Splenic hemangioendothelial neoplasm in a captive Northern Snakehead from the Potomac River.

OBJECTIVE: At the U.S. Geological Survey Leetown Research Laboratory in Kearneysville, West Virginia, an approximately 3-year-old, captive-held Northern Snakehead Channa argus with clinical signs of abdominal distention died and was necropsied 1 day after an examination under anesthesia. A mass discovered in the midcoelomic cavity, presumed to be deformed spleen, was comprised of large, pseudocystic structures that contained considerable volumes of opaque, straw-colored fluid. METHODS: A histopathological evaluation revealed that the tissue consisted of foci of small capillaries, nodular areas of proliferating, pleomorphic endothelial cells, and areas of necrosis within the pseudocyst wall. Positive nuclear and nonspecific immunolabeling with a vascular marker, cluster of differentiation 31, was concentrated in and around vascular spaces. RESULT: Based on these observations, the tumor has been putatively identified as a hemangioendothelial neoplasm. CONCLUSION: This would represent the first report of a vascular tumor in a Northern Snakehead and, globally, one of the few described neoplasms identified in a member of the Channidae family.

Influenza A Virus Detected in Native Bivalves in Waterfowl Habitat of the Delmarva Peninsula, USA.

We evaluated the prevalence of influenza A virus (IAV) in different species of bivalves inhabiting natural water bodies in waterfowl habitat along the Delmarva Peninsula and Chesapeake Bay in eastern Maryland. Bivalve tissue from clam and mussel specimens (Macoma balthica, Macoma phenax, Mulinia sp., Rangia cuneata, Mya arenaria, Guekensia demissa, and an undetermined mussel species) from five collection sites was analyzed for the presence of type A influenza virus by qPCR targeting the matrix gene. Of the 300 tissue samples analyzed, 13 samples (4.3%) tested positive for presence of influenza virus A matrix gene. To our knowledge, this is the first report of detection of IAV in the tissue of any bivalve mollusk from a natural water body.

Low Pathogenic Avian Influenza Viruses in Wild Migratory Waterfowl in a Region of High Poultry Production, Delmarva, Maryland.

Migratory waterfowl are natural reservoirs for low pathogenic avian influenza viruses (AIVs) and may contribute to the long-distance dispersal of these pathogens as well as spillover into domestic bird populations. Surveillance for AIVs is critical to assessing risks for potential spread of these viruses among wild and domestic bird populations. The Delmarva Peninsula on the east coast of the United States is both a key convergence point for migratory Atlantic waterfowl populations and a region with high poultry production (>4,700 poultry meat facilities). Sampling of key migratory waterfowl species occurred at 20 locations throughout the Delmarva Peninsula in fall and winter of 2013-14. Samples were collected from 400 hunter-harvested or live-caught birds via cloacal and oropharyngeal swabs. Fourteen of the 400 (3.5%) birds sampled tested positive for the AIV matrix gene using real-time reverse transcriptase PCR, all from five dabbling duck species. Further characterization of the 14 viral isolates identified two hemagglutinin (H3 and H4) and four neuraminidase (N2, N6, N8, and N9) subtypes, which were consistent with isolates reported in the Influenza Research Database for this region. Three of 14 isolates contained multiple HA or NA subtypes. This study adds to the limited baseline information available for AIVs in migratory waterfowl populations on the Delmarva Peninsula, particularly prior to the highly pathogenic AIV A(H5N8) and A(H5N2) introductions to the United States in late 2014.

Transforming growth factor-ß1 expression in endangered age-0 shortnose suckers (Chasmistes brevirostris) from Upper Klamath Lake, OR relative to histopathology, meristic, spatial, and temporal data.

During July-September of 2008, 2009, and 2010 endangered age-0 juvenile shortnose suckers were sampled from Upper Klamath Lake, OR in a health evaluation that included the measurement of transforming growth factor - beta (TGF-ß) expression in spleen in combination with a histopathology assessment. This analysis was performed to determine if the expression of this immuno-regulator could be used as a component of a larger health evaluation intended to identify potential risk-factors that may help to explain why very few of these fish survive to age-1. Potential associations between TGF-ß1 expression, histopathological findings, meristic data as well as temporal and spatial data were evaluated using analysis-of-variance. In this analysis, the absence or presence of opercula deformity and hepatic cell necrosis were identified as significant factors in accounting for the variance in TGF-ß1 expression observed in age-0 shortnose suckers (n = 122, squared multiple R = 0.989). Location of sample collection and the absence or presence of anchor worms (Lernaea spp.) were identified as significant cofactors. The actual mechanisms involved with these relationships have yet to be determined. The strength, however, of our findings support the concept of using TGF-ß1 expression as part of a broader fish health assessment and suggests the potential for using additional immunologic measures in future studies. Specifically, our results indicate that the measure of TGF-ß1 expression in age-0 shortnose sucker health assessments can facilitate the process of identifying disease risks that are associated with the documented lack of recruitment into the adult population.

In vitro immune functions in thiamine-replete and -depleted lake trout (Salvelinus namaycush).

In this study we examined the impacts of in vivo thiamine deficiency on lake trout leukocyte function measured in vitro. When compared outside the context of individual-specific thiamine concentrations no significant differences were observed in leukocyte bactericidal activity or in concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) stimulated leukocyte proliferation. Placing immune functions into context with the ratio of in vivo liver thiamine monophosphate (TMP--biologically inactive form) to thiamine pyrophosphate (TPP--biologically active form) proved to be the best indicator of thiamine depletion impacts as determined using regression modeling. These observed relationships indicated differential effects on the immune measures with bactericidal activity exhibiting an inverse relationship with TMP to TPP ratios, Con A stimulated mitogenesis exhibiting a positive relationship with TMP to TPP ratios and PHA-P stimulated mitogenesis exhibiting no significant relationships. In addition, these relationships showed considerable complexity which included the consistent observation of a thiamine-replete subgroup with characteristics similar to those seen in the leukocytes from thiamine-depleted fish. When considered together, our observations indicate that lake trout leukocytes experience cell-type specific impacts as well as an altered physiologic environment when confronted with a thiamine-limited state.

Impact of thiamine deficiency on T-cell dependent and T-cell independent antibody production in lake trout.

Lake trout Salvelinus namaycush on thiamine-replete and thiamine-depleted diets were evaluated for the effects of thiamine status on in vivo responses to the T-dependent antigen trinitophenol (TNP)-keyhole limpet hemocyanin (TNP-KLH), the T-independent antigen trinitrophenol-lipolysaccaharide (TNP-LPS), or Dulbecco's phosphate-buffered saline (DPBS; negative control fish). Plasma antibody concentrations were evaluated for possible differences in total anti-TNP activity as well as differences in response kinetics. Associations between anti-TNP activity and muscle and liver thiamine concentrations as well as ratios of muscle-to-liver thiamine to anti-TNP activity were also examined. Thiamine-depleted lake trout that were injected with TNP-LPS exhibited significantly more anti-TNP activity than thiamine-replete fish. The depleted fish injected with TNP-LPS also exhibited significantly different response kinetics relative to thiamine-replete lake trout. No differences in activity or kinetics were observed between the thiamine-replete and -depleted fish injected with TNP-KLH or in the DPBS negative controls. Anti-TNP activity in thiamine-depleted lake trout injected with TNP-KLH was positively associated with muscle thiamine pyrophosphate (thiamine diphosphate; TPP) concentration. A negative association was observed between the ratio of muscle-to-liver TPP and T-independent responses. No significant associations between anti-TNP activity and tissue thiamine concentration were observed in the thiamine-replete fish. We demonstrated that thiamine deficiency leads to alterations in both T-dependent and T-independent immune responses in lake trout.

Cytochemical and immunocytochemical characterization of blood cells and immunohistochemical analysis of spleen cells from 2 species of frog, Rana (Aquarana) catesbeiana and Xenopus laevis.

BACKGROUND: Mechanisms of amphibian diseases are not characterized as well as those in domestic mammalian species. Antemortem laboratory testing is limited in frogs, presenting a diagnostic challenge to zoos, laboratories, and exotic veterinarians. OBJECTIVE: This study aimed to characterize blood cells and splenic cells from 2 anuran species based on characteristics identified by Wright staining, cytochemical staining, and immunochemical analysis and on histologic examination of spleens. METHODS: Blood specimens and spleens were obtained from 2 species of frog, the American bullfrog (Rana [Aquarana] catesbeiana) and the African clawed frog (Xenopus laevis). Blood smears were evaluated after Wright staining and cytochemical staining for α-naphthyl butyrate esterase (NBE), chloroacetate esterase (CAE), myeloperoxidase (PER), Sudan black B (SBB), and leukocyte alkaline phosphatase (LAP) reactions and for immunoreactivity for antibodies against CD3ε, CD79a, and BLA.36 antigens. Histologic sections of spleen were evaluated after staining with H&E and for immunoreactivity for CD3ε, CD79a, and BLA.36 antigens. RESULTS: In bullfrogs, neutrophils, eosinophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; lymphocytes occasionally were positive for CAE. In clawed frogs, neutrophils, basophils, and monocytes were positive for some or all of the following: NBE, CAE, PER, and SBB; eosinophils occasionally were positive for CAE and PER, and lymphocytes were negative for all cytochemical stains. LAP was not a useful marker for any leukocyte type. In both species, peripheral blood lymphocytes were strongly immunoreactive for CD3ε, CD79a, and BLA.36. In splenic tissue, histologic patterns varied and there was diffuse immunoreactivity for CD79a and BLA.36 with focal reactivity for CD3ε, but with different distribution patterns in each species. CONCLUSION: Cytochemical and immunochemical analysis of cells may be helpful in identification and characterization of amphibian blood cells and splenic cells for evaluation of the health of these animals.

Diseases of amphibians.

The development and refinement of amphibian medicine comprise an ongoing science that reflects the unique life history of these animals and our growing knowledge of amphibian diseases. Amphibians are notoriously fastidious in terms of captive care requirements, and the majority of diseases of amphibians maintained in captivity will relate directly or indirectly to husbandry and management. Investigators have described many infectious and noninfectious diseases that occur among various species of captive and wild amphibians, and there is considerable overlap in the diseases of captive versus free-ranging populations. In this article, some of the more commonly reported infectious and noninfectious diseases as well as their etiological agents and causative factors are reviewed. Some of the more common amphibian diseases with bacterial etiologies include bacterial dermatosepticemia or "red leg syndrome," flavobacteriosis, mycobacteriosis, and chlamydiosis. The most common viral diseases of amphibians are caused by the ranaviruses, which have an impact on many species of anurans and caudates. Mycotic and mycotic-like organisms cause a number of diseases among amphibians, including chytridiomycosis, zygomycoses, chromomycoses, saprolegniasis, and ichthyophoniasis. Protozoan parasites of amphibians include a variety of amoeba, ciliates, flagellates, and sporozoans Common metazoan parasites include various myxozoans, helminths (particularly trematodes and nematodes), and arthropods. Commonly encountered noninfectious disease etiologies for amphibians include neoplasia, absolute or specific nutritional deficiencies or overloads, chemical toxicities, and inadequate husbandry or environmental management.

Calcein AM release-based cytotoxic cell assay for fish leucocytes.

A non-specific cytotoxic cell assay for fish is presented that is based on the release of the activated fluorochrome calcein AM from lysed carp epithelioma papulosum cyprini (EPC) cells. To establish the suitability of treating EPC cells with calcein AM the uptake and spontaneous release of the calcein AM by the EPC cells was evaluated. Incubation of 5 microM calcein AM in culture medium with 1x10(5)EPC cells well(-1)for a minimum of 3 h provided sufficient labelling. Spontaneous release of fluorescence from the labelled EPC cells during 10 h of post labelling incubation ranged from 30 to 39% of the total observed fluorescence. Cytotoxic activity of trout leucocytes was evaluated at three leucocyte to target cell ratios (10:1, 2:1 and 1:1) following incubation (4, 6, 8, and 10 h) with calcein AM-labelled EPC cells at 15 degrees C. In some instances, the monoclonal antibody specific for the NCC surface receptor NCCRP-1 (MAb5C.6) was included in the cultures. The activity of NCC cells was significantly inhibited in the presence of 0.25 microg well(-1)of MAb5C.6 relative to no antibody (P

Measurement of in vitro leucocyte mitogenesis in fish: ELISA based detection of the thymidine analogue 5'-bromo-2'-deoxyuridine.

In this study we present a method for the measurement of in vitro mitogenesis in fish leucocytes that is based on the incorporation of the thymidine analogue 5'-bromo-2'-deoxyuridine (BrdU) into the DNA of replicating cells, followed by ELISA-based detection. This technique, adapted from methods developed for mammalian cells, operates on a similar biological principle to (3)H-thymidine incorporation, but circumvents the logistical and safety issues inherent with the radioactive label. Because it directly measures DNA proliferation, the assay has advantages over other colorimetric methods that may be strongly influenced by leucocyte metabolic status. Using BrdU incorporation followed by ELISA, we evaluate the responsiveness of rainbow trout (Oncorhynchus mykiss [Walbaum]) leucocytes to the mammalian T-cell mitogen Concanavalin A (Con A) as well as the differential response of white perch (Morone americana [Gmelin]) leucocytes to Con A and pokeweed mitogen. Specific considerations intrinsic to the assay system are discussed, including the implications of utilising enzyme-based detection.

Effects of water temperature and substrate type on spore production and release in eastern Tubifex tubifex worms infected with Myxobolus cerebralis.

Eastern Tubifex tubifex worms were exposed to Myxobolus cerebralis spores at 9, 13, 17, and 20 C in 1-L jars that contained sand, mud, or leaf litter as substrata. Beginning 60 days after exposure, water from each jar was filtered daily and examined for the presence of waterborne triactinomyxon spores (TAMs). On discovering a single TAM from an experimental jar, 48 T. tubifex worms from that jar were placed individually into 24-well plates. Spores released from individual infected T. tubifex worms were quantified to determine the first day of TAM release from infected worms, the infection rate, the total number of TAMs released per worm, and the duration of release. No TAMs were found in any of the jars incubated at 20 C or in uninfected, control worms at any temperature. The total number of TAMs released by infected worms in mud and sand was highest at 13 C compared with other temperatures. Infection rates among individual worms increased with temperature between 9 and 17 C. Higher temperatures (up to 17 C) induced earlier TAM releases among infected worms, and substratum did not influence this production parameter. The average duration of TAM release decreased as the temperature increased from 9 to 17 C, and there was a significant effect of substratum in the groups maintained at 13 and 17 C. In all temperature treatments between 9 and 17 C, the duration of release was least in the worms maintained in leaf litter, as was the total number of TAMs released during the experimental period and the median number of TAMs per production day.