Neutralización de la actividad letal del veneno de serpiente Bothrops atrox por suero hiperinmune de llama (Lama glama) / Neutralization of the lethal activity from Bothrops atrox venom by hyperimune llama serum (Lama glama)

RESUMEN Objetivos: Evaluar la capacidad del suero hiperinmune de llama (Lama glama) para neutralizar la letalidad del veneno de la serpiente Bothrops atrox en ratones de laboratorio. Materiales y métodos: Se calculó la dosis letal media (DL50) de un pool de venenos de serpientes de Bothrops atrox de Perú, y se midieron los títulos de anticuerpos por ensayo ELISA; así como la potencia de neutralización del suero inmune por el cálculo de la dosis efectiva media (DE50) durante el periodo de inmunización. Resultados: La DL50 del veneno fue de 3,96 µg/g, similar a otros trabajos realizados en Bothrops atrox en Perú. Los títulos de anticuerpos contra el veneno se incrementan rápidamente en la llama mostrando una rápida respuesta inmune; sin embargo, la capacidad de neutralización se incrementa más lentamente y requiere de varias dosis y refuerzos de las inmunizaciones alcanzado una DE50 de 3,30 µL/g ratón y una potencia de neutralización 3,6 mg/mL después de 15 inmunizaciones. Conclusiones: El suero hiperinmune de llama es capaz de neutralizar la letalidad del veneno de la serpiente Bothrops atrox de Perú en ratones de laboratorio.

ABSTRACT Objectives: To evaluate the capacity of the hyperimmune llama serum (Lama glama) to neutralize the lethal activity of Bothrops atrox venom in laboratory mice. Materials and methods: Mean lethal dose (LD50) was calculated from a Bothrops atrox venom sample pool from Peru. The antibody titers were measured by ELISA assay; and the immune serum neutralization potency was measured by calculating the mean effective dose (ED50) during the immunization period. Results: The venom's LD50 was 3.96 μg/g; similar to what was found in other studies about Bothrops atrox carried out in Peru. The titers of antibodies against the venom increased rapidly in the llama, demonstrating a fast immune response; however, the neutralization capacity increased slowly and required several doses and immunization reinforcements, obtaining a ED50 of 3.30 μL/g mouse and a neutralization potency of 3.6 mg/mL after 15 immunizations. Conclusions: The hyperimmune llama serum is able to neutralize the lethality of the Bothrops atrox venom from Peru in laboratory mice.

Epidemiological and clinical aspects of snakebites in the upper Juruá River region, western Brazilian Amazonia

This study addresses the clinical and epidemiological aspects of envenoming cases resulting from snakebites treated at a hospital in Cruzeiro do Sul, in the upper Juruá River region, western Brazilian Amazonia. The specific identity of snakes that caused the envenomings was inferred (a) from the diagnosis of patient symptoms and signs upon hospital admission, (b) by enzyme immunoassay for detection of Bothrops atrox and Lachesis muta venom from serum samples taken from patients before antivenom therapy, or (c) by direct identification of the snake, when it was brought along to the hospital or photographed. There were 133 snakebites (76.2 cases per 100,000 inhabitants) registered during one year (July 2017 to June 2018). Most snakebites (88.7%) were caused by Bothrops spp., and the rest by non-venomous snakes or dry bites. Snakebites tended to occur more often during the rainy season, coinciding with the period of greater reproductive activity of the snakes and greater availability of their prey. In addition, the increase in the water level of rivers and lakes during the rainy season tends to concentrate snakes in dry places and, thus, to increase encounters with humans. Information campaigns on prevention and first aid, specially among the most vulnerable groups (indigenous people, farmers, and children and teenagers in rural areas), and the importance of using protective equipment (boots, leggings, leather gloves) in certain high risk activities (e.g. agriculture and extractivism in forests) are fundamental for the reduction of snakebite morbidity. (AU)

Venom complexity of Bothrops atrox (common lancehead) siblings

Variability in snake venoms is a well-studied phenomenon. However, sex-based variation of Bothrops atrox snake venom using siblings is poorly investigated. Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Differences in the venom composition of Bothrops genus have been linked to several factors such as ontogeny, geographical distribution, prey preferences and sex. Thus, in the current study, venom samples of Bothrops atrox male and female siblings were analyzed in order to compare their biochemical and biological characteristics. Methods: Venoms were collected from five females and four males born from a snake captured from the wild in São Bento (Maranhão, Brazil), and kept in the Laboratory of Herpetology of Butantan Intitute. The venoms were analyzed individually and as a pool of each gender. The assays consisted in protein quantification, 1-DE, mass spectrometry, proteolytic, phospholipase A2, L-amino acid oxidase activities, minimum coagulant dose upon plasma, minimum hemorrhagic dose and lethal dose 50%. Results: Electrophoretic profiles of male's and female's venom pools were quite similar, with minor sex-based variation. Male venom showed higher LAAO, PLA2 and hemorrhagic activities, while female venom showed higher coagulant activity. On the other hand, the proteolytic activities did not show statistical differences between pools, although some individual variations were observed. Meanwhile, proteomic profile revealed 112 different protein compounds; of which 105 were common proteins of female's and male's venom pools and seven were unique to females. Despite individual variations, lethality of both pools showed similar values. Conclusion: Although differences between female and male venoms were observed, our results show that individual variations are significant even between siblings, highlighting that biological activities of venoms and its composition are influenced by other factors beyond gender.(AU)

Comparative gender peptidomics of Bothrops atrox venoms: are there differences between them?

Bothrops atrox is known to be the pit viper responsible for most snakebites and human fatalities in the Amazon region. It can be found in a wide geographical area including northern South America, the east of Andes and the Amazon basin. Possibly, due to its wide distribution and generalist feeding, intraspecific venom variation was reported by previous proteomics studies. Sex-based and ontogenetic variations on venom compositions of Bothrops snakes were also subject of proteomic and peptidomic analysis. However, the venom peptidome of B. atrox remains unknown. Methods: We conducted a mass spectrometry-based analysis of the venom peptides of individual male and female specimens combining bottom-up and top-down approaches. Results: We identified in B. atrox a total of 105 native peptides in the mass range of 0.4 to 13.9 kDa. Quantitative analysis showed that phospholipase A2 and bradykinin potentiating peptides were the most abundant peptide families in both genders, whereas disintegrin levels were significantly increased in the venoms of females. Known peptides processed at non-canonical sites and new peptides as the Ba1a, which contains the SVMP BATXSVMPII1 catalytic site, were also revealed in this work. Conclusion: The venom peptidomes of male and female specimens of B. atrox were analyzed by mass spectrometry-based approaches in this work. The study points to differences in disintegrin levels in the venoms of females that may result in distinct pathophysiology of envenomation. Further research is required to explore the potential biological implications of this finding.(AU)

Ação cardiovascular da peçonha de Bothrops atrox em ratos anestesiados / Cardiovascular response to Bothrops atrox snake venom in anesthetized rats

Bothrops atrox (jararaca-do-norte) é a principal causa de envenenamento ofídico na região amazônica. Vários estudos têm investigado a bioquímica desta peçonha, bem como os efeitos locais (dor, edema, hemorragia e mionecrose) que ela causa. Por outro lado, os efeitos sistêmicos têm sido menos estudados. Neste trabalho, investigamos as alterações hemodinâmicas causadas por esta peçonha em ratos anestesiados bem como os possíveis mediadores envolvidas nestas respostas. Métodos: Ratos machos Wistar (300-400 g) anestesiados com isoflurano foram canulados para o registro da pressão arterial (carótida) e para a administração intravenosa (i.v.) de diferentes substâncias (peçonha, antagonistas e inibidores) (veia femoral esquerda). Em alguns experimentos, houve administração intramuscular (i.m.) de peçonha. A frequência respiratória foi determinada manualmente e o ECG foi monitorado via eletrodos introduzidos nas patas dianteiras e traseira. Em intervalos pré-estabelecidos, foram coletadas amostras de sangue arterial para análise bioquímica e determinação da cinética da peçonha. Ao término dos experimentos, foram coletados tecidos (rim, pulmão, coração, fígado e músculo) para análise histológica. A capacidade do antiveneno botrópico comercial em neutralizar algumas atividades enzimáticas e as alterações hemodinâmicas foi avaliada pré-incubando-se a peçonha com antiveneno antes de testar a atividade residual. A peçonha também foi fracionada por gel filtração e os picos testados quanto à sua atividade sobre a pressão arterial. Resultados: A peçonha (0,4 mg/kg, i.v.) causou hipotensão imediata (máxima aos 5 min) seguida por recuperação nos 20 min seguintes; não houve alteração na frequência cardíaca, ECG ou frequência respiratória, e não houve mortes (sobrevida até o final do experimento: 120 min). Uma dose maior (0,7 mg/kg, i.v.) causou hipotensão progressiva, bradicardia e falência respiratória, com morte de todos os ratos (n=6) em 20±4 min. A administração intramuscular (músculo gastrocnêmio) de peçonha (4 mg/kg) mostrou um perfil hemodinâmico semelhante àquele observado com a dose menor i.v. A análise histológica mostrou que a dose menor i.v. causou trombose e hemorragia pulmonares, além de dano renal (descamação epitelial, deposição proteica e microaneurismos); houve proteinúria e hemoglobinúria em urina coletada no final do experimento. Não houve alteração histológica nos outros tecidos examinados (fígado, coração)...(AU)

Bothrops atrox (jararaca-do-norte) is the main cause of snakebite in the Amazon region. Various studies have examined the biochemical aspects of this venom, as well as local effects such as pain, edema, hemorrhage and myonecrosis that this species causes. In contrast, the systemic effects caused by this venom have been less studied. In this work, we investigated the hemodynamic alterations caused by B. atrox venom in anesthetized rats, as well as the possible mediators involved in this response. Methods: Male Wistar rats (300-400 g) anesthetized with isoflurane were cannulated for arterial blood pressure measurements (carotid artery) and for the intravenous (i.v., femoral vein) administration of test substances (venom, antagonists and inhibitors). In some experiments, venom was injected intramuscularly (i.m.). Respiratory rate was determined manually and ECG was monitored using conventional electrodes. At pre-established intervals, arterial blood was drawn for biochemical analyses and determination of venom kinetics. At the end of the experiments, tissue samples were collected from heart, liver, lung and muscle for histological analysis. The ability of commercial bothropic antivenom to neutralize selected venom enzymatic activities and the venom-induced hemodynamic alterations was assessed by preincubating venom with antivenom prior to testing for residual activity. Venom was also fractionated by gel filtration and the resulting peaks were screened for their activity on blood pressure. Results: Venom (0.4 mg/kg, i.v.) caused immediate hypotension (maximal at 5 min) followed by recovery over 20 min; there were no changes in heart rate, ECG or respiratory rate and no deaths (survival for up to 120 min post-venom). A higher dose of venom (0.7 mg/kg, i.v.) caused progressive hypotension, bradycardia and respiratory failure, with all rats (n=6) dying in 20±4 min. A similar hemodynamic profile to the lower dose of venom i.v. was seen with venom (4 mg/kg) given i.m. (gastrocnemius muscle). Venom given i.v. (lower dose) caused pulmonary thrombosis and hemorrhage, in addition to renal damage (epithelial desquamation, deposition of protein and microaneurysms); proteinuria and hemoglobinúria were observed in urine collected at the end of the experiment. Venom given i.m. or i.v. (higher dose) caused only pulmonary thrombosis. Renal damage (epithelial desquamation, proteinuria and hemoglobinuria) was seen with venom i.v. (0.4 mg/kg); venom given i.m. caused local hemorrhage and necrosis, and proteinuria. There were no histological alterations in the other tissues examined (heart, liver)... (AU)

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A2 from Bothrops atrox snake venom

Background Snake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA 2 ) from Bothrops atroxvenom, and biochemically characterize these molecules to enable future functional studies.Methods To obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.Results The metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A 2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2 s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA 2 .Conclusions The present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA 2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease.

Purification procedure for the isolation of a P-I metalloprotease and an acidic phospholipase A 2 fromBothrops atrox snake venom

Background Snake venoms are complex mixtures of inorganic and organic components, mainly proteins and peptides. Standardization of methods for isolating bioactive molecules from snake venoms is extremely difficult due to the complex and highly variable composition of venoms, which can be influenced by factors such as age and geographic location of the specimen. Therefore, this study aimed to standardize a simple purification methodology for obtaining a P-I class metalloprotease (MP) and an acidic phospholipase A2 (PLA 2 ) from Bothrops atroxvenom, and biochemically characterize these molecules to enable future functional studies.Methods To obtain the toxins of interest, a method has been standardized using consecutive isolation steps. The purity level of the molecules was confirmed by RP-HPLC and SDS-PAGE. The enzymes were characterized by determining their molecular masses, isoelectric points, specific functional activity and partial amino acid sequencing.Results The metalloprotease presented molecular mass of 22.9 kDa and pI 7.4, with hemorrhagic and fibrin(ogen)olytic activities, and its partial amino acid sequence revealed high similarity with other P-I class metalloproteases. These results suggest that the isolated metalloprotease is Batroxase, a P-I metalloprotease previously described by our research group. The phospholipase A 2 showed molecular mass of 13.7 kDa and pI 6.5, with high phospholipase activity and similarity to other acidic PLA2 s from snake venoms. These data suggest that the acidic PLA2 is a novel enzyme from B. atrox venom, being denominated BatroxPLA 2 .Conclusions The present study successfully standardized a simple methodology to isolate the metalloprotease Batroxase and the acidic PLA 2 BatroxPLA2 from the venom of B. atrox, consisting mainly of classical chromatographic processes. These two enzymes will be used in future studies to evaluate their effects on the complement system and the inflammatory process, in addition to the thrombolytic potential of the metalloprotease.(AU)

Eficacia experimental de anticuerpos IgY producidos en huevos, contra el veneno de la serpiente peruana Bothrops atrox / Experimental efficacy of IgY antibodies produced in eggs against the venom of the Peruvian snake Bothrops atrox

Objetivos. Desarrollar un protocolo de inmunización para producir inmunoglobulinas IgY de origen aviar contra el veneno de la serpiente peruana Bothrops atrox y evaluar la capacidad neutralizante. Materiales y métodos. Se inmunizaron seis gallinas de postura de la raza hy line brown con 500 μg/dosis de veneno de B. atrox en un periodo de dos meses. Cada semana, los huevos fueron colectados para el aislamiento de inmunoglobulinas IgY a partir de la yema, usando dos pasos consecutivos con αcido caprνlico y sulfato de amonio. La detecciσn de anticuerpos se realizσ por inmunodifusiσn doble mientras que el tνtulo y reactividad cruzada se determinaron por las técnicas de ELISA y Western blot. El cálculo de DL50 y de la DE50 del antiveneno IgY producido se realizó utilizando el método de Probits. Resultados. La masa de anticuerpos aislados fue de 8,5 ± 1,35 mg de IgY/mL de yema. Asimismo, la DE50 del antiveneno aviar fue calculada en 575 μL de antiveneno/mg de veneno. Adicionalmente, los ensayos de reactividad cruzada mostraron que el veneno de B. atrox comparte mas epνtopes comunes con el veneno de B. brazili (47%) que con otros veneno del mismo género, en tanto que los venenos de Lachesis muta (19%) y Crotalus durissus (12%) mostraron una baja reactividad cruzada. Conclusiones. Se ha obtenido IgY purificada contra el veneno de B. atrox con capacidad neutralizante y se ha demostrado su utilidad como herramienta inmunoanalítica para evaluar la reactividad cruzada con venenos de otras especies.

Objectives. To develop an immunization protocol in order to produce avian IgY immunoglobulins against Bothrops atrox Peruvian snake venom and to evaluate its neutralizing capacity. Materials and methods. Six Hy Line Brown hens were immunized each two weeks using 500μg/doses of B. atrox venom in a period of two months. Each week, eggs were collected for IgY isolation from yolk using two consecutive steps with caprilic acid and ammonium sulfate. Detection of IgY anti-B. atrox were performed by double immunodiffusion, whereas title and cross-reactivity were analyzed using ELISA and Western Blot technics, respectively. Furthermore, letal dose (DL50) and Medium Effective Dose (DE50) were obtained by Probit analysis. Results. As a result of this protocol, chicken IgY’s were obtained in a concentration of 8,5 ± 1,35 mg/yolk mL. DE50 from avian antivenom was 575 μL/venom mg. Cross-reactivity studies showed Bothrops atrox venom share more commom epitopes with Bothrops brazili (47%) than others Bothrops venoms showing Lachesis muta (19%) and Crotalus durissus (12%) venoms a low crossing reactivity, instead. Conclusions. Using this procedure, we could purify chicken IgY with a neutralizant capacity of B. atrox venom which is comparable to the antivenom of equine origin and demonstrate its capacity as a immunoanalitical tool to evaluate the cross reactivity with others peruvian snakes.