ABSTRACT
The evolution of snake venoms resulted in multigene toxin families that code for structurally similar isoforms eventually harboring distinct functions. PLA2s are dominant toxins in viper venoms, and little is known about the impact of their diversity on human envenomings and neutralization by antivenoms. Here, we show the isolation of three distinct PLA2s from B. atrox venom. FA1 is a Lys-49 homologue, and FA3 and FA4 are catalytic Asp-49 PLA2s. FA1 and FA3 are basic myotoxic proteins, while FA4 is an acid non-myotoxic PLA2. FA3 was the most potent toxin, inducing higher levels of edema, inflammatory nociception, indirect hemolysis, and anticoagulant activity on human, rat, and chicken plasmas. FA4 presented lower anticoagulant activity, and FA1 had only a slight effect on human and rat plasmas. PLA2s presented differential reactivities with antivenoms, with an emphasis on FA3, which was not recognized or neutralized by the antivenoms used in this study. Our findings reveal the functional and antigenic diversity among PLA2s from B. atrox venom, highlighting the importance of assessing venom variability for understanding human envenomations and treatment with antivenoms, particularly evident here as the antivenom fails to recognize FA3, the most active multifunctional toxin described.
ABSTRACT
Differences in snake venom composition occur across all taxonomic levels and it has been argued that this variation represents an adaptation that has evolved to facilitate the capture and digestion of prey and evasion of predators. Bothrops atrox is a terrestrial pitviper that is distributed across the Amazon region, where it occupies different habitats. Using statistical analyses and functional assays that incorporate individual variation, we analyzed the individual venom variability in B. atrox snakes from four different habitats (forest, pasture, degraded area, and floodplain) in and around the Amazon River in Brazil. We observed venom differentiation between spatially distinct B. atrox individuals from the different habitats, with venom variation due to both common (high abundance) and rare (low abundance) proteins. Moreover, differences in the composition of the venoms resulted in individual variability in functionality and heterogeneity in the lethality to mammals and birds, particularly among the floodplain snakes. Taken together, the data obtained from individual venoms of B. atrox snakes, captured in different habitats from the Brazilian Amazon, support the hypothesis that the differential distribution of protein isoforms results in functional distinctiveness and the ability of snakes with different venoms to have variable toxic effects on different prey.
ABSTRACT
Bothrops (lance-head pit vipers) venoms are rich in weaponised metalloprotease enzymes (SVMP). These toxic enzymes are structurally diverse and functionally versatile. Potent coagulotoxicity is particularly important for prey capture (via stroke-induction) and relevant to human clinical cases (due to consumption of clotting factors including the critical depletion of fibrinogen). In this study, three distinct isoforms of P-III class SVMPs (IC, IIB and IIC), isolated from Bothrops neuwiedi venom, were evaluated for their differential capacities to affect hemostasis of prey and human plasma. Furthermore, we tested the relative antivenom neutralisation of effects upon human plasma. The toxic enzymes displayed differential procoagulant potency between plasma types, and clinically relevant antivenom efficacy variations were observed. Of particular importance was the confirmation the antivenom performed better against prothrombin activating toxins than Factor X activating toxins, which is likely due to the greater prevalence of the former in the immunising venoms used for antivenom production. This is clinically relevant as the enzymes displayed differential potency in this regard, with one (IC) in particular being extremely potent in activating Factor X and thus was correspondingly poorly neutralised. This study broadens the current understanding about the adaptive role of the SVMPs, as well as highlights how the functional diversity of SVMP isoforms can influence clinical outcomes.
ABSTRACT
Snakebite is a common occurrence for pet cats and dogs worldwide and can be fatal. In Australia the eastern brown snake (Pseudonaja textilis) is responsible for an estimated 76% of reported snakebite cases to domestic pets nationally each year, with the primary pathology being venom-induced consumptive coagulopathy. While only 31% of dogs survive P. textilis bites without antivenom, cats are twice as likely to survive bites (66%). Even with antivenom treatment, cats have a significantly higher survival rate. The reason behind this disparity is unclear. Using a coagulation analyser (Stago STA R Max), we tested the relative procoagulant effects of P. textilis venom—as well as 10 additional procoagulant venoms found around the world—on cat and dog plasma in vitro, as well as on human plasma for comparison. All venoms acted faster upon dog plasma than cat or human, indicating that dogs would likely enter coagulopathic states sooner, and are thus more vulnerable to procoagulant snake venoms. The spontaneous clotting time (recalcified plasma with no venom added) was also substantially faster in dogs than in cats, suggesting that the naturally faster clotting blood of dogs predisposes them to being more vulnerable to procoagulant snake venoms. This is consistent with clinical records showing more rapid onset of symptoms and lethal effects in dogs than cats. Several behavioural differences between cats and dogs are also highly likely to disproportionately negatively affect prognosis in dogs. Thus, compared to cats, dogs require earlier snakebite first-aid and antivenom to prevent the onset of lethal venom effects.
ABSTRACT
Variability in snake venom composition has been frequently reported and correlated to the adaptability of snakes to environmental conditions. Previous studies report plasticity for the venom phenotype. However, these observations are not conclusive, as the results were based on pooled venoms, which present high individual variability. Here we tested the hypothesis of plasticity by influence of confinement and single diet type in the venom composition of 13 adult specimens of Bothrops atrox snakes, maintained under captivity for more than three years. Individual variability in venom composition was observed in samples extracted just after the capture of the snakes. However, composition was conserved in venoms periodically extracted from nine specimens, which presented low variability restricted to the less abundant components. In a second group, composed of four snakes, drastic changes were observed in the venom samples extracted at different periods, mostly related to snake venom metalloproteinases (SVMPs), the core function toxins of B. atrox venom, which occurred approximately between 400 and 500 days in captivity. These data show plasticity in the venom phenotype during the lifetime of adult snakes maintained under captive conditions. Causes or functional consequences involved in the phenotype modification require further investigations.
ABSTRACT
Variability in the composition of snake venoms occurs in different taxa and is usually correlated to snake fitness. Here, we compared B. atrox venoms from three different geographic regions across the Brazilian Amazon and found remarkable functional differences particularly between venoms from two populations separated by the Amazon River, in specimens born, raised and maintained under the same conditions at Instituto Butantan serpentary. Venom from Presidente Figueiredo snakes induced stronger dermonecrosis, but was less procoagulant and lethal to mice; these activities were correlated to the presence of a PI-class SVMP and absence of a SVSP in the venom, respectively. Venom from São Bento snakes was more hemorrhagic, killed mice more efficiently, but induced lower signs of dermonecrosis, which was correlated to the higher proportion of SVMPs and the absence of a PI-class SVMP isoform. Belterra snakes, a reference of wild snakes, presented venoms with intermediate phenotypes. Commercial Bothrops antivenom was effective in neutralizing all biological activities evaluated in this study, including dermonecrosis and pro-coagulant, which are relevant for human snakebite accidents by B. atrox. Functional differences correlated to snake fitness may also imply in different symptomatology for B. atrox snakebite patients and deserve special attention from clinical toxicologists.
ABSTRACT
Variability in snake venom composition has been frequently reported and correlated to the adaptability of snakes to environmental conditions. Previous studies report plasticity for the venom phenotype. However, these observations are not conclusive, as the results were based on pooled venoms, which present high individual variability. Here we tested the hypothesis of plasticity by influence of confinement and single diet type in the venom composition of 13 adult specimens of Bothrops atrox snakes, maintained under captivity for more than three years. Individual variability in venom composition was observed in samples extracted just after the capture of the snakes. However, composition was conserved in venoms periodically extracted from nine specimens, which presented low variability restricted to the less abundant components. In a second group, composed of four snakes, drastic changes were observed in the venom samples extracted at different periods, mostly related to snake venom metalloproteinases (SVMPs), the core function toxins of B. atrox venom, which occurred approximately between 400 and 500 days in captivity. These data show plasticity in the venom phenotype during the lifetime of adult snakes maintained under captive conditions. Causes or functional consequences involved in the phenotype modification require further investigations.
ABSTRACT
Variability in the composition of snake venoms occurs in different taxa and is usually correlated to snake fitness. Here, we compared B. atrox venoms from three different geographic regions across the Brazilian Amazon and found remarkable functional differences particularly between venoms from two populations separated by the Amazon River, in specimens born, raised and maintained under the same conditions at Instituto Butantan serpentary. Venom from Presidente Figueiredo snakes induced stronger dermonecrosis, but was less procoagulant and lethal to mice; these activities were correlated to the presence of a PI-class SVMP and absence of a SVSP in the venom, respectively. Venom from São Bento snakes was more hemorrhagic, killed mice more efficiently, but induced lower signs of dermonecrosis, which was correlated to the higher proportion of SVMPs and the absence of a PI-class SVMP isoform. Belterra snakes, a reference of wild snakes, presented venoms with intermediate phenotypes. Commercial Bothrops antivenom was effective in neutralizing all biological activities evaluated in this study, including dermonecrosis and pro-coagulant, which are relevant for human snakebite accidents by B. atrox. Functional differences correlated to snake fitness may also imply in different symptomatology for B. atrox snakebite patients and deserve special attention from clinical toxicologists.
ABSTRACT
Lancehead pit-vipers (Bothrops genus) are an extremely diverse and medically important group responsible for the greatest number of snakebite envenomations and deaths in South America. Bothrops atrox (common lancehead), responsible for majority of snakebites and related deaths within the Brazilian Amazon, is a highly adaptable and widely distributed species, whose venom variability has been related to several factors, including geographical distribution and habitat type. This study examined venoms from four B. atrox populations (Belterra and Santarém, PA; Pres. Figueiredo, AM and São Bento, MA), and two additional Bothrops species (B. jararaca and B. neuwiedi) from Southeastern region for their coagulotoxic effects upon different plasmas (human, amphibian, and avian). The results revealed inter– and intraspecific variations in coagulotoxicity, including distinct activities between the three plasmas, with variations in the latter two linked to ecological niche occupied by the snakes. Also examined were the correlated biochemical mechanisms of venom action. Significant variation in the relative reliance upon the cofactors calcium and phospholipid were revealed, and the relative dependency did not significantly correlate with potency. Relative levels of Factor X or prothrombin activating toxins correlated with prey type and prey escape potential. The antivenom was shown to perform better in neutralising prothrombin activation activity than neutralising Factor X activation activity. Thus, the data reveal new information regarding the evolutionary selection pressures shaping snake venom evolution, while also having significant implications for the treatment of the envenomed patient. These results are, therefore, an intersection between evolutionary biology and clinical medicine.
ABSTRACT
Lancehead pit-vipers (Bothrops genus) are an extremely diverse and medically important group responsible for the greatest number of snakebite envenomations and deaths in South America. Bothrops atrox (common lancehead), responsible for majority of snakebites and related deaths within the Brazilian Amazon, is a highly adaptable and widely distributed species, whose venom variability has been related to several factors, including geographical distribution and habitat type. This study examined venoms from four B. atrox populations (Belterra and Santarém, PA; Pres. Figueiredo, AM and São Bento, MA), and two additional Bothrops species (B. jararaca and B. neuwiedi) from Southeastern region for their coagulotoxic effects upon different plasmas (human, amphibian, and avian). The results revealed inter– and intraspecific variations in coagulotoxicity, including distinct activities between the three plasmas, with variations in the latter two linked to ecological niche occupied by the snakes. Also examined were the correlated biochemical mechanisms of venom action. Significant variation in the relative reliance upon the cofactors calcium and phospholipid were revealed, and the relative dependency did not significantly correlate with potency. Relative levels of Factor X or prothrombin activating toxins correlated with prey type and prey escape potential. The antivenom was shown to perform better in neutralising prothrombin activation activity than neutralising Factor X activation activity. Thus, the data reveal new information regarding the evolutionary selection pressures shaping snake venom evolution, while also having significant implications for the treatment of the envenomed patient. These results are, therefore, an intersection between evolutionary biology and clinical medicine.
ABSTRACT
Venom variability is commonly reported for venomous snakes including Bothrops atrox. Here, we compared the composition of venoms from B. atrox snakes collected at Amazonian conserved habitats (terra-flrme upland forest and vcirzea) and human modified areas (pasture and degraded areas). Venom samples were submitted to shotgun proteomic analysis as a whole or compared after fractionation by reversed-phase chromatography. Whole venom proteomes revealed a similar composition among the venoms with predominance of SVMPs, CTLs, and SVSPs and intermediate amounts of PLA(2)s and LAAOs. However, when distribution of particular isoforms was analyzed by either method, the venom from varzea snakes showed a decrease in hemorrhagic SVMPs and an increase in SVSPs, and procoagulant SVMPs and PLA(2)s. These differences were validated by experimental approaches including both enzymatic and in vivo assays, and indicated restrictions in respect to antivenom efficacy to variable components. Thus, proteomic analysis at the isoform level combined to in silica prediction of functional properties may indicate venom biological activity. These results also suggest that the prevalence of functionally distinct isoforms contributes to the variability of the venoms and could reflect the adaptation of B. atrox to distinct prey communities in different Amazon habitats. Biological significance: In this report, we compared isoforms present in venoms from snakes collected at different Amazonian habitats. By means of a species venom gland transcriptome and the in silico functional prediction of each isoform, we were able to predict the principal venom activities in vitro and in animal models. We also showed remarkable differences in the venom pools from snakes collected at the floodplain (varzea habitat) compared to other habitats. Not only was this venom less hemorrhagic and more procoagulant, when compared to the venom pools from the other three habitats studied, but also this enhanced procoagulant activity was not efficiently neutralized by Bothrops antivenom. Thus, using a functional proteomic approach, we highlighted intraspecific differences in B. atrox venom that could impact both in the ecology of snakes but also in the treatment of snake bite patients in the region.
ABSTRACT
Frequently the venom composition has been considered as a reflex of snake phylogeny, with important implications for the production of antivenoms and treatment of envenomings. Snakes from Bothrops complex are responsible for most snake bites registered in Brazil, and their phylogenetic relationships are not yet completely clarified. Another important discussion is related to the efficacy of Bothrops Antivenom (SAB) in the treatment of envenoming by snakes whose venoms are not present in the immunization pool. Thus, the aim of this study was to analyze the composition of venoms from 6 species of snakes from Bothrops complex, and their correlation with snake phylogeny and reactivity with o SAB. In this way, venoms from Bothropoides jararaca, Bothropoides neuwiedi, Bothrops atrox, Bothrops jararacussu, Rhinocerophis cotiara and Rhinocerophis alternatus were analyzed by SDS-PAGE and reverse phase chromatography on HPLC C18 column. The reactivity of SAB with venoms and their fractions was performed by ELISA and western blot, followed by assessment of neutralization of lethality and hemorrhagic activity of the venoms of B. jararaca (present in immunization pool of SAB) and B. atrox (absent from the pool). The venoms showed distinct electrophoretic and chromatographic profiles, without apparent correlation with the snake phylogeny. Only venoms of the genus Rhinocerophis (R. alternatus and R. cotiara) showed relatively similar profiles. SAB recognized the venoms of different species with the same antibody titer of 640,000. Most of the fractions eluted from C18 column were recognized by SAB, especially those corresponding to snake venom metalloproteinases (SVMP) of class P-III. By SDS-PAGE, the venom of B. jararacussu presented the more distinct electrophoretic profile with predominance of phospholipase A2 (PLA2), while in the other venoms, the SVMPs were the predominant component. Two microlitters of antivenom were sufficient to protect 100 % of mice challenged with 3 LD50 of B. jararaca venom (105 µg), although for B. atrox venom (225 µg) the same effect was only obtained when doubling the antivenom amount. However, the SAB neutralized completely the hemorrhage induced by 10 µg of the both venoms, with the same volume of serum (4 µL). Thus, we conclude that SAB reacts similarly with 16 the same family of toxins from distinct venoms, regardless of the phylogeny of the snake or the presence of venom in the immunization pool employed in SAB production. This suggests that a Bothrops antivenom with greater efficiency and broad spectrum can be developed, if one considers the reactivity of antivenom to the different classes of toxins and not just the phylogeny of snakes.
Frequentemente a composição dos venenos tem sido considerada com um reflexo da filogenia das serpentes, com importantes implicações para a produção de antivenenos. As serpentes do complexo Bothrops são responsáveis pela grande maioria dos acidentes ofídicos registrados no Brasil e suas relações filogenéticas ainda não foram completamente esclarecidas. Outra discussão importante diz respeito à eficácia do Soro Antibotrópico (SAB) no tratamento dos envenenamentos ocasionados por serpentes cujos venenos não fazem parte do pool de imunização, utilizado em sua produção. Portanto, o objetivo deste trabalho foi analisar a composição dos venenos de 6 espécies de serpentes do complexo Bothrops e sua correlação com a filogenia da serpente e reatividade com o SAB. Para tanto, venenos de Bothropoides jararaca, Bothropoides neuwiedi, Bothrops jararacussu, Bothrops atrox, Rhinocerophis alternatus e Rhinocerophis cotiara foram analisados por SDS-PAGE e cromatografia de fase reversa em HPLC usando coluna C18. A reatividade do SAB com os venenos e suas frações foi analisada por ELISA e Western blotting, seguida por testes de neutralização da letalidade e da atividade hemorrágica induzida pelos venenos de B. jararaca (presente no pool de imunização do SAB) e B. atrox (ausente do pool). Os venenos mostraram distintos perfis eletroforéticos e cromatográficos, sem correlação aparente com a filogenia da serpente. Somente os venenos do gênero Rhinocerophis (R. alternatus e R. cotira) exibiram perfis relativamente semelhantes. O SAB reconheceu os venenos das seis espécies com um mesmo título de anticorpos (640.000). A maioria das frações eluídas da coluna C18 foram reconhecidas pelo SAB, principalmente as correspondentes à Metaloproteinases de Venenos de Serpentes (SVMPs) da classe P-III. Por SDS-PAGE, o veneno de B. jararacussu apresentou o perfil eletroforético mais distinto com predominância de PLA2, enquanto nos outros venenos as SVMPs foram os componentes predominantes. Dois microlitros de antiveneno foram suficientes para proteger 100 % dos camundongos desafiados com 3DL50 do veneno de B. jararaca (105 µg), embora para B. atrox (225 µg) o mesmo efeito tenha sido 14 obtido somente com o dobro do volume de antiveneno. Entretanto, o SAB neutralizou completamente a hemorragia induzida por 10 µg de ambos os venenos com um mesmo volume de soro (4 µL). Portanto, concluímos que o SAB reage similarmente com uma mesma família de toxinas de venenos distintos, independentemente da filogenia da serpente ou mesmo da presença do veneno no pool de imunização. Isso sugere que um antiveneno botrópico de maior espectro e eficiência pode ser desenvolvido, se for considerada a reatividade do antiveneno frente às diversas classes de toxinas e não apenas a filogenia da serpente.
