A follow-up study of selected biomarkers of health in cod Gadus morhua L. collected from the southern Baltic off the Polish coast.

Selected biomarkers of health were examined in 50 post-spawning cod Gadus morhua collected in November 2015 from the southern Baltic. The biomarkers included condition factor (CF), macroscopic lesions, histopathology of spleen, liver and gonads, and morphometry of follicular atresia and hepatic and splenic melanomacrophage cells (MMC). All fish appeared in good body condition. One fish had a dermal ulcer, and in seven, macroscopic nematodes were noted within body cavity. Microscopic lesions in the liver included biliary myxozoanosis, microsporidial and necrocentric granulomas, parasitic hepatitis, multifocal necrosis, foci of cellular alterations, spongiosis, peliosis and cytoplasmic fibrillar inclusions. The spleen and gonads had microsporidial and/or necrocentric granulomas. Some of the biomarkers showed differences as compared to spawning cod collected in May from the same location in 2012, most importantly values an order of magnitude lower for splenic MMC in post-spawning fish. In post-spawning fish, there were statistically significant correlations between MMC, CF, follicular atresia, parasitic hepatitis and microsporidiosis. This is the first comparison of biomarkers of health in post-spawning and spawning Baltic cod. Future studies need to examine the relationships of biomarkers to levels of pollutants in the environment and in tissues of cod.

Mortality due to a retained circle hook in a longfin mako shark Isurus paucus (Guitart-Manday).

A female longfin mako shark Isurus paucus (Guitart-Manday, 1966) was found moribund on the Atlantic Ocean beach near Canaveral National Seashore, Florida; the shark died shortly after stranding. Macroscopic lesions included a partially healed bite mark on the left pectoral fin, a clefted snout, pericardial effusion and a pericardial mass surrounding a 12/0 circle fishing hook. The heart, pericardial mass, gills, ovary, oviduct, shell gland, epigonal organ, liver, kidney and intrarenal and interrenal glands were processed for histopathology and examined by brightfield microscopy. Microscopic examination revealed chronic proliferative and pyogranulomatous pericarditis and myocarditis with rhabdomyolysis, fibrosis and thrombosis; scant bacteria and multifocal granular deposits of iron were found intralesionally. In addition, acute, multifocal infarcts within the epigonal organ and gill filaments were found in association with emboli formed by necrocellular material. The ovary had high numbers of atretic follicles, and the liver had diffuse, severe hepatocellular degeneration, multifocal spongiosis and moderate numbers of melanomacrophage cells. This report provides evidence of direct mortality due to systemic lesions associated with retained fishing gear in a prohibited shark species. Due to the large numbers of sharks released from both recreational and commercial fisheries worldwide, impact of delayed post-release mortality on shark populations is an important consideration.

The parasite-host interface in the zonetail butterfly ray, Gymnura zonura (Bleeker), infected with Hexacanalis folifer (Cestoda: Lecanicephalidea).

A wild-caught specimen of the zonetail butterfly ray, Gymnura zonura (Bleeker), harboured numerous specimens of Hexacanalis folifer Cielocha & Jensen, 2011 (Systematic Parasitology, 79, 1-16; Cestoda: Lecanicephalidea) within its spiral intestine. The cestodes were primarily attached in single rows along the base of mucosal folds, each associated with a nodular mucosal thickening. Microscopically, the scolex was embedded within the submucosa and muscularis; the attachment sites were marked by ulceration and necro-proliferative inflammation demarcating the parasite from normal host tissues. Physical attachment of the cestode was restricted to the anterior portion of its scolex where presumed tegumental secretions from the apical organ contributed to a cementing intermediate layer blending with necrotic host cells. The presence of tegumental differentiation between the apical organ and the scolex proper, associated with presumed different roles in attachment, correlated with ultrastructural observations of the surface modifications on the scolex. Despite the locally severe pathological change, insignificant morbidity owing to this particular host-parasite relationship is suggested.

Morphological features of coronary arteries and lesions in hearts from five species of sharks collected from the northwestern Atlantic Ocean.

Morphological features of coronary arteries and incidental lesions are reported from hearts in five species of sharks, the shortfin mako shark, Isurus oxyrhinchus Rafinesque, thresher shark Alopias vulpinus (Bonaterre), blue shark, Prionace glauca L., the smooth dogfish, Mustelus canis (Mitchill), and spiny dogfish, Squalus acanthias L. Sharks were collected from the northwestern Atlantic between June and August from 1996 to 2010. They were necropsied dockside and the hearts were preserved in buffered formalin. Routine sections including ventricle/conus arteriosus and the atrio-ventricular junctions were embedded in paraffin, stained with common histological and immunohistochemical methods and examined by brightfield microscopy. Myointimal hyperplasia, medial myo-myxomatous hyperplasia and bifurcation pads were observed commonly, and medial muscle reorientation and epicardial myeloid tissues were rare. All the above features differed in severity, prevalence and distribution depending on anatomical site and shark species/size. Morphometric analysis indicated that myomyxomatous hyperplasia is associated with luminal narrowing of blood vessels. As suggested previously, the described morphological features are most likely physiological responses to blood flow characteristics. Vascular and cardiac lesions were uncommon and included, granulomatous proliferative epicarditis with fibroepitheliomas, myxomatous epicardial expansions, medial arterial vacuolation, myocardial fibrosis, acute ventricular emboli and parasitic granulomas. The lesions of embolism, proliferative and granulomatous epicarditis and myocardial fibrosis were in all sharks associated with capture events including retained fishing hooks. The significance and aetiopathogenesis of medial vacuolation and epicardial myxomatous expansions remains unclear.

Lesions associated with plerocerci (Platyhelminthes: Cestoda: Trypanorhyncha) in the gastric wall of a cownose ray, Rhinoptera bonasus (Mitchill), (Myliobatiformes: Rhinopteridae) from the northern Gulf of Mexico.

We describe lesions associated with a seemingly intense infection of trypanorhynch plerocerci (Platyhelminthes: Cestoda: Trypanorhyncha) in the gastric wall of a female cownose ray, Rhinoptera bonasus (Myliobatiformes: Rhinopteridae) captured in the northern Gulf of Mexico. Grossly, the multitude of encapsulated, encysted plerocerci imparted a bumpy and cobbled appearance to the serosa of the stomach, and none was observed in any other tissue during routine parasitological necropsy. Histologically, the plerocerci were associated with severe intramural granulomatous gastritis, vascular ectasia and mesothelial polyposis with the exclusion of the mucosa. To our knowledge, this is the first published case study documenting platyhelminth-associated histopathological changes in the gastrointestinal tract of R. bonasus as well as that of the efficacy of immunocytochemical markers for smooth muscle actin, Factor VIII, S-100, and proliferating cell nuclear antigen in Myliobatiformes. It also may serve as a potential primer for much needed ecological investigations regarding the potential role of elasmobranchs as intermediate or 'paratenic' hosts in the life cycles of trypanorhynch cestodes.

Melanomacrophages in three species of free-ranging sharks from the northwestern Atlantic, the blue shark Prionacae glauca (L.), the shortfin mako, Isurus oxyrhinchus Rafinesque, and the thresher, Alopias vulpinus (Bonnaterre).

The melanomacrophage aggregates or cells (MMC) are commonly used as biomarkers of exposure to pollution in fish, albeit their numbers and morphological characteristics can be influenced not only by environmental toxins but also by a range of physiological parameters and pathological conditions. Accordingly, before we can use MMC as biomarkers in any fish species, their normal, 'background' characteristics have to be established in apparently healthy fish. The knowledge of MMC in sharks is minimal. The aim of this study was to characterize MMC from 51 free-ranging, large pelagic sharks from the northwestern Atlantic, including shortfin mako, Isurus oxyrhinchus, thresher, Alopias vulpinus and blue shark, Prionacae glauca. The rationale of this study was twofold. First, because, sharks have life histories predisposing them to the accumulation of environmental toxins they constitute good sentinel species of the health of the global marine ecosystem. Second, because many shark populations are in decline or threatened by extinction, we need to expand our understanding of their health status in order to develop appropriate protective measures. All sharks were collected by sports fishing gear between June and August in 2007. Their health condition was assessed by necropsy, morphometrics, and by microscopic examination of gonads, livers, spleens and kidneys. Routine, haematoxylin and eosin and/or Pearl's reagent-stained paraffin embedded sections were studied by light microscopy. Our results provide the first data on the morphometric and morphological characteristics of MMC in viscera of apparently healthy free-ranging sharks from the northwestern Atlantic.

Comparison of histological features, and description of histopathological lesions in thyroid glands from three species of free-ranging sharks from the northwestern Atlantic, the blue shark, Prionace glauca (L.), the shortfin mako, Isurus oxyrhinchus Rafinesque, and the thresher, Alopias vulpinus (Bonnaterre).

Abstract Histomorphological features of piscine thyroids are widely accepted and frequently used as bioindicators of environmental pollution. This despite the fact that there is marked variation in thyroid morphology resulting from numerous pathological and physiological conditions. Our hypothesis was that there will be variations in histological features in thyroids collected from different shark species during the summer season in the northwestern Atlantic. To test our hypothesis, we examined histological features encountered in grossly normal thyroids from three species of sharks, the shortfin mako, Isurus oxyrhinchus, thresher, Alopias vulpinus and blue sharks, Prionace glauca. In addition, microscopic lesions from these thyroids were described. Ninety-four sharks were collected in summer 2001, 2002 and 2004. Routine, haematoxylin and eosin-stained paraffin-embedded sections were studied by light microscopy. We found inter-species but not intra-species variation in histological patterns of thyroids, which were distinct enough to allow 'blind' assignment of a thyroid to the specific species. The most common lesions encountered were lymphofollicular hyperplasia and chronic thyroiditis. In addition, one case each of intravascular larval nematodes and a myxosporean infection was found. Our results provide the first data on species-specific morphology of thyroids collected during summer months from sharks. The results indicate that familiarity with normal thyroid morphology is crucial before using shark thyroids in biomonitoring of environmental contamination or interpreting data from this gland in shark-health studies.

Molecular markers of cancer in cartilaginous fish: immunocytochemical study of PCNA, p-53, myc and ras expression in neoplastic and hyperplastic tissues from free ranging blue sharks, Prionace glauca (L.).

Archival formalin-fixed tissues from wild-caught adult blue sharks, Prionace glauca (L.), were used for immunocytochemical detection of proliferating cell nuclear antigen (PCNA), two oncoproteins from the oncogenes c-myc and pan-ras, and a protein product from the tumour suppressor gene p-53. All sharks were caught during summer months between 2000 and 2006 by recreational fishermen off the USA coast in the northwestern Atlantic. The sharks were necropsied on landing and selected organ samples were collected into elasmobranch formalin and processed for paraffin embedding and light microscopy. Paraffin-embedded sections from collected tissue were both stained with haematoxylin and eosin and processed by immunocytochemical techniques using antibodies raised against the PCNA, p-ras, c-myc and p-53 proteins. The lesions examined in this study included two well differentiated adenomatous gastric polyps, a testicular capsular mesothelioma, a gingival fibropapilloma with elements of ameloblastoma, three liver tumours, two pericardial fibropapillomas and six cases of proliferative serositis (pericarditis and peritonitis). Normal and hyperplastic tissues from blue sharks, and human neoplastic tissues served as negative and positive controls, respectively. We detected upregulation of PCNA in many neoplastic, one dysplastic and in some hyperplastic lesions, and positive p-ras and c-myc signals in some of the neoplastic lesions. None of the examined tissues showed positive p-53 signalling. This is the first literature report on immunocytochemical detection of molecular markers of cancer in sharks and in fish of the class Chondrichthyes.

Mode of attachment and lesions associated with trypanorhynch cestodes in the gastrointestinal tracts of two species of sharks collected from coastal waters of Borneo.

Lesions associated with two species of tapeworms within the digestive tract of wild-caught specimens of the bull shark, Carcharhinus leucas, and the sicklefin weasel shark, Hemigaleus microstoma, from Malaysian Borneo are described. Portions of the glandular stomach and pyloric gut with parasites were removed and fixed in 10% formalin buffered in sea water. Whole mounts, histological sections of tissues with and without worms in situ, and scanning electron microscopy images of detached worms were examined. Both species of cestodes belonged to the trypanorhynch family Tentaculariidae. Heteronybelinia estigmena was found in large numbers parasitizing the pyloric gut of C. leucas; an unidentified tentaculariid was found in relatively small numbers in both the glandular stomach and pyloric gut of H. microstoma. Both species burrowed their scoleces deeply in the mucosa and attached via hooked tentacles and unciniform microtriches of the scolex. The lesions induced by the parasites were marked in both sharks and ranged from acute necrotizing to chronic granulomatous gastroenteritis. Regenerative hyperplasia and intestinal metaplasia of gastric epithelium were also present. The severity and character of pathology was causally linked to the intensity of infection, the attachment mode of the parasites, and to the anatomophysiological relationships within the gut of the host shark.

Hepatic cholangiocarcinoma and testicular mesothelioma in a wild-caught blue shark, Prionace glauca (L.).

An adult male blue shark, Prionace glauca (L.), caught in July 2000 by a recreational fisherman off Long Island, New York, USA, had a retained fishing hook from a previous capture. The hook penetrated the gastric wall and lacerated the right liver lobe. Macroscopic lesions consisted of transmural gastritis and peritonitis. Alteromonas sp. and Vibrio alginolyticus were isolated from the peritoneal fluid. In addition, a well delineated, sessile mass was found on the otherwise normal serosa of the right testis. Histopathological findings included mesothelial hyperplasia and hypertrophy involving diffusely the gastric, hepatic and parietal serosae, and forming a discrete testicular capsular mass compatible with mesothelioma. In the liver an intrahepatic cholangiocarcinoma, chronic hepatitis, biliary hyperplasia and increased numbers of melanomacrophages were found. In addition organisms compatible with histozoic and coelozoic myxosporeans were found within the skeletal muscle of the abdomen and intrahepatic bile ducts, respectively. This is the third literature report of a liver tumour and the first report of a coelomic mesothelioma from a shark.

Sarcoma of possible nerve sheath origin in a captive muskrat.

A captive adult female muskrat (Ondatra zibethicus) was found dead without previous signs of disease. At necropsy, abdominal organs were infiltrated with a poorly demarcated, soft, tan tissue. Microscopically this tissue was composed of neoplastic cells assuming two distinct growth characteristics consistent with Antoni A and B patterns. Ultrastructurally, the neoplastic cells were pleomorphic, lacked junctional devices, had abundant mitochondria and ergastoplasm, and frequently were closely associated with extracellular collagen. Immunocytochemical examination of tumor cells demonstrated sporadic expression of neuron specific enolase. Microscopic tumor metastases to the myocardium, ascending aorta, lungs and visceral pleura were present. This is the first report of a sarcoma compatible with a malignant peripheral nerve sheath tumor in a muskrat.

Disseminated granulomas associated with nematode larvae in a shortfin mako shark.

A shortfin mako shark (Isurus oxyrinchus) caught in 1996 by sportfishermen in Long Island (New York, USA) had many granulomas containing larval nematodes. Granulomas were present in the myocardium, spleen, pancreas, stomach, spiral intestine, hematopoietic tissue within the anterior kidney, and in the branchial septum and primary lamellae of the gills. Epicardial hyperplasia and granulomatous myocarditis were associated with the larvae. Although identification of the larvae was impossible due to lack of distinct morphological features, they resembled dracunculoid larvae previously reported from sharks.

Clinicopathological features and histopathology of experimental hepatic capillariasis in muskrats (Ondatra zibethicus).

Ten muskrats (Ondatra zibethicus) each were infected with 17,000 eggs (long-term study) and eight muskrats each were infected with 8,000 eggs (short-term study) of Capillaria hepatica (Nematoda). Food intake, body weight, and selected clinicopathological parameters were measured every 2 days for 28 days in the short-term study and every 14 days for 184 days in the long-term study. Muskrats in the short-term study had moderate to severe necrotizing granulomatous hepatitis associated with mild anorexia and weight loss, varying degrees of leukocytosis with eosinophilia and elevation of serum alanine and aspartate aminotransferases. No significant changes in packed cell volume, hemoglobin, total plasma protein, albumin, blood urea nitrogen, bilirubin, lactate dehydrogenase or alkaline phosphatase were found among animals from the short-term study. Muskrats in the long-term study had severe necrotizing granulomatous hepatitis associated with marked anorexia, weight loss and 60% mortality over 39 days post-inoculation (PI); animals that survived for 184 days did not return to pre-inoculation body weights despite returning to normal food intake. Hepatic lesions at 184 days PI consisted of minimal to severe liver replacement by C. hepatica eggs. No statistically significant differences in values of clinical parameters between inoculated animals and a non-inoculated control group from the long term study were found.

Brief characterization of muskrat (Ondatra zibethicus) immunoglobulin G (IgG) separated from serum on protein A.

Muskrat (Ondatra zibethicus) immunoglobulin fraction was separated from whole serum by Protein A Sepharose chromatography. In serum electrophoresis, this fraction had a gamma motility; when electrophoresed on a polyacrylamide gel with sodium dodecyl sulfate it resolved into two protein bands of approximately 52 and 25 kilodaltons, respectively. These bands were consistent with molecular weights of known heavy and light chains of immunoglobulin G (IgG) in other closely related species. Furthermore, the putative muskrat immunoglobulins had a strong cross-reactivity with mouse IgG1, IgG3, and kappa chain in an enzyme-linked immunosorbent assay. We propose, that the proteins bound to the Protein A Sepharose represent muskrat immunoglobulins of the IgG class.

Hepatic capillariasis in muskrats (Ondatra zibethicus).

We present the first prevalence data of hepatic capillariasis in muskrats in Pennsylvania and Connecticut (USA). The prevalence of hepatic capillariasis in five groups of muskrats coming from different locations and numbering 81, 229, 11, 19, and 20 animals, was 42%, 78%, 36%, 16%, and 0%, respectively. Liver lesions varied from minimal to severe multifocal granulomatous hepatitis, often containing adult worms or eggs.