Obtaining plasma to measure baseline corticosterone concentrations in reptiles: How quick is quick enough?

There is growing interest in the use of glucocorticoid (GC) hormones to understand how wild animals respond to environmental challenges. Blood is the best medium for obtaining information about recent GC levels; however, obtaining blood requires restraint and can therefore be stressful and affect GC levels. There is a delay in GCs entering blood, and it is assumed that blood obtained within 3 min of first disturbing an animal reflects a baseline level of GCs, based largely on studies of birds and mammals. Here we present data on the timing of changes in the principle reptile GC, corticosterone (CORT), in four reptile species for which blood was taken within a range of times 11 min or less after first disturbance. Changes in CORT were observed in cottonmouths (Agkistrodon piscivorus; 4 min after first disturbance), rattlesnakes (Crotalus oreganus; 2 min 30 s), and rock iguanas (Cyclura cychlura; 2 min 44 s), but fence lizards (Sceloporus undulatus) did not exhibit a change within their 10-min sampling period. In both snake species, samples taken up to 3-7 min after CORT began to increase still had lower CORT concentrations than after exposure to a standard restraint stressor. The "3-min rule" appears broadly applicable as a guide for avoiding increases in plasma CORT due to handling and sampling in reptiles, but the time period in which to obtain true baseline CORT may need to be shorter in some species (rattlesnakes, rock iguanas), and may be unnecessarily limiting for others (cottonmouths, fence lizards).

Physiological and behavioral effects of exogenous corticosterone in a free-ranging ectotherm.

In the face of global change, free-ranging organisms are expected to experience more unpredictable stressors. An understanding of how organisms with different life history strategies will respond to such changes is an integral part of biodiversity conservation. Corticosterone (CORT) levels are often used as metrics to assess the population health of wild vertebrates, despite the fact that the stress response and its effects on organismal function are highly variable. Our understanding of the stress response is primarily derived from studies on endotherms, leading to some contention on the effects of chronic stress across and within taxa. We assessed the behavioral and hormonal responses to experimentally elevated stress hormone levels in a free-ranging, arid-adapted ectotherm, the Southern Pacific rattlesnake (Crotalus helleri). Plasma CORT was significantly elevated in CORT-implanted snakes 15days after implantation. Implantation with CORT did not affect testosterone (T) levels or defensive behavior. Interestingly, we observed increased defensive behavior in snakes with more stable daily body temperatures and in snakes with higher plasma T during handling (tubing). Regardless of treatment group, those individuals with lower baseline CORT levels and higher body temperatures tended to exhibit greater increases in CORT levels following a standardized stressor. These results suggest that CORT may not mediate physiological and behavioral trait expression in arid-adapted ectotherms such as rattlesnakes.

PHARMACOKINETICS, EFFICACY, AND SAFETY OF VORICONAZOLE AND ITRACONAZOLE IN HEALTHY COTTONMOUTHS (AGKISTRODON PISCIVORUS) AND MASSASAUGA RATTLESNAKES (SISTRURUS CATENATUS) WITH SNAKE FUNGAL DISEASE.

Snake fungal disease (SFD; Ophidiomyces ophiodiicola) is posing a significant threat to several free-ranging populations of pitvipers. Triazole antifungals have been proposed for the treatment of mycoses in reptiles; however, data are lacking about their safety and efficacy in snakes with SFD. Study 1 investigated in vitro susceptibility, and identified that plasma concentrations >250 ng/ml (voriconazole) and >1,000 ng/ml (itraconazole) may be effective in vivo for SFD. In Study 2, the pharmacokinetics after a single subcutaneous voriconazole injection were assessed in apparently healthy free-ranging cottonmouths (Agkistrodon piscivorus). Based on pilot-study results, four snakes were administered a single injection of voriconazole (5 mg/kg). One pilot snake and three full-study snakes died within 12 hr of voriconazole administration. All surviving snakes maintained plasma concentrations >250 ng/ml for 12-24 hr. In Study 3, two Eastern massasaugas (Sistrurus catenatus) and a timber rattlesnake (Crotalus horridus horridus) diagnosed with SFD were treated with voriconazole delivered by subcutaneous osmotic pumps. The timber rattlesnake (12.1-17.5 mg/kg/hr) reached therapeutic concentrations, whereas the massasaugas (1.02-1.6 mg/kg/hr) did not. In Study 4, the pharmacokinetics of a single 10-mg/kg per-cloaca dose of itraconazole (Sporanox®) was evaluated in seven apparently healthy free-ranging cottonmouths. Similarly, the plasma and tissue concentrations did not meet therapeutic concentrations based on in vitro data. The data presented in this report serve as an initial step toward understanding the pharmacokinetics, efficacy, and safety of triazole antifungals in pitviper species with SFD. Further study is needed to determine the appropriate dose and route of administration of triazole antifungals in pitviper species.

Effects of long distance translocation on corticosterone and testosterone levels in male rattlesnakes.

Translocation is an increasingly common conservation tool used to augment declining populations or to remove nuisance animals from areas of human conflict. Studies show that venomous snakes translocated long distances may wander and experience increased mortality. However, potential sub-lethal physiological effects on translocated snakes remain unknown. We conducted an experimental study on free-ranging rattlesnakes to test the hypothesis that long distance translocation is stressful. The glucocorticoid response to translocation was variable among snakes. There was some evidence that translocation may be stressful, as baseline corticosterone levels in most snakes rose following translocation, whereas levels remained consistent in control snakes. Interestingly, testosterone levels rose dramatically following translocation, possibly reflecting effects of interaction with new environmental cues and/or resident snakes, or effects of navigation in a new environment. Corticosterone and testosterone were positively correlated. Our study shows that long distance translocation can affect steroid hormone concentrations in rattlesnakes, a result that should be taken into consideration when managing nuisance snakes or repatriating animals to the wild.

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.

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.

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.

Hematology, plasma biochemistry, and antibodies to select viruses in wild-caught Eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) from Illinois.

During the 2004 field season, blood was collected from Eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) in the Carlyle Lake (Carlyle, Illinois, USA) and Allerton Park (Monticello, Illinois, USA) populations to derive baseline complete blood count and plasma biochemistry data and to assess the prevalence of antibodies to West Nile virus (WNV) and ophidian paramyxovirus (OPMV). Massasaugas were located for sampling through visual encounter surveys. Body weight, snout-vent length, total protein, globulins, sodium, and potassium were normally distributed among the survey population. Aspartate aminotransferase, creatine kinase, albumin, calcium, uric acid, white blood cell count, heterophils, lymphocytes, monocytes, eosinophils, and basophils were non-normally distributed within these animals. Female snakes had significantly shorter tail lengths; lower blood glucose, packed cell volumes, and absolute azurophil counts; and higher plasma calcium and phosphorus concentrations than did males. None of the snakes tested (n=21) were seropositive for WNV, whereas all (n=20) were seropositive for OPMV.

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.

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.

Morphologic, cytochemical staining, and ultrastructural characteristics of blood cells from eastern diamondback rattlesnakes (Crotalus adamanteus).

OBJECTIVE: To evaluate light microscopic, cytochemical, and ultrastructural characteristics of blood cells from eastern diamondback rattlesnakes. ANIMALS: 10 healthy snakes. PROCEDURE: Various stains, including Wright-Giemsa, benzidine peroxidase, Sudan black B, chloroacetate esterase, alpha-naphthyl butyrate esterase, acid phosphatase, leukocyte alkaline phosphatase, periodic acid-Schiff with diastase, and toluidine blue, were used to stain leukocytes differentially on multiple blood smears. Electron microscopy also was performed. RESULTS: Lymphocytes were the most commonly observed leukocyte and could be distinguished from thrombocytes, using periodic acid-Schiff stain with diastase. Azurophils also were commonly observed; their granules stained with peroxidase. Eosinophils were not identified; however, 2 morphologic variations of heterophils were seen in the blood of all snakes and were considered the same cell type at different stages of cytoplasmic granule development. Heterophil granules were better preserved, using a one-step Wright-Giemsa method that did not require alcohol fixation prior to staining. Degranulated heterophils were observed in all preparations. CONCLUSIONS: Most leukocytes of eastern diamondback rattlesnakes can be identified easily on Wright-Giemsa-stained preparations. However, hematologic stains that do not require alcohol fixing prior to staining may be preferred for leukocyte evaluation in certain reptiles. A limited degree of heterophil maturation may continue in the blood of healthy snakes. This, along with degranulation of heterophils, may result in a variable staining pattern in this cell type, regardless of the stain used. CLINICAL RELEVANCE: Results provide baseline data for use in hematologic testing in diagnosis of disease and monitoring of treatment of sick or injured snakes.

Seasonal variation in hematology and blood plasma chemistry values of the timber rattlesnake (Crotalus horridus).

Hematology, biochemical analyses, and body condition indices are useful tools for describing animal health, especially when making management decisions for species of conservation concern. We report hematologic, biochemical, and body condition index data for 13 free-ranging timber rattlesnakes (Crotalus horridus) sampled repeatedly over an active season in Indiana, USA.

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.

Additional observations and notes on the natural history of the prairie rattlesnake (Crotalus viridis) in Colorado.

On account of their unique anatomy, physiology, natural history, ecology, and behavior, rattlesnakes make ideal subjects for a variety of different scientific disciplines. The prairie rattlesnake (Crotalus viridis) in Colorado was selected for investigation of its relationship to colonies of black-tailed prairie dogs (Cynomys ludovicianus) with regard to spatial ecology. A total of 31 snakes were anesthetized and had radiotransmitters surgically implanted. In addition, at the time of their capture, all snakes underwent the following: (1) they had bacterial culture taken from their mouths for potential isolation of pathogenic bacteria; (2) similarly, they had cloacal bacterial cultures taken to assess potentially harmful bacteria passed in the feces; and (3) they had blood samples drawn to investigate the presence of any zoonotic agents in the serum of the snakes. The results of the study and their implications are discussed here. Traditionally, a low incidence of bacterial wound infection has been reported following snakebite. Nevertheless, the oral cavity of snakes has long been known to house a wide variety of bacterial flora. In our study, 10 different bacterial species were isolated from the mouths of the rattlesnakes, 6 of which are capable of being zoonotic pathogens and inducing human disease. More studies are necessary to see why more rattlesnake bites do not become infected despite the presence of such pathogenic bacteria. The results of fecal bacteria isolated revealed 13 bacterial species, 12 of which can cause disease in humans. Of the snakes whose samples were cultured, 26% were positive for the presence of the pathogen Salmonella arizonae, one of the causative agents of reptile-related salmonellosis in humans. It has long been reported that captive reptiles have a much higher incidence than wild, free-ranging species. This study shows the incidence of Salmonella in a wild, free-ranging population of rattlesnakes. In addition, Stenotrophomonas maltophilia was isolated. This bacterium is associated with wound and soft tissue infections that can lead to sepsis, endocarditis, meningitis, and peritonitis. In addition, this bacterium has been increasingly implicated as an opportunistic pathogen to humans during pregnancies, hospitalizations, malignancies and chemotherapy, chronic respiratory diseases, and presurgical endotracheal intubation. Furthermore, S. maltophilia has an intense resistance to broad-spectrum antibiotics, the results of our study showed the bacterium was resistant to multiple antibiotics. Our results indicate that anyone working with snake feces, dead skin, or their carcasses must follow reasonable hygiene protocols. Rattlesnakes tested for West Nile antibodies had positive results but these were invalidated owing to possible cross-reactivity with other unknown viruses, interference with snake serum proteins, and the fact that the test was not calibrated for rattlesnake serum. Still, the interesting implication remains, should we be regularly testing these animals as sentinels against potentially zoonotic diseases. The results of this study clearly show the value of veterinarians in a multidisciplinary study of this sort and the particular skill set they can offer. Veterinarians must get involved in conservation studies if the biodiversity of the planet is to be preserved.

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 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)

Pathologic and hematologic responses to surgically implanted transmitters in eastern massasauga rattlesnakes (Sistrurus catenatus catenatus).

The study of secretive snakes, such as rattlesnakes, has benefited from the use of radiotelemetry. However, the principal assumption in telemetry studies is that the transmitter has no significant effect on the study animal. To test the validity of this assumption, the physiologic and pathologic effects of intracoelomic implants were examined in a group of 24 eastern massasauga rattlesnakes (Sistrurus catenatus catenatus) in a laboratory setting over a period of 58 wk between March 2005 and April 2006. Inflammation and infection were evaluated using gross examination, histopathology, bacteriology, hematology, and plasma protein electrophoresis. Inflammation and infection occurred despite careful surgical procedures and advanced veterinary care. Four of 12 (33%) snakes developed extensive inflammatory response to the transmitter and associated anaerobic and gram-negative bacterial infections. Another four (33%) snakes showed mild inflammatory responses without infection. Reaction to the transmitters was reflected in changes in values for heterophils, monocytes, alpha-1, and beta globulin levels. Some conclusions reached in field studies using implanted radiotransmitters in snakes may be invalid if the implant influences the behavior or survival of the subject. Advances in attachment methods and transmitter coating technology may prevent some of the adverse effects associated with surgically implanted transmitters.

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)

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.