Strategies in 'snake venomics' aiming at an integrative view of compositional, functional, and immunological characteristics of venoms

This work offers a general overview on the evolving strategies for the proteomic analysis of snake venoms, and discusses how these may be combined through diverse experimental approaches with the goal of achieving a more comprehensive knowledge on the compositional, toxic, and immunological characteristics of venoms. Some recent developments in this field are summarized, highlighting how strategies have evolved from the mere cataloguing of venom components (proteomics/venomics), to a broader exploration of their immunological (antivenomics) and functional (toxicovenomics) characteristics. Altogether, the combination of these complementary strategies is helping to build a wider, more integrative view of the life-threatening protein cocktails produced by venomous snakes, responsible for thousands of deaths every year.(AU)

Isolation, characterization and antigenic cross-reactivities of the major hemorrhagin from Cryptelytrops purpureomaculatus venom.

The major hemorrhagin from C. purpureomaculatus (mangrove pit viper) venom was purified to homogeneity and termed Maculatoxin. Maculatoxin has a molecular weight of 38 kDa as determined by SDS-PAGE. It is an acidic protein (pI= 4.2) and exhibited proteolytic and hemorrhagic activities (MHD10 = 0.84 μg in mice) but was not lethal to mice at a dose of 1 μg/g. The hemorrhagic activity of Maculatoxin was completely inactivated by EDTA and partially inhibited by ATP and citrate. The N-terminal sequence of Maculatoxin (TPEQQRFPPTYIDLGIFVDHGMYAT) shares a significant degree of homology with the metalloprotease domain of other venom hemorrhagins. Indirect ELISA showed anti-Maculatoxin cross reacted with protein components of many snake venoms. In the double-sandwich ELISA, however, anti-Maculatoxin cross-reacted only with venoms of certain species of the Trimeresurus (Asia lance-head viper) complex, and the results support the recent proposed taxonomy changes concerning the Trimeresurus complex

Perfil eco-epidemiológico de los accidentes por ofidios en Monagas, Venezuela (2002-2006) / Eco-epidemiological profile of snakebite accidents in Monagas state, Venezuela (2002-2006)

El accidente causado por animales venenosos representa un problema global, especialmente en las áreas subtropicales y tropicales del mundo. En Venezuela, es endémico para varias regiones incluyendo la zona nororiental. Con el objetivo de ampliar y actualizar el perfil eco-epidemiológico del ofidismo en el estado Monagas, se evaluó su comportamiento, en cada municipio, durante 5 años de registro (2002 - 2006). Se contabilizaron 339 accidentes ofídicos (con promedio de 68 anuales). El incidente ocurrió con mayor frecuencia en agricultores, adultos jóvenes y de género masculino, durante sus labores en el campo y en el periodo diurno. La incidencia siguió un patrón estacional bimodal con predominio en épocas de alta y baja pluviosidad. Los envenenamientos botrópicos (28,6%) fueron más frecuentes que los crotálicos (14,5%), con proporción 2:1. El 87,9% recibió sueroterapia específica. La incidencia promedio anual en la entidad se ubicó en 11,30 casos por 100.000 habitantes, siendo Punceres (46,29), Acosta (20,91) y Bolívar (19,52) los municipios con mayor impacto. El estado Monagas presentó un mapa de endemicidad distribuido en municipios de (1) muy alta endemicidad, (2) alta endemicidad, (3) mediana endemicidad, (4) baja endemicidad y (5) muy baja endemicidad. Durante el período analizado no se registró muerte por esta causa en el estado. Los hallazgos sugieren la importancia del accidente por serpientes en Monagas, con especial importancia en la mitad norte de la entidad.

The accidents caused by venomous animals are a global problem, especially in subtropical and tropical regions of the world. In Venezuela, they are endemic in several regions including the northeast. In order to expand and update the ecoepidemiological profile of snake bites in Monagas state, their behavior was assessed in each municipality for 5 years (2002 -2006). There were 339 ophidian accidents (on average 68 per year). The bites occurred more frequently in young adult male farmers, while working in the field and during daytime. The incidence followed a bimodal seasonal pattern with predominance in high and low rainfall periods. Bothropic envenoming (28.6%) were the most frequent followed by Crotalic ones (14.5%), with a 2:1 ratio. 87.9% received specific serum therapy. The annual average incidence in the state was 11.30 cases per 100,000 inhabitants. Punceres (46.29), Acosta (20.91) and Bolivar (19.52) were the municipalities with the highest impact. Monagas state showed an endemicity map with municipalities having (1) very high endemicity, (2) high endemicity, (3) medium endemicity, (4) low endemicity and (5) very low endemicity. In the studied period there were no deaths from this cause in the state. The findings suggest the importance of snake accidents in Monagas, especially in the northern half of the state.

Caracterização estrutural e funcional de uma proteína isolada do plasma de serpentes Bothrops jararaca, relacionada ao cininogênio de alta massa molecular de mamíferos / Structural and functional characterization of a protein isolated from Bothrops jararaca snake plasma, related to the mammalian high molecular weight kininogen

O plasma da serpente Bothrops jararaca é rico em inibidores de proteases, alguns dos quais com atividade inibitória sobre toxinas presentes no veneno de serpentes da mesma espécie. Um desses inibidores apresenta massa molecular de 110 kDa, é um potente inibidor de cisteíno-peptidase e libera um peptídeo que induz contração de musculatura lisa homóloga. Por estas características, essa proteína, denominada BjHK (Bothrops jararaca High Molecular Weight Kininogen), foi correlacionada ao cininogênio de alta massa molecular de mamíferos. Além dessas propriedades, verificou-se que essa proteína inibe metaloproteases presentes no veneno de B. jararaca. Esse efeito também foi observado no cininogênio de alta massa molecular humano e correlacionado a porções do domínio 5 dessa proteína. O objetivo do presente projeto é procurar possíveis homologias entre a BjHK e o cininogênio humano, além de possíveis atividades inibitórias sobre agregação plaquetária e adesão celular, atividades estas também descritas no cininogênio de alta massa molecular humano...

The Bothrops jararaca snake plasma is rich in protease inhibitors, some of which have inhibitory activity on toxins from its own venom. One of these, which has a molecular mass of 110 kDa, is a potent inhibitor of cysteine-peptidase and releases a peptide that induces contraction of homologous smooth musculature. For these characteristics this protein, named BjHK (Bothrops jararaca High Molecular Weight Kininogen) was correlated to mammalian high molecular weight kininogens. Moreover, it was found that this protein inhibits metalloproteases present in the B. jararaca venom. This effect was also observed in human high molecular weight kininogen and correlated to portions of the domain 5 of this protein. The aim of this project is to search for homologies between BjHK and the human kininogen, as well as a possible inhibitory activity of this protein on platelet aggregation and cells adhesion. These activities were also described in human high molecular weight kininogen...

Influence of ionizing radiation on Echis pyramidium snake venom: Biochemical and immunological aspects

The effect of a single LD[50] dose of native Echis pyramidum venom [27.69 micro g/mouse] on the activities of certain serum enzymes levels: aspartate aminotransferase [AST], alanine aminotransferase [ALT], alkaline phosphatase [ALP], urea, creatinine, lactate dehydrogenase [LDH], creatine phosphokinase [CPK], creatine kinase isoenzyme [CK-MB] were studied. Samples from the serum were collected 4hr following LD50 venom dose intraperitonealy injected in male Swiss albino mice. The activities of these enzymes showed significant elevation compared to the non-envenomated group. In contrast, an equivalent dose of 1.5 kGy gamma irradiated Echis pyramidum venom [27.69 micro g/mouse] did not cause any significant increase compared to non-envenomated group. The effect of a dose that is equivalent to ½ LD[50][13.8 micro g/50 micro l] of native Echis pyramidum venom on plasma creatine phosphokinase [CPK] induced a significant increase of creatine phosphokinase [CPK] level compared to normal control [P<0.01]. In contrast, an equivalent dose of 1.5 kGy gamma irradiated Echis pyramidum venom showed non significant difference in creatine phosphokinase activity when compared to the normal control. Light microscopic examinations of gastrocenemius muscles of mice injected with native Echis pyramidum venom [½ LD[50]; 13.8 micro g/50 micro l] showed fragmentation, disorganization, loss of myofibrils in some of the muscle fibers, hemorrhage in-between the muscle fibers and mononuclear cellular infiltration. While light microscopic examinations of gastrocenemius muscles of mice injected with 1.5 kGy gamma irradiated Echis pyramidum venom [13.8 micro g/50 micro l; a dose identical to that used from native venom] showed that most muscle fibers were of normal appearance except for small area of fragmentation and disorganized myofibrils and oedema of the intercellular connective tissue. Double immunodiffusion test revealed a similar reactivity for native, 1 kGy, 1.5 kGy and 3 kGy gamma irradiated Echis pyramidum venoms against a commercial polyvalent Egyptian antivenin. The visible lines obtained in the immunodiffusion reactions were identical and joined smoothly at the corners, indicating that there was no change in their antigenic reactivity. These results demonstrate that the ability of the venom antigens to react with its corresponding antibodies was maintained in spite of being exposed to radiation doses of 1 kGy, 1.5 kGy and 3 kGy. Both antivenins raised against native or 1.5 kGy gamma irradiated venoms recognized Echis pyramidum venom when submitted to protein blotting, but the anti 1.5 kGy gamma irradiated venom show a higher intensity bands than the antivenin raised against native Echis pyramidum venom, in spite of having less neutralizing activity [native neutralize 50 LD[50], 1.5 kGy gamma irradiated neutralize 40 LD[50]], this indicates that antibodies were formed not only for toxic fraction but also for non toxic fraction. Irradiation of the whole Echis Pyramidium Venom with 1.5KGy reduced its lethality 12.5 times though keeping its immunogenicity. The 1.5KGy dose was shown to be the best radiation dose to promote detoxification without significantly affecting its immunogenicity. Thus results of this study confirm the conclusion that gamma radiation is a suitable way to detoxify Echis Pyramidium Venom without affecting its immunogenicity provided that proper dose is used

Neutralización del efecto hemorrágico inducido por veneno de Bothrops asper (Serpientes : Viperidae) por extractos de plantas tropicales / Neutralization of hemorrhagic effect inducid by venom of Bothrops asper (Serpentes: Viperidae) for extracts of tropical plants

Se evaluó la capacidad de extractos orgánicos de 48 especies de plantas costarricense para neutralizar la actividad hemorrágica del veneno de la serpiente Bothrops asper (terciopelo). Los extractos se evaluaron mediante un bioensayo basado en inyecciones intradérmicas de veneno en ratones, o de mezclas veneno-extracto, seguidas de la cuantificación macroscópica de la hemorragia. Se observó una inhibición total de la hemorragia con los extractos etanólicos, de acetato de etilo y acuoso de Bursera simaruba, Clusia torresii, C. palmana, Croton draco, Persea americana, Phoebe brenesii, Pimenta dioica, Sapindus saponaria, Smilax cuculmeca y Virola koschnyi. El análisis químico de estos extractos permitió identificar catequinas, flavonas, antocianinas y taninos condensados, los cuales podrían jugar un papel en la inhibición del efecto hemorrágico debido a la capacidad de quelar el ion zinc requerido por las metaloproteínas hemorrágicas para su acción

Alteraciones cardiovasculares inducidas por el veneno de Lachesis muta (Serpientes: Viperidae) y por su enzima fibrinogenolítica / Cardiovascular alterations induced by the venom of Lachesis muta (Serpentes: Viperidae) and for its fibrinogenolytic enzime

Se estudió la actividad fibrinolítica del veneno Lachesis muta stenophyrs y de su enzima fibrinogenolítica. Ratas Wistar cateterizadas e la arteria carótida y vena yugular fueron inoculadas con el veneno crudo o la enzima. Se monitoreó los cambios en la presión arterial, frecuencia cardíaca y electrocardiograma. La enzima indujo una mayor reducción del fibrinógeno en el veneno crudo sin causar alteraciones cardiovasculares o histológicas. In vitro el veneno crudo coaguló la sangre mientras que la enzima redujo el fibrinógeno en un 23 por ciento. Los resultados sugieren el uso potencial de la enzima fibrinogenolítica como agente antitrombótico

Production of an effective anti-Bothrops-tetanus mixed hyperimmune serum of equine origin

The present investigation reveals the possibility of simultaneous immunization of horses with Bothrops or Crotalus snake venoms and Tetanus antigens for the production of anti-Bothrops-Tetanus or anti-Crotalus-Tetanus mixed serum, with high titers of the respective specific antibodies. Bothrops antivenoms with an average neutralizing titer of 4.16 mg venom/ml were obtained from plasma of horses with titers lower than 0.5 mg venom/ml when Tetanus antigens were not used. This suggests the existence of a synergism between Bothrops venoms and Tetanus antigens in the stimulation of the antibody response. The pooled plasma of the animal had a neutralizing titer of 21.0 mg/ml reference Bothrops venoms and 3,300 IU/ml to Tetanus antigens after purification by enzymatic digestion and ammonium sulphate precipitation. These experiments lead us to conclude that Bothrops envenomation therapy can be successfully performed using Anti-Bothrops-Tetanus serum also serving as Tetanus prophylaxis. anti-Crotalus-Tetanus serum can also be produced, although it is not of medical interest as Crotalus envenomation rarely results in local necrotizing lesions.

Preparación toxoide a partir de la fracción hemorrágica del veneno de Bothrops asper (serpiente de América Central y del Sur) / Toxoid preparation from hemorrhagic fraction of Bothrops asper venom

Se describe una técnica para la preparación de un toxoide a partir de la fracción hemorrágica del veneno de Bothrops asper. Este método conserva un alto grado de inmunogenecidad aunque elimina los efectos letales. Ninguno de los animales vacunados con el toxoide de esta fracción presentó lesiones hemorrágicas cuando les fue inyectado el veneno de la fracción hemorrágica

Alteraçöes neuroretinianas por picada de cobra / Neuroretinits alterations by snake-bite

Os autores apresentam um caso de neurorretinite bilateral pós-picada pro crotalus terrificus e discutem as açöes e os efeitos do veneno sobre o olho humano

Valores hematológicos y de enzimas séricas en caballos inoculados con venenos de serpientes para la produción de antivenenos en Costa Rica / Hematologic values and of serum enzymes in inoculated horses with snake venoms for the production of antivenins in Costa Rica

Se estudiaron algunos componentes sanguíneos en un grupo de seis caballos inmunizados por primera vez con venenos de serpientes para la producción de suero antiofídico polivalente en Costa Rica. No hubo cambios significativos en los valores de hematocrito y hemoglobina, aunque las proteínas totales sufrieron un pequeno pero significativo aumento en la segunda mitad del esquema de inmunización, probablemente relacionado con un aumento en la producción de globulinas. No hubo cambios significativos en los niveles de CK, mientras que las enzimas TGO y TGP aumentaron levemente, sin sobrepasar el límite superior normal. Estos resultados sugieren la ausencia de lesiones tisulares importantes en músculo esquelético, músculo cardíaco e hígado. Los caballos presentan únicamente alteraciones locales en el sitio de inoculación del veneno, caracterizadas por edema, abscesos y fístulas. No se observó ningún tipo de alteración sistémica. Se observó una gran variabilidad individual en el desarrollo de la respuesta de anticuerpos anti-fosfolipasa A2

Immunodiagnosis of snake venom poisoning.

Uncertainty as to the species diagnosis remains a serious problem in the management of snake venom poisoning. This is particularly so in areas inhabited by numerous species, the venoms of which elicit similar pharmacological effects and clinical symptoms and against which para-specific cross-neutralizing antivenom is not available. Attempts have been made to eliminate some of this ambiguity through the development of various immunodiagnostic tests. Tests based on ELISA are sensitive, specific and even quantitative and adaptable to field application. In the development of diagnostic tests for use in developing countries, however, practical consideration must be given to speed, cost, simplicity in terms of equipment and expertise, and stability to the climate and storage conditions. This may dictate further modification or selection of more suitable alternative methodologies. Furthermore, the test may have to allow more flexibility in accommodating local species distributions and to address probable complications of heterophile antibodies in test samples from rural people.