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Microbial flora can provide insight into the ecology and natural history of wildlife in addition to improving understanding of health risks. This study examines the anaerobic oral flora of hunter killed black bears (Ursus americanus) in eastern North Carolina. Oral swabs from the buccal and lingual supragingival tooth surfaces of the first and second mandibular and maxillary molars of 22 black bears were inoculated onto Brucella Blood Agar plates supplemented with hemin and vitamin K after transport from the field using reduced oxoid nutrient broth. Sixteen anaerobic bacterial species, representing nine genera were identified using the RapID ANA II Micromethod Kit system and a number of organisms grown that could not be identified with the system. The most frequently identified anaerobes were Peptostreptococcus prevotii, Streptococcus constellatus, and Porphyromonas gingivalis. The diversity in the anaerobic oral flora of black bear in eastern North Carolina suggests the importance of including these organisms in basic health risk assessment protocols and suggests a potential tool for assessment of bear/habitat interactions.

There is increased concern about the sublethal effects of organophosphorous (OP) compounds on human and animal health, including the potential role of OP compounds in the global decline of amphibian populations. Malathion is one of the most widely used OP pesticides with numerous agricultural and therapeutic applications, and exposure to environmentally applied malathion can lead to adverse systemic effects in anurans. Cutaneous absorption is considered a potentially important route of environmental exposure to OP compounds for amphibians, especially in aquatic environments. One in vitro system commonly used to determine the absorption kinetics of xenobiotics across the skin is the two-compartment Teflon flow-through diffusion cell system. To establish cutaneous absorption kinetics of malathion, six full thickness skin samples taken from both the dorsal and ventral surfaces of each of three bullfrogs (Rana catesbeiana) and three marine toads (Bufo marinus) were placed into two-compartment Teflon flow-through diffusion cells perfused with modified amphibian Ringer's solution. A 26μg/cm(2) dose of malathion-2,3-(14)C diluted in 100% ethanol was applied to each sample (0.44-0.45μCi). Perfusate was collected at intervals over a 6h period and analyzed for (14)C in a scintillation counter. At the end of 6h, surface swabs, tape strips, biopsy punches of the dosed area of skin, and peripheral samples were oxidized and analyzed for residue effects. Malathion absorption was greater across the ventral skin compared to dorsal skin in both bullfrogs and marine toads.

The objective of this study was to establish an accurate in vitro model for cutaneous absorption in anurans. The harvested perfused anuran pelvic limb (HPAPL) model maintains the anatomic and physiologic integrity of the skin from the pelvic limb, including the intact capillary network. Radiolabeled malathion was applied to the skin of the dorsal thigh, and perfusate was collected over a 6 h period. Residues from the skin surface, stratum externum, and dosed area beneath the stratum externum were analyzed. Kinetic parameters were calculated from these data. Absorption was significantly less for the HPAPL than previously reported for Teflon flow-through diffusion cells. However, partitioning effects were comparable. The HPAPL is an appropriate in vitro model for examining cutaneous absorption kinetics in the bullfrog.

Detection of enteric viral nucleic acids in preserved gorilla fecal specimens was investigated using reverse transcription polymerase chain reaction (rt-PCR). A commercially available viral RNA extraction kit was used to isolate nucleic acids from captive gorilla fecal samples seeded with rotavirus and stored in ethanol, formalin, a commercial RNA preservation solution, guanidine thiocyanate buffer (GT), and samples dried in tubes containing silica gel. Nucleic acids were extracted at 1, 7, 70 and 180 days and used for rt-PCR amplification of specific rotavirus RNA sequences. Successful rt-PCR amplification of the target product varied according to storage conditions, and storage time. Only samples stored in GT gave 100% positive results at 180 days. It is recommended that fecal samples be collected in GT for viral RNA analysis.

These studies showed sulfadimethoxine and ormetoprim to be stable at salinities of 0 and 30 ppt and at pHs of 2, 7, and 12 for a period of 1 year. Sulfadimethoxine was stable at 25°C and 37°C, but showed a marked decrease in concentration at 4°C. Warming of the 4°C sample resulted in a return to original drug levels indicating that the drug had redistributed out of the aquatic phase at the lower temperature. Ormetoprim concentrations were stable at all temperatures evaluated. The concentrations of both sulfadimethoxine and ormetoprim were unaffected by the presence of silica sand, high density poly-ethylene, or poly-vinyl chloride. The presence of bentonite clay caused a reduction in ormetoprim concentrations while sulfadimethoxine was unchanged by this substrate. Acidification of the sample containing the bentonite clay resulted in a return of ormetoprim concentration to original levels. From these studies, it is apparent that the potential environmental half-lives for these drugs must exceed 1 year and are likely to be several years in duration.

Imprinted gene identification in animals has been limited to eutherian mammals, suggesting a significant role for intrauterine fetal development in the evolution of imprinting. We report herein that M6P/IGF2R is not imprinted in monotremes and does not encode for a receptor that binds IGF2. In contrast, M6P/IGF2R is imprinted in a didelphid marsupial, the opossum, but it strikingly lacks the differentially methylated CpG island in intron 2 postulated to be involved in imprint control. Thus, invasive placentation and gestational fetal growth are not required for imprinted genes to evolve. Unless there was convergent evolution of M6P/ IGF2R imprinting and receptor IGF2 binding in marsupials and eutherians, our results also demonstrate that these two functions evolved in a mammalian clade exclusive of monotremes.

This study investigated the safety and efficacy of the EPG Masterflow Aquarium Management System with Aegis Microbe Shield™ (EPG-MAMS). Four different species of fish were used in the study. Ten fish of each species were placed in 75 l aquariums containing the EPG filter media, a commercially available filter media (Whisper®) and an aquarium with no filter material. At the end of the 45 day trial three fish from each tank were sacrificed and preserved in formalin for histopathology. Water quality parameters were routinely monitored. The EPG filter media was compared with the Whisper® filter media for efficacy against Aeromonas salmonicida using a shaker flask microbiological assay. The EPG filter proved to be clinically and histopathologically safe and reduced to some degree the number of A. salmonicida suspended in water in an in vitro study.

Metabolites, as the end products of metabolism, represent the functional responses of a cell. Their characterization can provide insight into the underlying mechanisms of genomic or environmental actions on metabolism. The aquatic environment is varied and dynamic, providing a vast diversity of physical and chemical challenges to metabolism, making the study of the metabolites of mollusks particularly fruitful for scientists interested in comparative physiology, pharmacology and toxicology. Organ specific metabolic fingerprints can establish time dependent assessments for interpreting functional adaptations to environmental and nutritional challenges using either invasive tissue extraction from multiple individuals or non-invasive longitudinal observation of the same individual. NMR spectroscopy and magnetic resonance imaging (MRI) permit non-invasive monitoring of the metabolome.The mollusks are a useful, robust model organism for tissue metabolism studies. Its relatively few organs are easily delineated and there is sufficient understanding of their functions based on classical assays to support interpretation of advanced spectroscopic approaches. Here we apply high-resolution proton and carbon nuclear magnetic resonance (1H and 13C NMR)-based metabolomic analysis to Eastern oyster C. virginica and freshwater mussel Eastern Elliptio E. Complanata to investigate the variation in the metabolic profile of different organ groups. MRI was used to non-invasively identify the morphology of the organs. In vivo MR spectra can be obtained from single region of interest (ROI or voxel) or multiple ROI simultaneously using the technique typically called chemical shift imaging (CSI). Here we also report applications of CSI to marine samples and describe the use of the technique to study in vivo glycine metabolism in oysters using 13C MRS.A biochemical schematic is presented that relates metabolites to biochemical pathways correlated with physiological organ functions.

A simple and sensitive liquid chromatographic method has been developed for the determination of therapeutic levels of ceftazidime in dolphin serum. The method involved an ultrafiltration of diluted serum with an equal amount of acetonitrile-ethanol-water (40:40:20, v/v/v) through a 10,000 daltons molecular mass cut-off filter. Separation of ceftazidime from the other serum components was performed by ion-paired (dodecanesulfonate) liquid chromatography using a reversed-phase column eluted with acetonitrile-water solution. The ultraviolet absorbance of the column effluent was monitored in the 200-340 nm range of a photodiode-array detector or at 258.8 nm on a variable-wavelength ultraviolet-visible detector. Recoveries of ceftazidime from dolphin serum spiked with 20 and 2 micrograms/ml were 92.9 and 91.1% with coefficients of variation of 5.5 and 5.7%, respectively. A correlation coefficient of 0.9994 occurred with ceftazidime in aqueous solutions (n = 6, in duplicates). The limit of detection for this antibiotic was estimated to be approximately 50 ppb (ng/ml). The unbound ceftazidime concentrations in dosed dolphin serum were determined to calculate the protein bindings of this antibiotic which yielded 32 +/- 2%. The ceftazidime peak identity in dosed dolphin serum was confirmed by thermospray liquid chromatography-mass spectrometry. The thermospray mass spectrum of ceftazidime exhibited only the fragment ions, involving the opening of the beta-lactam ring, at m/z 237, 255 and 315 when positive-ion detection mode was employed and the fragment ions at m/z 235, 253 and 313 when negative-ion detection mode was used.

NMR imaging and localized 1H spectroscopy of a variety of aquatic organisms in vivo is described for the first time. The practical consideration of life support, water volume, salinity, and anesthesia are discussed and solutions presented. Such animal studies shape our understanding of physiology, biochemistry, and biology, and provide models of human disease and normal function. These studies also have economic and ecological importance.

The effects of dietary administration of equimolar doses (5 mmol/kg body wt per day) of trimethylene oxide, trimethylene sulfide, coumaran, benzofuran, indole, and indole-3-carbinol on the activities of microsomal epoxide hydrolase and several other xenobiotic metabolizing enzymes were measured in the liver of female CD-1 mouse. Every compound, with the exception of indole, caused a significant increase (P less than 0.01) of the styrene oxide epoxide hydrolase activity over controls in hepatic microsomes. These results indicate that the enzyme activity is elevated in vivo by several heterocyclic compounds with strained bond angles to a nucleophilic hetero-atom. In addition, the ability of sulfur-containing trimethylene sulfide and nitrogen-containing indole-3-carbinol to elevate the enzyme activity indicates that the heterocyclic oxygen atom is not an absolute requirement for this effect. Data from the other xenobiotic metabolizing enzymes indicate that trimethylene oxide and trimethylene sulfide enhance the epoxide hydrolase activity rather specifically, while not affecting the activities of the other enzymes measured. While the oxygen-containing coumaran and benzofuran both increased the NADH: quinone reductase activity in hepatic cytosol, the nitrogen-containing indole and indole-3-carbinol did not. This indicated a specific requirement for the oxygen atom in elevating the quinone reductase activity, which was not the case for the elevation of microsomal epoxide hydrolase activity.

A novel class of glycolipids, assigned the trivial name blaberosides, was isolated from whole head tissues of the giant cockroach (Blaberus colosseus). The class consists of two closely related families, blaberoside I and blaberoside II, each containing species differing by 26 atomic mass units. The structure of these gentiobiose-based glycoglycerolipids was elucidated by chromatographic behavior, nuclear magnetic resonance spectroscopy, mass spectrometry, and analysis of chemical degradation products and derivatives. Species in the blaberoside I family have been identified as 2-O-[6'-O-(6"-O-3-hydroxy-11-eicosenoyl-beta-D-glucopyranosyl)-bet a-D- glucopyranosyl]-3-(hexadecyloxy)-1-(3-hydroxy-11-eicosenoyl)-1,2-p ropanediol (blaberoside Ia) and 2-O-[6'-O-(6"-O-3-hydroxy-11-eicosenoyl-beta-D-glucopyranosyl)-bet a- D-glucopyranosyl]-3-(6-octadeceloxy)-1-(3-hydroxy-11-eicosenoyl )-1,2- propanediol (blaberoside Ib). Two smaller homologs of the blaberoside II family were discerned to be 2-O-[6'-O-(6"-O-3-hydroxy-11- eicosenoyl-beta-D-glucopyranosyl)-beta-D-glucopyranosyl]-3-(hex ade cyloxy)- 1,2-propanediol (blaberoside IIa), and 2-O-[6'-O-(6"-O-3-hydroxy-11-eicosenoyl-beta-D- glucopyranosyl)-beta-D-glucopyranosyl]-3-(4-octadeceloxy)-1,2-prop anediol (blaberoside IIb). These compounds are unique because they are animal origin glyceroglycolipids with a highly flexible gentiobiose backbone, and a beta-linkage of the carbohydrate to the glycerol ether at the 2 position rather than the usual 1 position.

We have studied the lipid composition of brain (optic and cerebral lobes), stellate ganglia and fin nerves of the squid. Cholesterol, phosphatidylethanolamine and phosphatidylcholine were the major lipids in these nervous tissues. Phosphatidylethanolamine contained about 3% of its amount in [corrected] plasmalogen form. Phosphatidylserine and -inositol, sphingomyelin and ceramide 2-aminoethylphosphonate were also present in significant amounts. In addition, cardiolipin and free fatty acids were detected in brain (each 2-3% of total lipids) and stellate ganglia (about 1% each), but not in fin nerves. Phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol from brain contained large amounts of polyunsaturated fatty acids, namely 20:4, 20:5 and 22:6 in the n-3 family. On the other hand, phosphatidylcholine, cardiolipin, and sphingomyelin, and ceramide 2-aminoethylphosphonate contained only saturated or monounsaturated C16-C18 fatty acids. The aldehyde moieties of ethanolamine plasmalogen were also C16-C18 saturated or monounsaturated. These lipid compositions are compared with those in other invertebrate nervous systems.