Patterns of energetic substrate modifications in response to feeding in boas, Boa constrictor (Serpentes, Boidae).

Ambush-foraging snakes that ingest large meals might undergo several months without eating when they use the internal reserves to support the energetic costs of living. Then, morphological and physiological processes might be orchestrated during the transition from fasting to the postprandial period to rapidly use the energetic stores while the metabolic rate is elevated in response to food intake. To understand the patterns of substrates deposition after feeding, we accessed the morphological and biochemical response in Boa constrictor snakes after two months of fasting and six days after feeding. We followed the plasma levels of glucose, total proteins, and total lipids, and we performed the stereological ultrastructural analysis of the liver and the proximal region of the intestine to quantify glycogen granules and lipid droplets. In the same tissues and stomach, we measured the activity of the enzyme fructose-1,6-biphosphatase (FBPase1) involved in the gluconeogenic pathway, and we measured pyruvate kinase (PK) and lactate dehydrogenase (LDH) enzymatic activities involved in the anaerobic pathway in the liver. Briefly, our results indicated an increase in boas' plasma glucose one day after meal intake compared to unfed snakes. The hepatic glycogen reserves were continuously restored within days after feeding. Also, the enzymes involved in the energetic pathways increased activity six days after feeding in the liver. These findings suggest a quick restoring pattern of energetic stores during the postprandial period.

Reproductive state and water deprivation increase plasma corticosterone in a capital breeder.

Plasma corticosterone (CORT) concentrations fluctuate in response to homeostatic demands. CORT is widely recognized as an important hormone related to energy balance. However, far less attention has been given to the potential role of CORT in regulating salt and water balance or responding to osmotic imbalances. We examined the effects of reproductive and hydric states on CORT levels in breeding Children's pythons (Antaresia childreni), a species with substantial energetic and hydric costs associated with egg development. Using a 2 × 2 experimental design, we examined how reproduction and water deprivation, both separately and combined, impact CORT levels and how these changes correlate with hydration (plasma osmolality) and energy levels (blood glucose). We found that reproduction leads to increased CORT levels, as does dehydration induced by water deprivation. The combined impact of reproduction and water deprivation led to the largest increases in CORT levels. Additionally, we found significant positive relationships among CORT levels, plasma osmolality, and blood glucose. Our results provide evidence that both reproductive activity and increased plasma osmolality can lead to increased plasma CORT in an ectotherm, which could be explained by either CORT having a role as a mineralocorticoid or CORT being elevated as part of a stress response to resource imbalances.

PROTEIN ELECTROPHORESIS OF PLASMA SAMPLES FROM BOA CONSTRICTORS WITH AND WITHOUT REPTARENAVIRUS INFECTION.

Reptarenaviruses infect a variety of boid and pythonid snake species worldwide and have been shown to be the cause of inclusion body disease (IBD). Little is known about the correlations between virus infection and clinical disease, as well as the effects of viral infection on the immune system and the blood protein fractions. The goal of this study was to examine the differences in the plasma protein fractions in reptarenavirus reverse transcription polymerase chain reaction (RT-PCR)-negative and -positive tested snakes with and without clinical signs of disease. Blood from a total of 111 boa constrictors (Boa constrictor) was evaluated. Reverse transcription PCRs and H&E staining for inclusion bodies were carried out on each sample for the detection of reptarenavirus, and the plasma protein fractions were evaluated by capillary zone electrophoresis (CZE). Thirty four of the 111 evaluated snakes were positive by RT-PCR and 19 of the 34 showed clinical signs of disease. In comparison with IBD-negative healthy boa constrictors, the positive snakes with clinical signs had significantly lower albumin levels (P = 0.0052), lower A: G ratios (P = 0.0037), and lower α-globulin levels (P = 0.0073), while their γ-globulin levels were significantly higher (P = 0.0004). In the same comparison, clinically healthy arenavirus-positive boas showed only significantly lower α-globulin (P = 0.0124) and higher γ-globulin levels (P = 0.0394). The results of the present study indicate that reptarenavirus infection may influence plasma protein fractions in boa constrictors.

Pharmacokinetics of midazolam and its major metabolite 1-hydroxymidazolam in the ball python (Python regius) after intracardiac and intramuscular administrations.

Midazolam is a benzodiazepine with sedative, muscle relaxant, anxiolytic, and anticonvulsant effects. Twelve ball pythons (Python regius) were used in a parallel study evaluating the pharmacokinetics of 1 mg/kg midazolam following a single intracardiac (IC) or intramuscular (IM) administration. Blood was collected from a central venous catheter placed 7 days prior, or by cardiocentesis, at 15 time points starting just prior to and up to 72 hr after drug administration. Plasma concentrations of midazolam and 1-hydroxymidazolam were determined by the use of high-performance liquid chromatography tandem-mass spectrometry and pharmacokinetic parameters were estimated using noncompartmental analysis. The mean ± SD terminal half-lives of IC and IM midazolam were 12.04 ± 3.25 hr and 16.54 ± 7.10 hr, respectively. The area under the concentration-time curve extrapolated to infinity, clearance, and apparent volume of distribution in steady-state of IC midazolam were 19,112.3 ± 3,095.9 ng*hr/ml, 0.053 ± 0.008 L hr-1  kg-1 , and 0.865 ± 0.289 L/kg, respectively. The bioavailability of IM midazolam was estimated at 89%. Maximum plasma concentrations following an IM administration were reached 2.33 ± 0.98 hr and 24.00 ± 14.12 hr postinjection for midazolam and 1-hydroxymidazolam, respectively, and 22.33 ± 20.26 hr postinjection for 1-hydroxymidazolam following IC administration.

ARTIFICIAL ULTRAVIOLET B RADIATION RAISES PLASMA 25-HYDROXYVITAMIN D3 CONCENTRATIONS IN BURMESE PYTHONS ( PYTHON BIVITTATUS).

Deficiency of vitamin D can contribute to health complications that present as metabolic bone disease. The aim of this small-scale study was to determine if a high UVb irradiance would affect an increase in plasma vitamin D3 concentrations in Burmese pythons ( Python bivittatus). There have been inconsistent results throughout the literature concerning the usefulness of UVb radiation regarding vitamin D3 synthesis. Blood samples of four healthy Burmese pythons were taken at day 0 and day 310. After the first blood sample was taken, an Arcadia Superzoo T5 ASZ01 lamp was fitted in the enclosure. For 310 days, the pythons were exposed to UVb radiation. Blood plasma vitamin D3 concentrations were considerably higher after UVb exposure. This study indicates that a period of 10 mo of UVb exposure can result in an increased vitamin D3 status in Burmese pythons. Answering whether these elevated levels have health benefits for Burmese pythons (and possibly other snake species) requires further studies.

Reduction of blood oxygen levels enhances postprandial cardiac hypertrophy in Burmese python (Python bivittatus).

Physiological cardiac hypertrophy is characterized by reversible enlargement of cardiomyocytes and changes in chamber architecture, which increase stroke volume and via augmented convective oxygen transport. Cardiac hypertrophy is known to occur in response to repeated elevations of O2 demand and/or reduced O2 supply in several species of vertebrate ectotherms, including postprandial Burmese pythons (Python bivittatus). Recent data suggest postprandial cardiac hypertrophy in P. bivittatus is a facultative rather than obligatory response to digestion, though the triggers of this response are unknown. Here, we hypothesized that an O2 supply-demand mismatch stimulates postprandial cardiac enlargement in Burmese pythons. To test this hypothesis, we rendered animals anemic prior to feeding, essentially halving blood oxygen content during the postprandial period. Fed anemic animals had heart rates 126% higher than those of fasted controls, which, coupled with a 71% increase in mean arterial pressure, suggests fed anemic animals were experiencing significantly elevated cardiac work. We found significant cardiac hypertrophy in fed anemic animals, which exhibited ventricles 39% larger than those of fasted controls and 28% larger than in fed controls. These findings support our hypothesis that those animals with a greater magnitude of O2 supply-demand mismatch exhibit the largest hearts. The 'low O2 signal' stimulating postprandial cardiac hypertrophy is likely mediated by elevated ventricular wall stress associated with postprandial hemodynamics.

A critical evaluation of automated blood gas measurements in comparative respiratory physiology.

Precise measurements of blood gases and pH are of pivotal importance to respiratory physiology. However, the traditional electrodes that could be calibrated and maintained at the same temperature as the experimental animal are increasingly being replaced by new automated blood gas analyzers. These are typically designed for clinical use and automatically heat the blood sample to 37°C for measurements. While most blood gas analyzers allow for temperature corrections of the measurements, the underlying algorithms are based on temperature-effects for human blood, and any discrepancies in the temperature dependency between the blood sample from a given species and human samples will bias measurements. In this study we review the effects of temperature on blood gases and pH and evaluate the performance of an automated blood gas analyzer (GEM Premier 3500). Whole blood obtained from pythons and freshwater turtles was equilibrated in rotating Eschweiler tonometers to a variety of known P(O2)'s and P(CO2)'s in gas mixtures prepared by Wösthoff gas mixing pumps and blood samples were measured immediately on the GEM Premier 3500. The pH measurements were compared to measurements using a Radiometer BMS glass capillary pH electrode kept and calibrated at the experimental temperature. We show that while the blood gas analyzer provides reliable temperature-corrections for P(CO2) and pH, P(O2) measurements were substantially biased. This was in agreement with the theoretical considerations and emphasizes the need for critical calibrations/corrections when using automated blood gas analyzers.

A novel protein from the serum of Python sebae, structurally homologous with type-γ phospholipase A(2) inhibitor, displays antitumour activity.

Cytotoxic and antitumour factors have been documented in the venom of snakes, although little information is available on the identification of cytotoxic products in snake serum. In the present study, we purified and characterized a new cytotoxic factor from serum of the non-venomous African rock python (Python sebae), endowed with antitumour activity. PSS (P. sebae serum) exerted a cytotoxic activity and reduced dose-dependently the viability of several different tumour cell lines. In a model of human squamous cell carcinoma xenograft (A431), subcutaneous injection of PSS in proximity of the tumour mass reduced the tumour volume by 20%. Fractionation of PSS by ion-exchange chromatography yielded an active protein fraction, F5, which significantly reduced tumour cell viability in vitro and, strikingly, tumour growth in vivo. F5 is composed of P1 (peak 1) and P2 subunits interacting in a 1:1 stoichiometric ratio to form a heterotetramer in equilibrium with a hexameric form, which retained biological activity only when assembled. The two peptides share sequence similarity with PIP {PLI-γ [type-γ PLA(2) (phospholipase A(2)) inhibitor] from Python reticulatus}, existing as a homohexamer. More importantly, although PIP inhibits the hydrolytic activity of PLA(2), the anti-PLA(2) function of F5 is negligible. Using high-resolution MS, we covered 87 and 97% of the sequences of P1 and P2 respectively. In conclusion, in the present study we have identified and thoroughly characterized a novel protein displaying high sequence similarity to PLI-γ and possessing remarkable cytotoxic and antitumour effects that can be exploited for potential pharmacological applications.

Pharmacokinetics of a long-acting ceftiofur formulation (ceftiofur crystalline free acid) in the ball python (Python regius).

The objective of this study was to determine the pharmacokinetics of a long-acting formulation of ceftiofur crystalline-free acid (CCFA) following intramuscular injection in ball pythons (Python regius). Six adult ball pythons received an injection of CCFA (15 mg/kg) in the epaxial muscles. Blood samples were collected by cardiocentesis immediately prior to and at 0.5, 1, 2, 4, 8, 12, 18, 24, 48, 72, 96, 144, 192, 240, 288, 384, 480, 576, 720, and 864 hr after CCFA administration. Plasma ceftiofur concentrations were determined by high-performance liquid chromatography. A noncompartmental pharmacokinetic analysis was applied to the data. Maximum plasma concentration (Cmax) was 7.096 +/- 1.95 microg/ml and occurred at (Tmax) 2.17 +/- 0.98 hr. The area under the curve (0 to infinity) for ceftiofur was 74.59 +/- 13.05 microg x h/ml and the elimination half-life associated with the terminal slope of the concentration-time curve was 64.31 +/- 14.2 hr. Mean residence time (0 to infinity) was 46.85 +/- 13.53 hr. CCFA at 15 mg/kg was well tolerated in all the pythons. Minimum inhibitory concentration (MIC) data for bacterial isolates from snakes are not well established. For MIC values of < or =0.1 microg/ml, a single dose of CCFA (15 mg/kg) provides adequate plasma concentrations for at least 5 days in the ball python. For MICs > or =0.5 microg/ml, more frequent dosing or a higher dosage may be required.

BoaγPLI: Structural and functional characterization of the gamma phospholipase A2 plasma inhibitor from the non-venomous Brazilian snake Boa constrictor.

Plasma in several organisms has components that promote resistance to envenomation by inhibiting specific proteins from snake venoms, such as phospholipases A2 (PLA2s). The major hypothesis for inhibitor's presence would be the protection against self-envenomation in venomous snakes, but the occurrence of inhibitors in non-venomous snakes and other animals has opened new perspectives for this molecule. Thus, this study showed for the first time the structural and functional characterization of the PLA2 inhibitor from the Boa constrictor serum (BoaγPLI), a non-venomous snake that dwells extensively the Brazilian territory. Therefore, the inhibitor was isolated from B. constrictor serum, with 0.63% of recovery. SDS-PAGE showed a band at ~25 kDa under reducing conditions and ~20 kDa under non-reducing conditions. Chromatographic analyses showed the presence of oligomers formed by BoaγPLI. Primary structure of BoaγPLI suggested an estimated molecular mass of 22 kDa. When BoaγPLI was incubated with Asp-49 and Lys-49 PLA2 there was no severe change in its dichroism spectrum, suggesting a non-covalent interaction. The enzymatic assay showed a dose-dependent inhibition, up to 48.2%, when BoaγPLI was incubated with Asp-49 PLA2, since Lys-49 PLA2 has a lack of enzymatic activity. The edematogenic and myotoxic effects of PLA2s were also inhibited by BoaγPLI. In summary, the present work provides new insights into inhibitors from non-venomous snakes, which possess PLIs in their plasma, although the contact with venom is unlikely.

Anticancer Properties of Asian Water Monitor Lizard (Varanus salvator), Python (Malayopython reticulatus) and Tortoise (Cuora kamaroma amboinensis).

BACKGROUND: Cancer contributes to significant morbidity and mortality despite advances in treatment and supportive care. There is a need for the identification of effective anticancer agents. Reptiles such as tortoise, python, and water monitor lizards are exposed to heavy metals, tolerate high levels of radiation, feed on rotten/germ-infested feed, thrive in unsanitary habitat and yet have prolonged lifespans. Such species are rarely reported to develop cancer, suggesting the presence of anticancer molecules/mechanisms. METHODS: Here, we tested effects from sera of Asian water monitor lizard (Varanus salvator), python (Malayopython reticulatus) and tortoise (Cuora kamaroma amboinensis) against cancer cells. Sera were collected and cytotoxicity assays were performed using prostate cancer cells (PC3), Henrietta Lacks cervical adenocarcinoma cells (HeLa) and human breast adenocarcinoma cells (MCF7), as well as human keratinized skin cells (Hacat), by measuring lactate dehydrogenase release as an indicator for cell death. Growth inhibition assays were performed to determine the effects on cancer cell proliferation. Liquid chromatography mass spectrometry was performed for molecular identification. RESULTS: The findings revealed that reptilian sera, but not bovine serum, abolished viability of Hela, PC3 and MCF7 cells. Samples were subjected to liquid chromatography mass spectrometry, which detected 57 molecules from V. salvator, 81 molecules from Malayopython reticulatus and 33 molecules from C. kamaroma amboinensis and putatively identified 9 molecules from V. salvator, 20 molecules from Malayopython reticulatus and 9 molecules from C. kamaroma amboinensis when matched against METLIN database. Based on peptide amino acid composition, binary profile, dipeptide composition and pseudo-amino acid composition, 123 potential Anticancer Peptides (ACPs) were identified from 883 peptides from V. salvator, 306 potential ACPs from 1074 peptides from Malayopython reticulatus and 235 potential ACPs from 885 peptides from C. kamaroma amboinensis. CONCLUSION: To our knowledge, for the first time, we reported comprehensive analyses of selected reptiles' sera using liquid chromatography mass spectrometry, leading to the identification of potentially novel anticancer agents. We hope that the discovery of molecules from these animals will pave the way for the rational development of new anticancer agents.

Dehydration enhances innate immunity in a semiaquatic snake from the wet-dry tropics.

Dehydration is considered a physiological challenge, and many organisms live in environments that undergo periods of reduced water availability that can lead to dehydration. Recent studies have found a positive relationship between dehydration and innate immune function in animals adapted to xeric or semixeric environments. To explore the generality of this relationship, we examined the impact of dehydration on innate immune performance in water pythons (Liasis fuscus), a semiaquatic snake from the wet-dry tropics of Australia. We collected blood samples from male and female water pythons held in the laboratory without food and water for 4 weeks. We also collected blood from free-ranging snakes throughout the Austral dry-season. We evaluated plasma osmolality and innate immune function (agglutination, lysis, and bacterial-killing ability) and found that increased osmolality, whether manipulated in the laboratory or as a result of natural water limitation, resulted in enhanced aspects of innate immune performance. Counter-intuitively, snakes in the wild became more hydrated as the dry season progressed, suggesting the dehydrated snakes move to water sources periodically to rehydrate. Comparing our data with those from previous studies, we suspect species divergence in the level of dehydration (i.e., hyperosmolality) that triggers enhanced immune capabilities.

Unraveling avian and reptilian hematology: An optical and electron microscopic study of the buffy coat.

BACKGROUND: Blood centrifugation and buffy coats are at the cornerstone of hematology. In mammals, the buffy coat has a layered disposition (from bottom to top) with neutrophils on top of erythrocytes, followed by monocytes/lymphocytes, and platelets. In nonmammals, this distribution is unknown. Recently, the cell tube block (CTB) technique was developed to study the buffy coat, but it was never applied to nonmammal buffy coats. OBJECTIVES: This study aimed to evaluate using the CTB technique to study reptilian and avian buffy coats and to propose its use for clinical applications. METHODS: Blood from five birds and eight reptiles of different species was obtained to make CTBs that were processed for optical/electron microscopy. H&E, Sirius red, and immunohistochemistry staining against CD3 (to label T lymphocytes) were applied to the CTBs. RESULTS: In birds, the buffy coat had a layered appearance with the granulocyte layer containing granulocytes (heterophils and eosinophils) and nucleated erythrocytes followed by a mononuclear cell layer containing lymphocytes, monocytes, and thrombocytes. In some animals, a nucleated erythrocyte layer was observed admixed with the granulocyte/mononuclear cell layer. A small clot within the buffy coat was seen in seven reptiles, and less definition of layers occurred in reptiles, with only one or two layers. Lymphocytes appeared toward the top of the buffy coat. CONCLUSIONS: From a comparative hematology perspective, the buffy coat of mammals differs from that of birds and more from that of reptiles. The CTB technique can be used to study these differences in avian and reptilian hematology, especially to study atypical circulating cells, hemoparasites, or blood cell proportions in health and disease.

Seasonal influence on hematologic values and hemoglobin electrophoresis in Brazilian boa constrictor amarali.

As ectothermic animals, snakes depend exclusively on the environment for proper temperature maintenance, which may greatly influence their activity. Twenty-five adult Boa constrictor amarali snakes maintained in captivity were used to determine the influence of seasons on their hematologic values and electrophoretic profile of hemoglobin. A complete blood cell count (CBC) and examination for hemoparasites were performed in the summer and winter of 2004. Hemoglobin was stored for later electrophoresis. Significant differences (P < 0.05) were obtained in RBC, WBC, lymphocyte, thrombocyte, and monocyte counts, demonstrating the importance of the period of the year in the interpretation of reference values in these animals. Two snakes were detected with blood parasites (Hepatozoon sp.) in the winter and four in the summer, although it appears that their presence did not cause any significant alterations in the CBC. The electrophoretic analysis of the samples demonstrated two-four hemoglobin bands in this species.

Preliminary single-dose pharmacokinetics of marbofloxacin in ball pythons (Python regius).

Pharmacokinetics of marbofloxacin in two male and four female adult ball pythons (Python regius) was determined after i.v. and p.o. administration of a single dose. Using a crossover design, each snake was given a single 10 mg/kg dose of marbofloxacin i.v. and p.o. Blood samples were collected prior to and 0.5, 1, 1.5, 3, 6, 12, and 24 hr after marbofloxacin administration. Marbofloxacin was quantitated by use of liquid chromatography-mass spectrometry. Following p.o. administration, marbofloxacin had a peak plasma concentration (Cmax) of 9.40 microg/ml and a time to Cmax (Tmax) of 9.0 hr. Based on the plasma pharmacokinetics generated in this study and pending any further studies to evaluate potential toxicity and multi-dose pharmacokinetics, we suggest a dosage for marbofloxacin in ball pythons of 10 mg/kg p.o. at least every 48 hr, depending on the sensitivity of the pathogen and as a basis for further research.

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.

Temporal effects of 3 commonly used anticoagulants on hematologic and biochemical variables in blood samples from macaws and Burmese pythons.

BACKGROUND: Few studies have been done to evaluate anticoagulants for use with blood samples from birds and reptiles. Heparin currently is the most commonly used anticoagulant in practice, but may adversely affect blood cell staining and quantitation. OBJECTIVE: The purpose of this study was to evaluate the effects of lithium heparin, K3-EDTA, and sodium citrate, with and without the addition of albumin, on hematologic variables in macaw (Ara sp) and python (Python molurus bivittatus) blood samples. METHODS: Blood samples from 10 macaws and 10 Burmese pythons were collected in heparin-coated syringes and placed into tubes containing either lithium heparin, K3-EDTA, or sodium citrate with and without the addition of 0.25 mL of a 22% bovine serum albumin solution. Cell lysis was determined by counting the number of lysed cells/200 WBCs in Wright's-Giemsa-stained blood smears and by qualitative evaluation of pink plasma in microhematocrit tubes. A CBC was done after 3, 12, and 24 hours of storage at 4 degrees C in anticoagulant-containing tubes and results were compared with those obtained at 0 hour for the heparin-coated syringe sample. A biochemical panel also was done at each time point in similarly stored lithium-heparin samples. RESULTS: Hemolysis was significantly increased in citrated samples from both macaws and pythons beginning at 12 hours. At 24 hours, 19 of 30 (63%) macaw samples in all anticoagulants had >100 lysed cells/200 WBCs. There were no significant differences in hematologic values in samples from pythons collected in heparin or EDTA at any time point. No significant differences were found in the number of lysed cells or in other hematologic data in samples with albumin. Glucose concentration decreased and potassium concentration increased significantly over time in heparinized blood samples. CONCLUSIONS: Based on the results of this study, whole blood samples anticoagulated with lithium heparin or EDTA should be evaluated within 12 hours (macaws) or 24 hours (pythons) of collection and stored at 4 degrees C for best results. Citrate should be avoided as it may result in increased cell lysis. The addition of albumin does not prevent cell lysis.

Characterization of carbonic anhydrase XIII in the erythrocytes of the Burmese python, Python molurus bivittatus.

Carbonic anhydrase (CA) is one of the most abundant proteins found in vertebrate erythrocytes with the majority of species expressing a low activity CA I and high activity CA II. However, several phylogenetic gaps remain in our understanding of the expansion of cytoplasmic CA in vertebrate erythrocytes. In particular, very little is known about isoforms from reptiles. The current study sought to characterize the erythrocyte isoforms from two squamate species, Python molurus and Nerodia rhombifer, which was combined with information from recent genome projects to address this important phylogenetic gap. Obtained sequences grouped closely with CA XIII in phylogenetic analyses. CA II mRNA transcripts were also found in erythrocytes, but found at less than half the levels of CA XIII. Structural analysis suggested similar biochemical activity as the respective mammalian isoforms, with CA XIII being a low activity isoform. Biochemical characterization verified that the majority of CA activity in the erythrocytes was due to a high activity CA II-like isoform; however, titration with copper supported the presence of two CA pools. The CA II-like pool accounted for 90 % of the total activity. To assess potential disparate roles of these isoforms a feeding stress was used to up-regulate CO2 excretion pathways. Significant up-regulation of CA II and the anion exchanger was observed; CA XIII was strongly down-regulated. While these results do not provide insight into the role of CA XIII in the erythrocytes, they do suggest that the presence of two isoforms is not simply a case of physiological redundancy.

Laboratory reference values for a group of captive Ball Pythons (Python regius)

OBJECTIVE: Laboratory reference values, including hematologic and serum biochemical variables, and oropharyngeal bacteria flora, were determined in a group of captive Ball Pythons (Python regius). ANIMALS: 20 adult Ball Pythons, weighing between 700 and 1,510 g, were allowed to acclimate at the recommended temperature range for the species (25 C night-time, up to 30 C daytime), then were evaluated for internal parasites and treated with appropriate medication prior to the start of the study. PROCEDURE: Hematologic values determined included WBC, hemoglobin, hematocrit, plasma protein, and differential cell count. Clinical biochemical analysis included determination of glucose, uric acid, calcium, phosphorus, total protein, alanine transaminase, alkaline phosphatase, and aspartate transaminase values. In addition to blood values, oropharyngeal swab specimens of the mouth were submitted for culture to determine the species of bacteria found in this population. Descriptive statistics were calculated for each hematologic and clinical biochemical value. Mean, SEM, and ranges were calculated. RESULTS: Hematologic values were similar to those reported in other snake species, except the hematocrit, which was lower. Clinical biochemical values different from those of other species were alkaline phosphatase activity, which was lower, and calcium and phosphorus concentrations, which were lower than values in other species. Bacteria isolated from the oropharynx were principally gram-negative organisms. CONCLUSION: Reference intervals reported in this study are important for establishing a database for comparative studies of Ball Pythons in other locations and under different husbandry conditions. CLINICAL RELEVANCE: Accumulated laboratory reference values will assist veterinarians in assessing the health status of Ball Pythons.

Comparative cell morphology in the peripheral blood film from exotic and native animals.

Haematological investigation is an important part of disease diagnosis. This is particularly so when investigating individual animal disease. It may also be important when investigating diseases in groups of animals, but the opportunity to perform necropsies to directly detect diseases processes often diminishes its usefulness. Haematological investigation is essentially similar for all species. The presence of nucleated erythrocytes and thrombocytes in nonmammals requires alteration of haemoglobin measurement and cell counting. In addition, it may cause some confusion in identification of cells in peripheral blood films. Examination of blood films is an important component of haematological investigation and provides useful information on erythroid, leukocytic and platelet/thrombocytic alterations. Interpretation of alterations is essentially similar for all species. However, cell identification can at times be difficult. There are five basic leukocytes in all species: neutrophil (mammals) or heterophil (nonmammals), eosinophil, basophil, lymphocyte and monocyte. In nonmammals it may be difficult at times to distinguish between heterophils and eosinophils. In addition, lymphocytes may be confused with thrombocytes. However, a common-sense approach to the examination of the peripheral blood film will minimise this confusion.