Aspectos citoquímicos e morfológicos de elementos sanguíneos das serpentes dos gêneros Bothrops e Crotalus mantidas em cativeiro no serpentário do Instituto Vital Brasil / Cytochemical and morphological aspects of blood cells from Bothrops and Crotalus kept in captivity in the serpentarium of the Institute Vital Brazil

A criação de serpentes peçonhentas em cativeiro vem se tornando prática cada vez mais difundida no país. Dessa forma, o conhecimento do manejo e da clínica de serpentes se torna prioritário, a fim de permitir maior sobrevida dos animais. No que concerne a serpentes peçonhentas, dados hematológicos já foram descritos na literatura, no entanto, apesar dos recursos utilizados, os dados analisados ainda são insipientes. Com o objetivo de caracterizar, morfologicamente, as células sanguíneas e de esclarecer diferenças morfológicas e funcionais, foram coletadas amostras de sangue de 50 serpentes pertencentes ao plantel do Instituto Vital Brasil. Foram confeccionados e analisados por microscopia óptica e citoquimicamente os esfregaços sanguíneos corados por métodos de Romanowsky e citoquímicos. Foi possível diferenciar as células, caracterizar e confirmar a existência de eosinófilo em B. atrox e C. durissus. Concluiu-se que a caracterização celular pode fornecer evidências indispensáveis ao entendimento da fisiologia de serpentes.(AU)

Breeding of venomous snakes in captivity is becoming increasingly widespread in the country, so clinical and management knowledge on these animals has become priority to increase survival of animals. Regarding venomous snakes, hematological data have been described in some studies; however, despite the resources used, data analyzed are still unrecognized. Aiming to characterize morphology of blood cells and clarify morphological and functional differences, blood samples were collected from 50 snakes belonging to the Instituto Vital Brazil squad. Blood smears were prepared and analyzed by optical microscopy and cytochemistrically, stained by Romanowsky and cytochemical methods. Cell differentiation was possible as well as characterization and confirmation of eosinophil in B. atrox and C. durissus. In conclusion, cell characterization can provide vital evidence to the understanding of the physiology of snakes.(AU)

Aspectos citoquímicos e morfológicos de elementos sanguíneos das serpentes dos gêneros Bothrops e Crotalus mantidas em cativeiro no serpentário do Instituto Vital Brasil / Cytochemical and morphological aspects of blood cells from Bothrops and Crotalus kept in captivity in the serpentarium of the Institute Vital Brazil

A criação de serpentes peçonhentas em cativeiro vem se tornando prática cada vez mais difundida no país. Dessa forma, o conhecimento do manejo e da clínica de serpentes se torna prioritário, a fim de permitir maior sobrevida dos animais. No que concerne a serpentes peçonhentas, dados hematológicos já foram descritos na literatura, no entanto, apesar dos recursos utilizados, os dados analisados ainda são insipientes. Com o objetivo de caracterizar, morfologicamente, as células sanguíneas e de esclarecer diferenças morfológicas e funcionais, foram coletadas amostras de sangue de 50 serpentes pertencentes ao plantel do Instituto Vital Brasil. Foram confeccionados e analisados por microscopia óptica e citoquimicamente os esfregaços sanguíneos corados por métodos de Romanowsky e citoquímicos. Foi possível diferenciar as células, caracterizar e confirmar a existência de eosinófilo em B. atrox e C. durissus. Concluiu-se que a caracterização celular pode fornecer evidências indispensáveis ao entendimento da fisiologia de serpentes.(AU)

Breeding of venomous snakes in captivity is becoming increasingly widespread in the country, so clinical and management knowledge on these animals has become priority to increase survival of animals. Regarding venomous snakes, hematological data have been described in some studies; however, despite the resources used, data analyzed are still unrecognized. Aiming to characterize morphology of blood cells and clarify morphological and functional differences, blood samples were collected from 50 snakes belonging to the Instituto Vital Brazil squad. Blood smears were prepared and analyzed by optical microscopy and cytochemistrically, stained by Romanowsky and cytochemical methods. Cell differentiation was possible as well as characterization and confirmation of eosinophil in B. atrox and C. durissus. In conclusion, cell characterization can provide vital evidence to the understanding of the physiology of snakes.(AU)

Aspectos morfológicos e ultraestruturais de células sanguíneas de Crotalus durissus terrificus / Morphological and ultrastructural aspects of Crotalus durissus terrificus blood cells

A avaliação hematológica, de importância comprovada como um meio auxiliar de diagnóstico ao clínico de pequenos animais domésticos, vem se tornando comum em animais selvagens não apenas para a clínica, mas para a avaliação do manejo e como estudo auxiliar para a fisiologia das várias espécies. Tendo em vista o aumento da demanda para a produção de várias drogas de importância farmacêutica, a criação de serpentes peçonhentas vem se tornando comum a ponto destes animais já serem reconhecidos como sendo de produção. O conhecimento do manejo e da clínica destes animais ainda é escasso e a mortalidade é elevada nos criatórios, tornando urgente a ampliação destes. Embora alguns estudos hematológicos já tenham sido realizados em cascavéis (Crotalus durissus) os dados analisados ainda são insipientes, notadamente em relação à caracterização das células do sangue e poucos estudos em microscopia eletrônica foram realizados em serpentes. Com o objetivo de caracterizar as células sanguíneas morfologicamente, sob microscopia óptica e ultraestrutural, foram coletadas amostras de sangue de 52 de indivíduos da subespécie Crotalus durissus terrificus para a realização de esfregaços sanguíneos e avaliação ultraestrutural. Concluiu-se que a coloração hematológica de Giemsa permite a avaliação morfológica e a diferenciação das células sanguíneas em serpentes assim como a visualização de hemoparasitos. A avaliação ultraestrutural permite evidenciar as organelas celulares e a diferenciação entre as células, inclusive entre os tipos leucocitários, porém ainda são necessários outros estudos para que seja elucidada a hipótese da existência dos eosinófilos na espécie estudada assim como é necessária melhor caracterização dos grânulos dos azurófilos para que se confirme uma possível diferença entre os monócitos típicos e os azurófilos.(AU)

Hematological evaluation, important for the diagnostic by the small domestic animal clinician, has become common in wildlife clinic, and for handling and study of the physiology of various species. Given the increased demand for drug production of pharmaceutical importance, the breeding of venomous snakes has become common and is already recognized as production. Knowledge of the management and clinics of snakes is still insufficient and their mortality is high. Although some hematological studies have already been conducted in the rattlesnake (Crotalus durissus), the analyzed data are still insufficient, especially with respect to the characterization of blood cells, and few electron microscopy studies have been performed on snakes. In order to characterize morphologically blood cells with light and ultrastructural microscopy, blood samples from 52 individuals of subspecies of Crotalus durissus terrificus were collected to perform blood smears and ultrastructural evaluation. It was concluded that hematologic Giemsa staining allows morphological evaluation and differentiation of the blood cells as well as of snake hemoparasites. The ultrastructural evaluation will highlight the cell organelles and differentiation between cells, including leukocyte types; although still further studies are needed to elucidate the hypothesis of eosinophils in the species studied as also is necessary a better characterization of azurophilic beads to confirm a possible difference between the typical monocyte and the azurophilic.(AU)

Aspectos morfológicos e ultraestruturais de células sanguíneas de Crotalus durissus terrificus / Morphological and ultrastructural aspects of Crotalus durissus terrificus blood cells

Hematological evaluation, important for the diagnostic by the small domestic animal clinician, has become common in wildlife clinic, and for handling and study of the physiology of various species. Given the increased demand for drug production of pharmaceutical importance, the breeding of venomous snakes has become common and is already recognized as production. Knowledge of the management and clinics of snakes is still insufficient and their mortality is high. Although some hematological studies have already been conducted in the rattlesnake (Crotalus durissus), the analyzed data are still insufficient, especially with respect to the characterization of blood cells, and few electron microscopy studies have been performed on snakes. In order to characterize morphologically blood cells with light and ultrastructural microscopy, blood samples from 52 individuals of subspecies of Crotalus durissus terrificus were collected to perform blood smears and ultrastructural evaluation. It was concluded that hematologic Giemsa staining allows morphological evaluation and differentiation of the blood cells as well as of snake hemoparasites. The ultrastructural evaluation will highlight the cell organelles and differentiation between cells, including leukocyte types; although still further studies are needed to elucidate the hypothesis of eosinophils in the species studied as also is necessary a better characterization of azurophilic beads to confirm a possible difference between the typical monocyte and the azurophilic.(AU)

A avaliação hematológica, de importância comprovada como um meio auxiliar de diagnóstico ao clínico de pequenos animais domésticos, vem se tornando comum em animais selvagens não apenas para a clínica, mas para a avaliação do manejo e como estudo auxiliar para a fisiologia das várias espécies. Tendo em vista o aumento da demanda para a produção de várias drogas de importância farmacêutica, a criação de serpentes peçonhentas vem se tornando comum a ponto destes animais já serem reconhecidos como sendo de produção. O conhecimento do manejo e da clínica destes animais ainda é escasso e a mortalidade é elevada nos criatórios, tornando urgente a ampliação destes. Embora alguns estudos hematológicos já tenham sido realizados em cascavéis (Crotalus durissus) os dados analisados ainda são insipientes, notadamente em relação à caracterização das células do sangue e poucos estudos em microscopia eletrônica foram realizados em serpentes. Com o objetivo de caracterizar as células sanguíneas morfologicamente, sob microscopia óptica e ultraestrutural, foram coletadas amostras de sangue de 52 de indivíduos da subespécie Crotalus durissus terrificus para a realização de esfregaços sanguíneos e avaliação ultraestrutural. Concluiu-se que a coloração hematológica de Giemsa permite a avaliação morfológica e a diferenciação das células sanguíneas em serpentes assim como a visualização de hemoparasitos. A avaliação ultraestrutural permite evidenciar as organelas celulares e a diferenciação entre as células, inclusive entre os tipos leucocitários, porém ainda são necessários outros estudos para que seja elucidada a hipótese da existência dos eosinófilos na espécie estudada assim como é necessária melhor caracterização dos grânulos dos azurófilos para que se confirme uma possível diferença entre os monócitos típicos e os azurófilos.(AU)

Flow cytometric characterization of peripheral blood leukocyte populations of 3 neotropical snake species: Boa constrictor, Bothrops jararaca, and Crotalus durissus.

BACKGROUND: The reptilian immune system is represented by innate, humoral, and cell-mediated mechanisms, involving different types of blood leukocytes. The development of optimized methods for the advanced study of origin and function of reptilian blood leukocytes is needed. OBJECTIVES: The purpose of the study was to optimize leukocyte density gradient isolation protocols from snake peripheral blood samples, and characterize recovered cells by flow cytometry based on size and internal complexity for a qualitative and semi-quantitative assessment of leukocyte populations in one boa (Boa constrictor), and 2 viper species (Bothrops jararaca, Crotalus durissus). METHODS: Blood samples from 30 snakes (10 from each species, 5 males and 5 females) were collected in tubes with sodium heparin. Fresh blood was centrifuged with either ficoll-paque PLUS or percoll density gradients for leukocyte isolation. Flow cytometric leukocyte gates were defined based on size (forward scatter [FSC]) and internal complexity (side scatter [SSC]). Relative leukocyte differential counts after sorting the cells in these gates in one snake for each species were compared to conventional light microscopic differential counts on unsorted isolated leukocytes. RESULTS: There was no statistical difference in the relative leukocyte populations, including heterophils, azurophils, and small and large lymphocytes between samples isolated by ficoll or percoll. Four leukocyte gates were identified based on their location in FSC/SSC cytograms. The relative leukocyte differential counts after sorting in single animals showed some agreement with the light microscopy differential count on unsorted cells. CONCLUSIONS: Based on FSC and SSC, 4 distinct leukocyte populations were found in ficoll or percoll density gradient isolated leukocytes from peripheral blood from boa and viper species. Further optimization of the technique should allow the performance of functional assays.

Blood oxygen affinity increases during digestion in the South American rattlesnake, Crotalus durissus terrificus.

Digesting snakes experience massive increases in metabolism that can last for many days and are accompanied by adjustments in the oxygen transport cascade. Accordingly, we examined the oxygen-binding properties of the blood in the South American rattlesnake (Crotalus durissus terrificus) during fasting and 24 and 48h after the snakes have ingested a rodent meal corresponding to 15% (±2%) of its own body mass. In general, oxygen-hemoglobin (Hb-O2) affinity was significantly increased 24h post-feeding, and then returned toward fasting values within 48h post-feeding. Content of organic phosphates ([NTP] and [NTP]/[Hb]), hemoglobin cooperativity (Hill's n), and Bohr Effect (ΔlogP50/ΔpH) were not affected by feeding. The postprandial increase in Hb-O2 affinity in the South American rattlesnake can be almost entirely ascribed by the moderate alkaline tide that follows meal ingestion. In general, digesting snakes were able to regulate blood metabolites at quite constant levels (e.g., plasma osmolality, lactate, glucose, and total protein levels). The level of circulating lipids, however, was considerably increased, which may be related to their mobilization, since lipids are known to be incorporated by the enterocytes after snakes have fed. In conclusion, our results indicate that the exceptional metabolic increment exhibited by C. d. terrificus during meal digestion is entirely supported by the aerobic pathways and that among the attending cardiorespiratory adjustments, pulmonary Hb-O2 loading is likely improved due to the increment in blood O2 affinity.

Determinação das concentrações plasmáticas de proteínas e metabólitos de cascavéis em cativeiro / Determination of proteins and metabolites concentrations in plasma of rattlesnakes in captivity

Para determinar as concentrações plasmáticas de proteínas e metabólitos de cascavéis em cativeiro, foram utilizadas 60 serpentes adultas, sendo 30 machos e 30 fêmeas. O sangue foi coletado através de punção do seio venoso paravertebral cervical e armazenado em tubos com heparina. As análises bioquímicas foram processadas colorimetricamente em Analisador Automático de Bioquímica Chemwell (Awareness Technology®, Inc). Foram calculadas as médias e desvios padrão dos seguintes constituintes: proteínas totais, albumina, globulinas, relação albumina/globulinas, ácido úrico, creatinina, ureia, colesterol, colesterol HDL e triglicérides. Os valores obtidos foram semelhantes aos descritos na literatura para repteis e serpentes, sendo as diferenças observadas provavelmente decorrentes da diferença entre espécies, clima, estação do ano e metodologia utilizada. Não houve diferenças significativas entre machos e fêmeas para os parâmetros estudados. Estes resultados podem ser úteis no estabelecimento de valores de referência para planos de conservação destes ofídios em cativeiro.(AU)

For determining plasma concentrations of proteins and metabolites of rattlesnakes in captivity, 60 adult snakes, 30 males and 30 females were used. Blood was collected by puncture of the cervical paravertebral venous sinus and stored in tubes with heparin. Biochemical analyzes were colorimetrically processed using an Automatic Biochemistry Analyzer Chemwell (Awareness Technology®, Inc). The mean and standard deviation were calculated for the following constituents: total protein, albumin, globulin, albumin/globulin ratio, uric acid, creatinine, urea, cholesterol, HDL cholesterol and triglycerides. The values were similar to those previously reported for reptiles and snakes, with the differences observed probably due to the difference between species, climate, season and the methodology used. There were no significant differences between males and females for the parameters studied. These results may be useful in establishing normal biochemical values for conservation plans for these snakes in captivity.(AU)

Isolation and biochemical characterization of a γ-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum.

In the present work, we describe the isolation and partial structural and biochemical characterization of the first phospholipase A2 inhibitor (γPLI) from Crotalus durissus collilineatus (Cdc) snake serum. Initially, the Cdc serum was subjected to a Q-Sepharose ion exchange column, producing six peaks at 280 nm absorbance (Q1-Q6). Subsequently, Q4 fraction was submitted to affinity chromatography with immobilized PLA2 BnSP-7, a step that resulted in two fractions (NHS-1 and NHS-2). The latter contained the inhibitor, denominated γCdcPLI. The molecular mass of γCdcPLI, determined by Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF), was 22,340 Da. Partial sequences obtained by Edman degradation and by mass spectrometry (MALDI-TOF/TOF), showed similarity, as expected, to other related inhibitors. Circular dichroism (CD) analysis showed the presence of approximately 22% alpha helices and 29% beta sheets in the protein secondary structure. Additionally, CD studies also indicated no significant changes in the secondary structure of γCdcPLI when it is complexed to BpPLA2-TXI. On the other hand, dynamic light scattering (DLS) assays showed a temperature-dependent oligomerization behavior for this inhibitor. Biochemical analyses showed γCdcPLI was able to inhibit the enzymatic, cytotoxic and myotoxic activities of PLA2s. Structural and functional studies performed on this inhibitor may elucidate the action mechanisms of PLA2 inhibitors. In addition, we hope this study may contribute to investigating the potential use of these inhibitors for the treatment of snakebite or inflammatory diseases in which PLA2s may be involved.

Avaliação do efeito da crotoxina em animais portadores de encefalomielite autoimune experimental, um modelo de animais de esclerose múltipla / Evaluation of crotoxin effect in the experimental autoimune encephalomyelitis, an animal model of multiple sclerosis

A esclerose múltipla é uma doença inflamatória crônica, de origem autoimune, que acarreta diversas alterações motoras, cognitivas e sensitivas. Dentre as alterações sensitivas, a dor é um dos graves problemas que afetam pessoas portadoras desta doença, interferindo com diversos aspectos da vida do paciente. É importante ressaltar que a esclerose múltipla não tem cura, sendo que a terapêutica se concentra nas ações que retardam a progressão da doença e promovem o alívio dos sintomas, melhorando a qualidade da vida do paciente...

Multiple sclerosis is a Central Nervous System Inflamatory demyelinating disease that has as primary symptomps losses of sensory, cognitive and motor functions. Among the sensory alternations, pain is one of the major concern, afecting various aspects of the patients lives...

Hepatozoon spp. (Apicomplexa: Hepatozoidae) infection and selected hematological values of the neotropical rattlesnake, Crotalus durissus collilineatus (Linnaeus, 1758) (Serpentes: Viperidae), from Brazil.

This study aims to establish the hematological values of Crotalus durissus collilineatus snakes captured in Brazil as well as to verify the effects of hematozoan infection on these snakes. Eighty-three blood samples were drawn from C. d. collilineatus specimens for analysis. The sample set was composed of 30 males and 30 females, recently caught from the wild, and 11 males and 12 females bred in captivity. Blood samples were used to determine red blood cell counts, white blood cell counts, thrombocyte counts, hematocrit values, hemoglobin concentration, and total plasma protein. Blood smears were used to diagnose Hepatozoon spp. infection and to calculate the parasitic load in the sample as well as the percentage of immature red cells. Results obtained for the wild-caught animals, with and without parasites, were compared among themselves and with the values obtained for the captive-bred animals. Hematological values for C. durissus were established. Wild-caught snakes had an infection rate of 38.3%, while no Hepatozoon sp. infection was detected in the captive-bred animals. The snakes which were not infected by the Hepatozoon sp. exhibited average weight, length, and weight-length ratios higher than those of the infected animals. An increase in immature red cells was noted in the Hepatozoon-infected snakes.

Avaliação da resposta inflamatória hematológica em cascavéis (Crotalus durissus Linnaeus, 1758) inoculadas com BCG / Assessment of blood inflammatory response in BCG stimulated rattlesnakes (Crotalus durissus Linnaeus, 1758)

A criação de serpentes peçonhentas em cativeiro para produção de soros antipeçonhas possui crescente importância para a saúde pública devido ao aumento do número de notificações de acidentes ofídicos a cada ano no Brasil. Iniciado no século XX, ainda hoje essa atividade apresenta alguns desafios como a instalação de doenças no plantel. O hemograma é um exame de triagem clínica que auxilia no diagnóstico de diversas moléstias que acometem diferentes espécies de animais, no entanto ainda pouco estudado em serpentes. A caracterização das alterações hematológicas em cascavéis inoculadas experimentalmente com BCG pode servir de base na utilização deste exame no auxílio ao diagnóstico de infecções bacterianas na espécie. Dessa forma, foram realizados exames hematológicos em 10 serpentes da espécie Crotalus durissus pertencentes ao plantel da Divisão de Herpetologia do Instituto Vital Brazil. Os animais foram divididos em dois grupos (Grupos 1 e 2), homogêneos entre si em relação ao peso e proporção sexual. Os dois grupos foram inoculados com BCG e submetidos à coleta de sangue antes da inoculação e em três momentos pós-inoculação (3º, 5º, e 7º dias para o Grupo 1 e 11º, 17º e 21º dias para o Grupo 2). O hemograma foi realizado por método semidireto pela utilização de líquido de Natt e Herrick e as lâminas foram coradas pelo Giemsa. Observou-se anemia discreta, com redução dos valores de concentração de hemoglobina corpuscular média e da hemoglobina globular média no Grupo 1 que foi relacionada à doença inflamatória. A trombocitopenia observada no Grupo 2 sugeriu a atuação deste tipo celular em processos inflamatórios. Um único animal do Grupo 1 apresentou granulocitose e alguns animais apresentaram discreta azurofilia. Observaram-se alterações morfológicas nos leucócitos. Os granulócitos apresentaram granulações grosseiras e os azurófilos apresentaram aumento de tamanho e grandes vacúolos. De forma geral, a inoculação de BCG em cascavéis desencadeia ...

The high demand for anti-venom production in response to the increased number of cases of snakebite envenomation highlights the importance of raising and breeding venomous snakes in captivity. Knowledge of types of venoms and anti-venoms is of great interest to public health. The maintenance of venomous serpents in captivity started in the early twentieth century, but still nowadays it is a challenge to manage and prevent diseases in captive fauna. Hematology is commonly used for general health assessment and illness detection. However, data on serpent blood analysis are still scarce. Alterations in hematological parameters were experimentally induced in rattlesnakes by the inoculation of BCG. Based on this, hemograms can be used as a health auxiliary diagnosis method for bacterial diseases in this species. In this study, blood samples were taken from 10 healthy specimens of rattlesnakes (Crotalus durissus) born and bred in captivity in the Herpetological Division of Vital Brazil Institute. Animals were divided into two groups (group 1 and 2) with similar live weight and sex proportion, and were then inoculated subcutaneously with BCG (Bacillus Calmette-Guérin). Blood samples were taken before and after inoculation at three experimental times (days 3, 5 and 7 for group 1 and days 11, 17 and 21 for group 2). Hematological analysis was performed through semi-direct technique, blood samples were diluted in Natt and Herrick solution and smears were stained by Giemsa. Serpents from group 1 developed discrete anemia due to the inflammatory syndrome, and showed significant decrease of MCH and MCHC. Granulocytes were characterized by the presence of rough granules. The azurophils varied in shape and size and showed large amount of cytoplasmic vacuoles. The thrombocytopenia observed in group 2 suggests that these cells participate in the inflammatory process. A single individual from group 1 showed granulocytosis and a few animals showed a discrete ...

Avaliação da resposta inflamatória hematológica em cascavéis (Crotalus durissus Linnaeus, 1758) inoculadas com BCG / Assessment of blood inflammatory response in BCG stimulated rattlesnakes (Crotalus durissus Linnaeus, 1758)

A criação de serpentes peçonhentas em cativeiro para produção de soros antipeçonhas possui crescente importância para a saúde pública devido ao aumento do número de notificações de acidentes ofídicos a cada ano no Brasil. Iniciado no século XX, ainda hoje essa atividade apresenta alguns desafios como a instalação de doenças no plantel. O hemograma é um exame de triagem clínica que auxilia no diagnóstico de diversas moléstias que acometem diferentes espécies de animais, no entanto ainda pouco estudado em serpentes. A caracterização das alterações hematológicas em cascavéis inoculadas experimentalmente com BCG pode servir de base na utilização deste exame no auxílio ao diagnóstico de infecções bacterianas na espécie. Dessa forma, foram realizados exames hematológicos em 10 serpentes da espécie Crotalus durissus pertencentes ao plantel da Divisão de Herpetologia do Instituto Vital Brazil. Os animais foram divididos em dois grupos (Grupos 1 e 2), homogêneos entre si em relação ao peso e proporção sexual. Os dois grupos foram inoculados com BCG e submetidos à coleta de sangue antes da inoculação e em três momentos pós-inoculação (3º, 5º, e 7º dias para o Grupo 1 e 11º, 17º e 21º dias para o Grupo 2). O hemograma foi realizado por método semidireto pela utilização de líquido de Natt e Herrick e as lâminas foram coradas pelo Giemsa. Observou-se anemia discreta, com redução dos valores de concentração de hemoglobina corpuscular média e da hemoglobina globular média no Grupo 1 que foi relacionada à doença inflamatória. A trombocitopenia observada no Grupo 2 sugeriu a atuação deste tipo celular em processos inflamatórios. Um único animal do Grupo 1 apresentou granulocitose e alguns animais apresentaram discreta azurofilia. Observaram-se alterações morfológicas nos leucócitos. Os granulócitos apresentaram granulações grosseiras e os azurófilos apresentaram aumento de tamanho e grandes vacúolos. De forma geral, a inoculação de BCG em cascavéis desencadeia ...(AU)

The high demand for anti-venom production in response to the increased number of cases of snakebite envenomation highlights the importance of raising and breeding venomous snakes in captivity. Knowledge of types of venoms and anti-venoms is of great interest to public health. The maintenance of venomous serpents in captivity started in the early twentieth century, but still nowadays it is a challenge to manage and prevent diseases in captive fauna. Hematology is commonly used for general health assessment and illness detection. However, data on serpent blood analysis are still scarce. Alterations in hematological parameters were experimentally induced in rattlesnakes by the inoculation of BCG. Based on this, hemograms can be used as a health auxiliary diagnosis method for bacterial diseases in this species. In this study, blood samples were taken from 10 healthy specimens of rattlesnakes (Crotalus durissus) born and bred in captivity in the Herpetological Division of Vital Brazil Institute. Animals were divided into two groups (group 1 and 2) with similar live weight and sex proportion, and were then inoculated subcutaneously with BCG (Bacillus Calmette-Guérin). Blood samples were taken before and after inoculation at three experimental times (days 3, 5 and 7 for group 1 and days 11, 17 and 21 for group 2). Hematological analysis was performed through semi-direct technique, blood samples were diluted in Natt and Herrick solution and smears were stained by Giemsa. Serpents from group 1 developed discrete anemia due to the inflammatory syndrome, and showed significant decrease of MCH and MCHC. Granulocytes were characterized by the presence of rough granules. The azurophils varied in shape and size and showed large amount of cytoplasmic vacuoles. The thrombocytopenia observed in group 2 suggests that these cells participate in the inflammatory process. A single individual from group 1 showed granulocytosis and a few animals showed a discrete ...(AU)

Identification of proteins similar to Bothrops jararaca coagulation inhibitor (BjI) in the plasmas of Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrificus snakes.

Bothrops jararaca coagulation inhibitor (BjI), a protein isolated from B. jararaca plasma, specifically inhibits the coagulant activity of thrombin. Our group previously identified proteins similar to BjI in the plasma of other snakes [Tanaka-Azevedo, A.M., Tanaka, A.S., Sano-Martins I.S., 2003. A new blood coagulation inhibitor from the snake Bothrops jararaca plasma: isolation and characterization. Biochem Biophys Res Commun 308, 706-712.]. In the present study, we analyzed the presence of BjI-like proteins in the plasmas of three different species of viperid snakes, Bothrops alternatus, Bothrops jararacussu and Crotalus durissus terrificus. These proteins exhibited 109 and/or 138 kDa and were immunologically related to BjI. They also inhibited the coagulant activity of thrombin, evaluated by the thrombin time test. These findings demonstrate the presence of proteins similar to BjI in these three species, although such inhibitor could not be observed in all samples of the specimens tested. Moreover, the presence of these proteins in the plasma is related to prolongation of thrombin time, implying a relationship between these proteins and their inhibitory coagulant activity upon thrombin. Our results suggest that BjI-like proteins are widely distributed among Crotalinae snakes found in Brazil.

Arterial acid-base status during digestion and following vascular infusion of NaHCO(3) and HCl in the South American rattlesnake, Crotalus durissus.

Digestion is associated with gastric secretion that leads to an alkalinisation of the blood, termed the "alkaline tide". Numerous studies on different reptiles and amphibians show that while plasma bicarbonate concentration ([HCO(3)(-)](pl)) increases substantially during digestion, arterial pH (pHa) remains virtually unchanged, due to a concurrent rise in arterial PCO(2) (PaCO(2)) caused by a relative hypoventilation. This has led to the suggestion that postprandial amphibians and reptiles regulate pHa rather than PaCO(2). Here we characterize blood gases in the South American rattlesnake (Crotalus durissus) during digestion and following systemic infusions of NaHCO(3) and HCl in fasting animals to induce a metabolic alkalosis or acidosis in fasting animals. The magnitude of these acid-base disturbances were similar in magnitude to that mediated by digestion and exercise. Plasma [HCO(3)(-)] increased from 18.4+/-1.5 to 23.7+/-1.0 mmol L(-1) during digestion and was accompanied by a respiratory compensation where PaCO(2) increased from 13.0+/-0.7 to 19.1+/-1.4 mm Hg at 24 h. As a result, pHa decreased slightly, but were significantly below fasting levels 36 h into digestion. Infusion of NaHCO(3) (7 mmol kg(-1)) resulted in a 10 mmol L(-1) increase in plasma [HCO(3)(-)] within 1 h and was accompanied by a rapid elevation of pHa (from 7.58+/-0.01 to 7.78+/-0.02). PaCO(2), however, did not change following HCO(3)(-) infusion, which indicates a lack of respiratory compensation. Following infusion of HCl (4 mmol kg(-1)), plasma pHa decreased by 0.07 units and [HCO(3)(-)](pl) was reduced by 4.6 mmol L(-1) within the first 3 h. PaCO(2), however, was not affected and there was no evidence for respiratory compensation. Our data show that digesting rattlesnakes exhibit respiratory compensations to the alkaline tide, whereas artificially induced metabolic acid-base disturbances of same magnitude remain uncompensated. It seems difficult to envision that the central and peripheral chemoreceptors would experience different stimuli during these conditions. One explanation for the different ventilatory responses could be that digestion induces a more relaxed state with low responsiveness to ventilatory stimuli.

The role of nitric oxide in the regulation of the systemic and pulmonary vasculature of the rattlesnake, Crotalus durissus terrificus.

The functional role of nitric oxide (NO) was investigated in the systemic and pulmonary circulations of the South American rattlesnake, Crotalus durissus terrificus. Bolus, intra-arterial injections of the NO donor, sodium nitroprusside (SNP) caused a significant systemic vasodilatation resulting in a reduction in systemic resistance (Rsys). This response was accompanied by a significant decrease in systemic pressure and a rise in systemic blood flow. Pulmonary resistance (Rpul) remained constant while pulmonary pressure (Ppul) and pulmonary blood flow (Qpul) decreased. Injection of L-Arginine (L-Arg) produced a similar response to SNP in the systemic circulation, inducing an immediate systemic vasodilatation, while Rpul was unaffected. Blockade of NO synthesis via the nitric oxide synthase inhibitor, L-NAME, did not affect haemodynamic variables in the systemic circulation, indicating a small contribution of NO to the basal regulation of systemic vascular resistance. Similarly, Rpul and Qpul remained unchanged, although there was a significant rise in Ppul. Via injection of SNP, this study clearly demonstrates that NO causes a systemic vasodilatation in the rattlesnake, indicating that NO may contribute in the regulation of systemic vascular resistance. In contrast, the pulmonary vasculature seems far less responsive to NO.

Hematological values of some Bothrops species (Ophidia - Crotalidae) in captivity

Blood samples of 50 healthy specimens from each of the following species: Bothrops alternatus, Bothrops jararacussu, Bothrops moojeni, and Bothrops neuwiedi diporus all kept in captivity were taken to determine the hematocrit (PCV) value, red blood cell count (RBC), total leukocyte (WBC) and differential leukocyte count, thrombocyte count, mean corpuscular volume (MCV), hemoglobin concentration (HbC), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC). These hematological parameters were compared to those obtained from other Bothrops species. PCV values, RBC, hemoglobin, WBC count, and differential leukocyte count are within the range of values reported for other Bothrops species, while the thrombocyte count was significantly lower. All the hematological parameters obtained from the four studied Bothrops species were higher than those described for B. ammodytoides.

Studies on the specificity of CNF, a phospholipase A2 inhibitor isolated from the blood plasma of the South American rattlesnake (Crotalus durissus terrificus). I. Interaction with PLA2 from Lachesis muta muta snake venom.

A phospholipase A2 inhibitor has been previously purified and cloned from the blood plasma of the South American rattlesnake, Crotalus durissus terrificus. This inhibitor, named CNF for Crotalus neutralizing factor, interacts with crotoxin, the main neurotoxin from C. d. terrificus venom, abolishing its phospholipase A2 activity. Crotoxin is a heterodimer of an acidic subunit (CA) and a basic phospholipase A2 (CB). CNF acts by forming a stable non-toxic complex with CB, replacing CA in the toxic CA-CB of crotoxin. In the present investigation, we have shown that CNF has a broader specificity. It is able to inhibit the PLA2 activity of the whole venom from the bushmaster snake (Lachesis muta muta), a species evolutionary related to Crotalus. Inhibition experiments have been carried out with four PLA2 active components isolated from L. m. muta venom, one basic and three acidic ones. CNF inhibition is not restricted to the basic PLA2, but extended to the three acidic forms as well.

A phospholipase A2 inhibitor from the plasma of the South American rattlesnake (Crotalus durissus terrificus). Protein structure, genomic structure, and mechanism of action.

The lethal toxicity of the South American rattlesnake (Crotalus durissus terrificus) venom can be attributed mainly to the presence of a pre-synaptic neurotoxin, crotoxin, with phospholipase A2 activity. Crotoxin is a heterodimer of an acidic protein (CA) and a basic phospholipase A2 (CB). An anti-toxic protein of subunit molecular mass 23.6 kDa that neutralizes both lethal and PLA2 activity of crotalid venom and crotoxin has been previously purified from the plasma of this snake (Fortes-Dias, C., Fonseca, B. C. B., Kochva, E., and Diniz, C. R. (1991) Toxicon 29, 997-1008). The protein has been named CNF for Crotalus neutralizing factor. In the present study, we have shown that CNF exists as an oligomeric aggregate of (CNF)n, where n = 6-8, and when it interacts with crotoxin, it replaces the acidic protein CA of crotoxin to form a stable near stoichiometric complex of CNF.CB. The CNF.CB complex no longer exhibits PLA2 activity and is inert in vivo. Thus, the exchange reaction between CA.CB of crotoxin and CNF to form CNF.CB and free CA is reminiscent of the interaction of crotoxin with its target receptor at the neuromuscular transmission site in the presynaptic cells. A cDNA encoding CNF has been isolated from a liver cDNA library using an appropriate nucleotide probe. The nucleotide sequence codes for a 19-residue signal peptide, followed by a 181-residue protein of which 16 are half-cystines. Calculated molecular mass is 20.06 kDa, and there is a putative N-linked carbohydrate site at Asn157.