Influence of sexual dimorphism on venom composition in Bothrops asper and Crotalus simus (Serpentes: Viperidae) and its potential implications on the snake antivenom production.

This work is aimed to bring insights on the potential sexual dimorphism differences on the venom composition of Bothrops asper and Crotalus simus to expand the knowledge of the venom variability that might improve the antivenom design. Biological characterization of venoms of each sex in both species did not show significant qualitative differences. Considerations on the sexual venom variations in these species are not relevant for choosing the snake donors for venom production.

Intraspecific variability of the Central American rattlesnake (Crotalus simus) venom and its usefulness to obtain a representative standard venom.

Snake venoms are mixtures of proteins whose physicochemical features confer them toxicity and immunogenicity. Animals (e.g., horses or sheep) immunized with snake venoms produce antibodies towards the venom proteins. Since these antibodies can neutralize the venom toxicity, they have been used to formulate snake antivenoms. The efficacy of the antivenoms is widely accepted, and standard venoms are expected to be representative of the snake's population that inhabit in the region where the antivenom is intended to be used. The representativeness of a single venom collected from a Crotalus simus snake, and its usefulness as standard venom to produce an antivenom is evaluated. The use of an "average venom" might be as representative of the population intended to be used, as the standard venom composed by many venom samples. Variations in the relative abundance concentration of crotoxin in the C. simus leads to different clinical manifestations, as well as differences in the neutralization efficacy of the antivenoms. A monovalent anti-Cs antivenom was produced from a single venom C. simus specimen, and its efficacy in neutralizing the lethal activity of 30 C. simus snakes was tested. Despite the variations in the relative abundance content of crotoxin found in the proteomes, the monovalent anti-Cs antivenom was successful in neutralize the toxicity caused by the variations on the venom composition of three different snake population used. Interestingly, it seems that the sex is not a key factor in the lethality of the venoms tested. The concept of representative venom mixtures for immunization should be revised for the case of C. simus on the populations found in Costa Rica, since it might use as less as one representative individual whose venom covers the mainly toxic enzymes.

Expanding the neutralization scope of the Central American antivenom (PoliVal-ICP) to include the venom of Crotalus durissus pifanorum.

The proteomic, enzymatic, toxicological, and immunogenic profiles of the venom of C. d. pifanorum were studied. It was found that venom of C. d. pifanorum is composed of 63% phospholipases A2 (PLA2s), 13% serine proteinases (SVSPs), 8% bradykinin-potentiating peptides (BPPs), 4% L-amino acid oxidases (LAAOs), 3% metalloproteinases (SVMPs), and other minor components. This composition allows the venom to exert lethal, PLA2, myotoxic, coagulant and defibrinogenating activities, but no azocaseinolytic or hemorrhagic activities. The addition of C. d. pifanorum venom to the group of venoms used as immunogens to produce the Central American antivenom PoliVal-ICP (i.e., venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys) resulted in 1) the expansion of the neutralization scope of the antivenom to cover the venom of C. d. pifanorum and other antigenically related venom (i.e., C. s. scutulatus venom), 2) improvement of the neutralizing potency towards the venom of C. simus, and 3) no significant reduction of the neutralization of venoms of B. asper and L. stenophrys. It was concluded that supplementation of the immunogens used to produce PoliVal-ICP with the venom of C. d. pifanorum is a viable alternative to expand the neutralization scope of the antivenom. BIOLOGICAL SIGNIFICANCE: The venom of Crotalus durissus pifanorum from Venezuela has a proteomic profile like those of other subspecies of the South American rattlesnake C. durissus, with predominance of phospholipases A2 (especially crotoxin) and serine proteinases. This explains a toxicological profile characterized by neurotoxicity, myotoxicity, and coagulopathies, but being devoid of hemorrhagic activity. The antivenom used in Central America (PoliVal-ICP) includes the venom of C. simus, which has a different composition, in the immunizing strategy. Accordingly, this antivenom does not neutralize C. d. pifanorum venom. The addition of C. d. pifanorum venom to the immunizing mixture of PoliVal-ICP expands the neutralizing scope of this antivenom, to cover additional rattlesnake venoms, while not affecting the response to C. simus, Bothrops asper and Lachesis stenophrys venoms. This represents an improvement of the current PoliVal-ICP.

Morphological alterations caused by manual venom extraction on the main venom gland of Bothrops asper and Crotalus simus snakes (Serpentes: Viperidae): Long-term implications for antivenom production.

The only scientifically validated treatment for snakebite envenomation is the administration of antivenoms. For their production, small quantities of snake venom are injected in animals to elicit a specific antibody response. Snakes are kept in captivity, and their venom is regularly extracted to assure antivenom access. It has already been reported that the pressure exerted upon the venom gland during this extraction can cause tissue damage and fibrosis, leading to a decrease in the venom yield. We described the histopathology of venom glands for B. asper and C. simus snakes used for antivenom production. Based on these reported tissue abnormalities, we quantify the tissue injury by a generated damage-SCORE and fibrosis. A variety of histopathological damages were found such as fibrosis, edema, necrosis, hemorrhage, and formation of anomalous structures, especially in C. simus, which is more prone to suffer severe damage. The level and severity of the damage depend on the frequency and the number of venom extractions. Furthermore, we design an experimental intensive venom extraction scheme with which we could confirm the causality of these effects. In addition to the histopathological damages, the LD50 and biochemical venom composition were also affected giving experimental evidence that the venom extraction not only causes tissue damage but also affects the composition stability and toxicity of the venom. In order to produce quality and effective antivenoms, an improvement of the management of snake collections could be established, such as rotation groups to assure the quality of the venom yielded.

Differences in plasmatic butyrylcholinesterases (BChE) values between Pacific and Caribbean populations of terciopelo (Bothrops asper) in Costa Rica.

The terciopelo (Bothrops asper) inhabits human-modified environments such as agricultural areas, becoming more prone to be exposed to organophosphate insecticides. These chemicals can inhibit plasmatic butyrylcholinesterases (BChE) activity in B. asper. Caribbean and Pacific populations of B. asper belong to two divergent lineages that exhibit robust genetic partitioning; however, differences across versants in biochemistry and hematology have not yet been examined, especially in BChE variations. This study aims to evaluate the differences of BChE plasmatic values, hematology parameters, and some biochemical analytes as biomarkers in the presence of organophosphates between the Pacific and Caribbean population of B. asper in Costa Rica. A total of 89 snakes (41 Pacific and 48 Caribbean) were used, and hematology parameter, albumin, aspartate aminotransferase (AST), total protein and BChEs were evaluated. Differences in hemoglobin content, thrombocytes, white cell count, AST, and BChE values were found between both versants. Intrinsic genetic factors might influence the variation found in BChE and AST values in the snakes sampled from both versants; moreover, understanding this variation in BChE and AST values across the B. asper's distribution can be useful in future ecotoxicology, biomonitoring, genetic and other clinical/health studies.

First look into the venom of Roatan Island's critically endangered coral snake Micrurus ruatanus: Proteomic characterization, toxicity, immunorecognition and neutralization by an antivenom.

A proteomic and toxicological study of the venom from one specimen of Micrurus ruatanus, a critically endangered coral snake species endemic to Roatan Island, Honduras, was carried out. Immunorecognition and neutralization of venom lethality by an anticoral antivenom was also evaluated. Forty peaks were collected from RP-HPLC fractionation of the venom. After SDS-PAGE analysis, fifty-eight bands were examined by MALDI-TOF/TOF mass spectrometry. Micrurus ruatanus venom displayed a three-finger toxin (3FTx)-rich venom phenotype, as well as a significant amount of phospholipases A2 (PLA2s). Various other proteins were identified, including Kunitz-type inhibitor proteins, L-amino acid oxidases, C-type lectin/lectin-like, metalloproteinases, serine proteinases, vespryn/ohanin, 5'-nucleotidases, glutathione peroxidases, and phosphodiesterases. Micrurus ruatanus venom displayed significant PLA2 activity in vitro and myotoxicity in vivo. The venom showed high lethal potency in mice, being one of the most lethal in Central America. The anticoral antivenom (SAC-ICP) produced by Instituto Clodomiro Picado neutralized the lethal activity of the venom. Major fractions with relevant lethal activity were also identified by a screening analysis. SIGNIFICANCE: The proteomic characterization, toxicity, immunorecognition and neutralization of Micrurus ruatanus venom have been determined for the first time. This coral snake is endemic to Roatan Island and contains a three-finger toxin-rich venom that displayed a potent lethal activity in mice. The anticoral antivenom produced by Instituto Clodomiro Picado neutralized the lethal activity of this venom in vivo, and therefore should be effective in the treatment of envenomings by this snake.

Effect of exposition to chlorpyrifos upon plasmatic cholinesterases, hematology and blood biochemistry values in Bothrops asper (Serpentes: Viperidae).

The terciopelo (Bothrops asper), is one of the most important venomous snakes in Costa Rica and common on agriculture where insecticides are frequently used for pest control. To assess the exposure to organophosphates on captive B. asper, an experiment using chlorpyrifos and butyrylcholinesterase (BChE), as a biomarker was conducted. In addition to BChE, hematology, aspartate aminotransferase (AST), total proteins (TP) and albumin were measured after exposure. Different concentrations of chlorpyrifos were used in Group A (0.1%) and B (1%), while the Control Group received distilled water; each group was composed of 5 snakes. Values of BChE, AST, TP, and albumin were measured before exposure, and at 6, 12, 24, 196, 360 and 528 h post-exposure. Hematology values were measured after 24 h post-exposure. As result, an important variation between subjects in all groups before exposure was obtained. Moreover, BChE activity showed 37% inhibition of Group A when compared to Control Group at 12 h post-exposure, and a higher inhibition of Group B (97%) related to Control Group, at 6 h post-exposure. Recovery of BChE occurred towards 528 h, never reaching initial values. Despite some variation in the rest of parameters used, a marked relative lymphopenia and monocytosis occurred at 24 h, assuming stress as the main cause.

Contributions of the snake venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys to the paraspecificity of the Central American polyspecific antivenom (PoliVal-ICP).

PoliVal-ICP antivenom is produced from plasma of horses immunized toward the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys. The antibody response induced by these venoms confers PoliVal-ICP the capacity to neutralize the venoms of the most important Central American viperids, including not only homologous venoms (i.e., venoms used as immunogen), but many heterologous venoms (i.e., venoms not used as immunogen). In this work, the individual contributions of homologous venoms to the paraspecificity of PoliVal-ICP were inferred from the capacity of experimental monospecific antivenoms toward venoms of B. asper (anti-Ba), C. simus (anti-Cs) and L. stenophrys (anti-Ls), and an experimental polyspecific antivenom (anti-Ba/Cs/Ls) to neutralize the lethality induced by different venoms in mice. It was found that all antivenoms neutralized their corresponding homologous venoms. Moreover, the anti-Ba antivenom cross-neutralized the venoms of Agkistrodon howardgloydi, Atropoides picadoi, Bothriechis lateralis, Bothriechis supraciliaris and Porthidium ophryomegas; the anti-Cs antivenom cross-neutralized the venoms of B. lateralis, B. supraciliaris, Cerrophidion sasai and Porthidium nasutum; and the anti-Ls antivenom cross-neutralized the venoms of B. lateralis, B. supraciliaris, C. sasai and Lachesis melanocephala. All venoms neutralized by any monospecific antivenom were also neutralized by the anti-Ba/Cs/Ls antivenom. Venoms of Atropoides mexicanus, Bothriechis nigroviridis and Bothriechis schlegelii were not neutralized by any experimental antivenom, thus explaining the limitations of PoliVal-ICP to neutralize these venoms. Consequently, an enlargement of the neutralization scope of PoliVal-ICP could be achieved by including these venoms in the group of those used as immunogens.

Integrated "omics" profiling indicates that miRNAs are modulators of the ontogenetic venom composition shift in the Central American rattlesnake, Crotalus simus simus.

BACKGROUND: Understanding the processes that drive the evolution of snake venom is a topic of great research interest in molecular and evolutionary toxinology. Recent studies suggest that ontogenetic changes in venom composition are genetically controlled rather than environmentally induced. However, the molecular mechanisms underlying these changes remain elusive. Here we have explored the basis and level of regulation of the ontogenetic shift in the venom composition of the Central American rattlesnake, Crotalus s. simus using a combined proteomics and transcriptomics approach. RESULTS: Proteomic analysis showed that the ontogenetic shift in the venom composition of C. s. simus is essentially characterized by a gradual reduction in the expression of serine proteinases and PLA2 molecules, particularly crotoxin, a ß-neurotoxic heterodimeric PLA2, concominantly with an increment of PI and PIII metalloproteinases at age 9-18 months. Comparison of the transcriptional activity of the venom glands of neonate and adult C. s. simus specimens indicated that their transcriptomes exhibit indistinguisable toxin family profiles, suggesting that the elusive mechanism by which shared transcriptomes generate divergent venom phenotypes may operate post-transcriptionally. Specifically, miRNAs with frequency count of 1000 or greater exhibited an uneven distribution between the newborn and adult datasets. Of note, 590 copies of a miRNA targeting crotoxin B-subunit was exclusively found in the transcriptome of the adult snake, whereas 1185 copies of a miRNA complementary to a PIII-SVMP mRNA was uniquely present in the newborn dataset. These results support the view that age-dependent changes in the concentration of miRNA modulating the transition from a crotoxin-rich to a SVMP-rich venom from birth through adulthood can potentially explain what is observed in the proteomic analysis of the ontogenetic changes in the venom composition of C. s. simus. CONCLUSIONS: Existing snake venom toxins are the result of early recruitment events in the Toxicofera clade of reptiles by which ordinary genes were duplicated, and the new genes selectively expressed in the venom gland and amplified to multigene families with extensive neofunctionalization throughout the approximately 112-125 million years of ophidian evolution. Our findings support the view that understanding the phenotypic diversity of snake venoms requires a deep knowledge of the mechanisms regulating the transcriptional and translational activity of the venom gland. Our results suggest a functional role for miRNAs. The impact of specific miRNAs in the modulation of venom composition, and the integration of the mechanisms responsible for the generation of these miRNAs in the evolutionary landscape of the snake's venom gland, are further challenges for future research.

Relación entre la ganancia del peso materno y la macrosomía fetal en el Hospital Nacional Arzobispo Loayza. Lima - Perú. Julio 2010 - Junio 2011 / Relationship between the maternal weight gain and fetal macrosomia at the National Hospital Arzobispo Loayza. Lima - Peru. July 2010 - June 2011

Objetivos: Determinar la relación la ganancia de peso de la madre y el nacimiento de un feto macrosómico con peso mayor o igual a 4000 g en el Servicio de Obstetricia del Hospital Nacional Arzobispo Loayza. Material y métodos: Se realizó un estudio observacional, diseño retrospectivo, transversal. Se revisaron 263 historias clínicas de pacientes recién nacidos vivos atendidos en el Servicio de Obstetricia del Hospital Nacional Arzobispo Loayza, nacidos entre el 01 de Julio del 2010 al 31 de Junio del 2011 y que registraron un peso mayor o igual a 4000 gramos, y que tuvieran sus datos completos consignados en la historia clínica materna y libro de registro. Resultados: La prevalencia de nacimientos macrosómicos en el Hospital es de 6.22 por ciento. Hubo diferencias estadísticamente significativas en relación al peso habitual, las obesas tenían una media del peso habitual de 80.2+/-6kilos, y las que tuvieron peso ideal pregestacional tuvieron una media de 58.6+/-5.7 kilos. También observamos que las pacientes con obesidad pregestacional tuvieron recién nacidos de 4343.9+/-206.8 gramos, mucho más en relación a las pacientes con peso ideal[4179.3+/-268.5] y con sobrepeso [4113.8+/-118.4]. Observamos que las pacientes con variación ponderal mayor de 10 kilos tuvieron recién nacidos con un mayor peso [4222.2+/-218.6 versus 4171.7+/-250.6 gramos] sin haber una diferencia estadísticamente significativa [P>0.05]. Conclusiones: Existe relación entre la ganancia de peso de la madre (>10 kilos) y el nacimiento de un feto macrosómico con peso mayor o igual a 4200 g en el Servicio de Obstetricia del Hospital Nacional Arzobispo Loayza. Los recién nacidos del sexo femenino muestran asociación con el peso al nacer mayor de 4500 gramos [P<0.05]. En los pacientes neonatos con más de 4500 gramos encontramos que en las gestantes hubo una mayor edad [31.8+/-3.3 versus 29.2+/-5.8 años], y mayor número de gestaciones [4 versus 2] [P<0.05].

Maintaining Coral Snakes (Micrurus nigrocinctus, Serpentes: Elapidae) for venom production on an alternative fish-based diet.

American Elapid snakes (Coral Snakes) comprise the genera Leptomicrurus, Micruroides and Micrurus, which form a vast taxonomic assembly of 330 species distributed from the South of United States to the southern region of South America. In order to obtain venom for animal immunizations aimed at antivenom production, Coral Snakes must be kept in captivity and submitted periodically to venom extraction procedures. Thus, to maintain a snake colony in good health for this purpose, a complete alternative diet utilizing an easily obtained prey animal is desirable. The development of a diet based on fish is compared to the wild diet based on colubrid snakes, and assessed in terms of gain in body weight rate (g/week), longevity (weeks), venom yield (mg/individual), venom median lethal dose (LD50) and venom chromatographic profiles. The animals fed with the fish-based diet gained more weight, lived longer, and produced similar amount of venom whose biological and biochemical characteristics were similar to those of venom collected from specimens fed with the wild diet. This fish-based diet appears to be suitable (and preferable to the wild diet) to supply the nutritional requirements of a Micrurus nigrocinctus snake collection for the production of antivenom.