality.

Intraspecific Differences in the Venom of Crotalus durissus cumanensis from Colombia.

Biochemical and biological differences in the venom of Crotalus durissus cumanensis from three ecoregions of Colombia were evaluated. Rattlesnakes were collected from the geographic areas of Magdalena Medio (MM), Caribe (CA) and Orinoquía (OR). All three regionally distributed venoms contain proteases, PLA2s and the basic subunit of crotoxin. However, only crotamine was detected in the CA venom. The highest lethality, coagulant, phospholipase A2 and hyaluronidase activities were found in the MM venom. Also, some differences, observed by western blot and immunoaffinity, were found in all three venoms when using commercial antivenoms. Furthermore, all three eco-regional venoms showed intraspecific variability, considering the differences in the abundance and intensity of their components, in addition to the activity and response to commercial antivenoms.

Differential Antivenom and Small-Molecule Inhibition of Novel Coagulotoxic Variations in Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium American Viperid Snake Venoms.

Within Neotropical pit-vipers, the Mexican/Central-American clade consisting of Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium is a wide-ranging, morphologically and ecologically diverse group of snakes. Despite their prevalence, little is known of the functional aspects of their venoms. This study aimed to fill the knowledge gap regarding coagulotoxic effects and to examine the potential of different therapeutic approaches. As a general trait, the venoms were shown to be anticoagulant but were underpinned by diverse biochemical actions. Pseudo-procoagulant activity (i.e., thrombin-like), characterized by the direct cleavage of fibrinogen to form weak fibrin clots, was evident for Atropoides picadoi, Cerrophidiontzotzilorum, Metlapilcoatlus mexicanus, M. nummifer, M. occiduus, M. olmec, and Porthidium porrasi. In contrast, other venoms cleaved fibrinogen in a destructive (non-clotting) manner, with C. godmani and C. wilsoni being the most potent. In addition to actions on fibrinogen, clotting enzymes were also inhibited. FXa was only weakly inhibited by most species, but Cerrophidion godmani and C. wilsoni were extremely strong in their inhibitory action. Other clotting enzymes were more widely inhibited by diverse species spanning the full taxonomical range, but in each case, there were species that had these traits notably amplified relatively to the others. C. godmani and C. wilsoni were the most potent amongst those that inhibited the formation of the prothrombinase complex and were also amongst the most potent inhibitors of Factor XIa. While most species displayed only low levels of thrombin inhibition, Porthidium dunni potently inhibited this clotting factor. The regional polyvalent antivenom produced by Instituto Picado Clodomiro was tested and was shown to be effective against the diverse anticoagulant pathophysiological effects. In contrast to the anticoagulant activities of the other species, Porthidium volcanicum was uniquely procoagulant through the activation of Factor VII and Factor XII. This viperid species is the first snake outside of the Oxyuranus/Pseudonaja elapid snake clade to be shown to activate FVII and the first snake venom of any kind to activate FXII. Interestingly, while small-molecule metalloprotease inhibitors prinomastat and marimastat demonstrated the ability to prevent the procoagulant toxicity of P. volcanicum, neither ICP antivenom nor inhibitor DMPS showed this effect. The extreme variation among the snakes here studied underscores how venom is a dynamic trait and how this can shape clinical outcomes and influence evolving treatment strategies.

Neutralization of black widow spider (Latrodectus mactans) venom with rabbit polyclonal serum hyperimmunized with recombinant alpha-latrotoxin fragments.

Alpha-latrotoxin (ɑLTx) is the component responsible for causing the pathophysiology in patients bitten by spiders from the genus Latrodectus, commonly known as black widow spiders. The current antivenom used to treat these envenomations in Mexico is produced using the venom of thousands of spiders, obtained through electrical stimulation. This work aimed to produce this protein as well as two of its fragments in a bacterial model, to evaluate their use as immunogens to produce neutralizing hyperimmune sera, in rabbits. ɑLTx is a 130 kDa protein which has not yet been obtained in a soluble active form using bacterial models. In the present work, ɑLTx and two of its fragments, ankyrin domain and amino terminal domain (LTxAnk and LTxNT) were produced in bacteria and solubilized from inclusion bodies using N-lauroyl sarcosine. These three proteins were used for hyperimmunization in order to evaluate their potential as immunogens for the production of neutralizing hyperimmune sera against the complete venom of Latrodectus mactans. The hyperimmune sera obtained using the complete ɑLTx as well as the LTxNT, was capable of preventing death of mice envenomated with 3 LD50s of venom, both in preincubation and rescue experiments. Conversely, the serum obtained using the LTxAnk fragment, generated only partial protection and a delay in the time of death, even with a maximum dose of 450 µL. We therefore conclude that the produced proteins show great potential for their use as immunogens and should be further tested in large animals, such as horses.

Proteomic and toxicological characterization of the venoms of the most enigmatic group of rattlesnakes: The long-tailed rattlesnakes.

The most enigmatic group of rattlesnakes is the long-tailed rattlesnake group, consisting of three species: Crotalus ericsmithi, Crotalus lannomi and Crotalus stejnegeri. These species have been the least studied rattlesnakes in all aspects, and no study on the characterization of their venoms has been carried out to date. Our main objective was to investigate the proteomic composition, as well as some of the biochemical and toxic activities of these venoms, and their neutralization by commercial antivenom. The venom proteome of C. ericsmithi mainly contains metalloproteinases (SVMP; 49.3%), phospholipases A2 (PLA2; 26.2%), disintegrins (Dis; 12.6%), and snake venom serine proteases (SVSP; 6.8%), while C. lannomi venom mainly consists of SVMP (47.1%), PLA2 (19.3%), Dis (18.9%), SVSP (6%) and l-amino acid oxidase (LAAO; 2.6%). For these venoms high lethality was recorded in mice, the most potent being that of C. lannomi (LD50 of 0.99 µg/g body weight), followed by C. ericsmithi (1.30 µg/g) and finally C. stejnegeri (1.79 µg/g). The antivenoms Antivipmyn® from SILANES and Fabotherapic polyvalent antiviperin® from BIRMEX neutralized the lethal activity of the three venoms. Although this group of snakes is phylogenetically related to the C. viridis group, no neurotoxic components (crotoxin or crotoxin-like proteins) common in rattlesnakes were found in their venoms. This study expands current knowledge on the venoms of understudied snake species of the Mexican herpetofauna.

Intraspecific venom variation of Mexican West Coast Rattlesnakes (Crotalus basiliscus) and its implications for antivenom production.

Intraspecific variation in snake venoms has been widely documented worldwide. However, there are few studies on this subject in Mexico. Venom characterization studies provide important data used to predict clinical syndromes, to evaluate the efficacy of antivenoms and, in some cases, to improve immunogenic mixtures in the production of antivenoms. In the present work, we evaluated the intraspecific venom variation of Crotalus basiliscus, a rattlesnake of medical importance and whose venom is used in the immunization of horses to produce one of the Mexican antivenoms. Our results demonstrate that there is variation in biological and biochemical activities among adult venoms and that there is an ontogenetic change from juvenile to adult venoms. Juvenile venoms were more lethal and had higher percentages of crotamine and crotoxin, while adult venoms had higher percentages of snake venom metalloproteases (SVMPs). Additionally, we documented crotoxin-like PLA2 variation in which specimens from Zacatecas, Sinaloa and Michoacán (except 1) lacked the neurotoxin, while the rest of the venoms had it. Finally, we evaluated the efficacy of three lots of Birmex antivenom and all three were able to neutralize the lethality of four representative venoms but were not able to neutralize crotamine. We also observed significant differences in the LD50 values neutralized per vial among the different lots. Based on these results, we recommend including venoms containing crotamine in the production of antivenom for a better immunogenic mixture and to improve the homogeneity of lots.

Neutralization of crotamine by polyclonal antibodies generated against two whole rattlesnake venoms and a novel recombinant fusion protein.

Crotamine is a paralyzing toxin (MW: ~5 kDa) found in different proportions in some rattlesnake venoms (up to 62%). Mexican pit viper antivenoms have shown low immunoreactivity against crotamine, which is an urgent quality to be improved. The objective of this work was to evaluate the ability of a novel recombinant fusion protein composed of sphingomyelinase D and crotamine, and two whole venoms from Crotalus molossus nigrescens and C. oreganus helleri to produce neutralizing antibodies against crotamine. These immunogens were separately used for immunization procedures in rabbits. Then, we generated three experimental antivenoms to test their cross-reactivity via western-blot against crotamine from 7 species (C. m. nigrescens, C. o. helleri, C. durissus terrificus, C. scutulatus salvini, C. basiliscus, C. culminatus and C. tzabcan). We also performed pre-incubation neutralization experiments in mice to measure the neutralizing potency of each antivenom against crotamine induced hind limb paralysis. Our antivenoms showed broad recognition across crotamine from most of the tested species. Also, neutralization against crotamine paralysis symptom was successfully achieved by our three antivenoms, albeit with different efficiencies. Our results highlight the use of crotamine enriched venoms and our novel recombinant fusion protein as promising immunogens to improve the neutralizing potency against crotamine for the improvement of Mexican antivenoms.

Protein composition and biochemical characterization of venom from Sonoran Coral Snakes (Micruroides euryxanthus).

The elapid genus, Micruroides, is considered the sister clade of all New World coral snakes (Genus Micrurus), is monotypic, and is represented by Sonoran Coral Snakes, Micruroides euryxanthus. Coral snakes of the genus Micrurus have been reported to have venoms that are predominantly composed of phospholipases A2 (PLA2) or three finger toxins (3FTx), but the venoms of the genus Micruroides are almost completely unstudied. Here, we present the first description of the venom of M. euryxanthus including identification of some proteins as well as transcriptomic, and biological activity assays. The most abundant components within M. euryxanthus venom are 3FTxs (62.3%) and there was relatively low proportion of PLA2s (14.2%). The venom phenotype supports the hypothesis that the common ancestor of Micrurus and Micruroides had a 3FTx-dominated venom. Within the venom, there were two nearly identical α-neurotoxins (α-Ntx), one of which was designated Eurytoxin, that account for approximately 60% of the venom's lethality to mice. Eurytoxin was cloned, expressed in a soluble and active form, and used to produce rabbit hyperimmune serum. This allowed the analysis of its immunochemical properties, showing them to be different from the recombinant αNTx D.H., present in the venoms of some species of Micrurus. Finally, we observed that the commercial antivenom produced in Mexico for coral snake envenomation is unable to neutralize the lethality from M. euryxanthus venom. This work allowed the classification of Micruroides venom into the 3FTx-predominant group and identified the main components responsible for toxicity to mice.