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The genus Megalocytivirus is the most recently described member of the family Iridoviridae; as such, little is known about the genetic diversity of this genus of globally emerging viral fish pathogens. We sequenced the genomes of 2 megalocytiviruses (MCVs) isolated from epizootics involving South American cichlids (oscar Astronotus ocellatus and keyhole cichlid Cleithracara maronii) and three spot gourami Trichopodus trichopterus sourced through the ornamental fish trade during the early 1990s. Phylogenomic analyses revealed the South American cichlid iridovirus (SACIV) and three spot gourami iridovirus (TSGIV) possess 116 open reading frames each, and form a novel clade within the turbot reddish body iridovirus genotype (TRBIV Clade 2). Both genomes displayed a unique truncated paralog of the major capsid protein gene located immediately upstream of the full-length parent gene. Histopathological examination of archived oscar tissue sections that were PCR-positive for SACIV revealed numerous cytomegalic cells characterized by basophilic intracytoplasmic inclusions within various organs, particularly the anterior kidney, spleen, intestinal lamina propria and submucosa. TSGIV-infected grunt fin (GF) cells grown in vitro displayed cytopathic effects (e.g. cytomegaly, rounding, and refractility) as early as 96 h post-infection. Ultrastructural examination of infected GF cells revealed unenveloped viral particles possessing hexagonal nucleocapsids (120 to 144 nm in diameter) and electron-dense cores within the cytoplasm, consistent with the ultrastructural morphology of a MCV. Sequencing of SACIV and TSGIV provides the first complete TRBIV Clade 2 genome sequences and expands the known host and geographic range of the TRBIV genotype to include freshwater ornamental fishes traded in North America.

The present study describes, for the first time, histopathological alterations in the gills of Macrobrachium amazonicum caused by infestation of Probopyrus bithynis (Isopoda: Bopyridae). In every case (100%), the infestation by P. bithynis was by a single pair of parasites (male and female) and occurred in the right or left side of the branchial chamber; the gill structures were visibly compressed due to the presence of parasites. The gills of M. amazonicum parasitized by P. bithynis exhibited a chronic inflammatory response, with the presence of edema, greater quantities of hemocytes, necrosis, epithelial cell hyperplasia, rupture of the pillar cells at the ends of the gill lamellae, desquamation of the cuticle, lamellar fusion and rupture of the lamellar epithelium. Tissue lesions were found in the histological sections of the gills of the parasitized M. amazonicum. Structural alterations in the branchial chamber of the hosts caused by the presence of P. bithynis can lead to physiological changes that can impair host respiratory performance. Finally, histopathological alterations in the branchial chamber of hosts suggest that P. bithynis feed directly on the gill tissues of this shrimp.

Aeromonas dhakensis (Ad) CAIM 1873 growth was evaluated at different conditions and antibiotic susceptibility. Mortality and histopathological damages in hybrid tilapia Oreochromis niloticus × O. mossambicus, and virulence factors caused by Ad bacterial cells and extracellular products (ECPs) were evaluated, and the whole genome was obtained. Ad grew between 0.0 and 5.5% NaCl at a pH of between 4 and 10 and from 4 to 37°C. The lowest minimum inhibitory concentration was found for enrofloxacin (<5 µg ml-1), and bacteria were resistant to erythromycin, amoxicillin and ampicillin. Ad bacterial cells (1.86 × 105 cells g-1) and ECPs (0.462 µg protein fish-1) were highly virulent to challenged hybrid tilapia and caused over 80% mortality at 24 h. The primary clinical sign caused was haemorrhage, and damage was most marked in the spleen, liver, kidney and brain of fish challenged with bacterial cells. To our knowledge, this is the first report that Ad causes pyknotic and karyorrhectic nuclei of erythrocytes in the internal organs of hybrid tilapia, which was the most striking histopathological observation. The virulence of Ad to hybrid tilapia may be primarily related to the activity of haemolysins (hlyA genes) and cytotoxins (aerolysin aerA), along with the production of siderophores and proteases. We also found β-lactamase, tetracycline and multiple antibiotic resistance genes, as well as adherence, iron acquisition, toxins (aerolysin family, haemolysins) and diverse protease genes.

Among the Saprolegnia species found in aquaculture facilities, S. parasitica is recognized as the primary fish pathogen and remains an ongoing concern in fish health management. Until recently, these pathogens were kept in check by use of malachite green; due to its toxicity, this chemical has now been banned from use in many countries. It is difficult to predict and control S. parasitica outbreaks in freshwater systems and there is a need to understand the population genetic structure of this pathogen. Genetic characterization of this species in aquaculture systems would provide information to track introductions and determine possible sources of inoculum. Degenerate PCR primers containing short sequence repeats were used to create microsatellite-associated genetic markers (random amplified microsatellites) for the comparison of S. parasitica isolates collected primarily from commercial Atlantic salmon aquaculture systems in British Columbia, Canada, over a 15 mo period to describe their spatial and temporal variability. The frequencies of amplified products were compared and the population genetic diversity was measured using Nei's genetic distance and Shannon's information index, while the species population structure was evaluated by phylogenetic analysis. S. parasitica was detected in all facilities sampled. Genetic diversity was low but not clonal, most likely due to repeated introduction events and a low level of sexual recombination over time. A better understanding of pathogen population structure will assist the development of effective preventative measures and targeted treatments for disease outbreaks.

This study evaluated the hematological profile and condition factor (Kn) of tui chub Siphateles bicolor and fathead minnow Pimephales promelas and their associations with larvae of Contracaecum sp. infection of the heart. A total of 30 tui chub and 17 fathead minnow were collected from Upper Klamath Lake, Oregon, USA, measured, and weighed and blood was drawn for hematological analysis. Nematode larvae parasitized tui chub with a prevalence of 50% and mean intensity of 1.40, while 11.8% of fathead minnow were parasitized at a mean intensity of 1.0. Non-parasitized tui chub were significantly larger than the parasitized fish, indicating that small fish could be easily predated by the definitive host, a piscivorous bird. Although the relatively large worm occupied a large portion of the atrium, the presence of the larvae did not affect tui chub Kn, possibly associated with low parasite intensity and a harmonic co-evolution. Only parasitized fathead minnow showed significant differences in red blood cell measurements (greater cell width and larger nuclei) compared to non-parasitized fish. Lymphocytes were the most common white blood cells found in tui chub, followed by neutrophils, monocytes, and periodic acid-Schiff positive granular leukocytes; in fathead minnow lymphocytes were followed by heterophils, monocytes, neutrophils and eosinophils. This study is the first report of Kn and description of blood cells and hematological parameters in these fish species.

Goldfish Carassius auratus is the most popular ornamental species, widely present in private and public aquaria. In the present case, 2 goldfish exhibited bilateral, multiple, variably sized, round, pale-white, soft, protruding masses on the body. The microscopic examination of the masses revealed well-differentiated adipocytes infiltrating the subcutaneous skeletal muscle bundles. The histological lesions were consistent with infiltrative lipoma. To our knowledge, this is the first report of cutaneous infiltrative lipoma in goldfish.

We report the spontaneous occurrence of oral neoplasms in pickhandle barracuda Sphyraena jello Cuvier, 1829 from Parangipettai, on the southeast coast of India. A total of 11736 fish were examined, of which 43 were affected with oral tumours, with an overall prevalence of 0.37%. Gross and clinical symptoms included reddish to grayish-white distended tumourous growths on the gingiva, intra-oral bones and tongue. The tumours exhibited delayed eruption and intra- or extra-oral swelling, varied in consistency from extremely firm to fleshy and released mucinous material. The majority of tumours consisted of numerous clumped toothlets, but some included hardened tissues. Local area invasion/transmission was observed in most cases; however, necropsy examination revealed no gross evidence of distant metastasis into visceral organs. Radiographic examination of compound odontomas revealed distinct unilocular radio-opaque mini-toothlets surrounded by defined radiolucency, whereas complex odontomas exhibited unilocular and indistinct radio-opaque masses within a much more extensive radiolucent zone. Histopathologically, the intra-oral tumour lesions were characterized by numerous imperfect (germ) toothlets consisting of a disorganized combination of dental tissues: pulp tissues with manifested and predominantly mixed hard dental tissues of immature dentine and enamel, numerous small to large and round to polyhedral ossicles embedded in hypocellular fibrous stromal tissues and sparsely spaced bland spindloid cells with cleft-like spaces of loose mucoid stroma. Histochemically, the neoplastic lesions stained positive for periodic acid-Schiff and Masson's trichrome. Based on the clinical and histological findings, the tumours were diagnosed as compound odontomas, complex odontomas, odontogenic myxomas, lingual myxomas and psammomatoid ossifying fibromas.

Proliferative kidney disease (PKD) is an emerging disease threatening wild salmonid populations, with the myxozoan parasite Tetracapsuloides bryosalmonae as the causative agent. Species differences in parasite susceptibility and disease-induced mortality seem to exist. The aim of the present study was to compare incidence, pathology and mortality of PKD in grayling Thymallus thymallus and brown trout Salmo trutta under identical semi-natural conditions. Young-of-the-year grayling and brown trout, free of T. bryosalmonae, were jointly exposed in cage compartments in a river in the northeast of Switzerland during 3 summer months. Wild brown trout were caught by electrofishing near the cage, and PKD status was compared with that of caged animals. Cage-exposed grayling showed a PKD incidence of 1%, regardless of whether parasite infection was determined by means of real-time PCR or histopathology/immunohistochemistry. In contrast, PKD incidence of caged brown trout was 77%. This value was not significantly different to PKD prevalence of wild brown trout caught above and below the cage (60 and 91%, respectively). Mortality in grayling was significantly higher compared with that of brown trout (40 versus 23%); however, grayling mortality was not considered to be associated with PKD. Mortality of caged and infected brown trout was significantly higher than mortality of non-infected caged trout. Histopathology indicated an ongoing mostly acute or chronic active infection in brown trout, which survived until the end of exposure. The results suggest that grayling are less susceptible to infection with T. bryosalmonae compared with brown trout under the tested field conditions.

Fish die-offs are important signals in tropical marine ecosystems. In 2010, a mass mortality of pufferfish in Hawaii (USA) was dominated by Arothron hispidus showing aberrant neurological behaviors. Using pathology, toxinology, and field surveys, we implicated a series of novel, polar, marine toxins as a likely cause of this mass mortality. Our findings are striking in that (1) a marine toxin was associated with a kill of a fish species that is itself toxic; (2) we provide a plausible mechanism to explain clinical signs of affected fish; and (3) this epizootic likely depleted puffer populations. Whilst our data are compelling, we did not synthesize the toxin de novo, and we were unable to categorically prove that the polar toxins caused mortality or that they were metabolites of an undefined parent compound. However, our approach does provide a template for marine fish kill investigations associated with marine toxins and inherent limitations of existing methods. Our study also highlights the need for more rapid and cost-effective tools to identify new marine toxins, particularly small, highly polar molecules.

A pharyngeal tumor was found in a wild European bullhead. The tumor mass appeared underneath the operculum and was bilobed. The major neoplastic component showed diffuse epithelial squamous differentiation. Crossmonn's trichrome allowed identification of connective tissues whereas no neoplastic cells were stained. Periodic acid-Schiff was negative within the mass, and Giemsa did not show any further diagnostic significance. Immunohistochemistry showed diffuse positive cytoplasmic staining of the neoplastic population with an anti-human pancytokeratin antibody. Vimentin was negative and exclusively stained the stroma. On the basis of the morphological and immunohistochemical results, a squamous cell carcinoma was diagnosed.

The morphogenesis of large icosahedral viruses associated with lymphocystis-like lesions in the skin of parore Girella tricuspidata is described. The electron-lucent perinuclear viromatrix comprised putative DNA with open capsids at the periphery, very large arrays of smooth endoplasmic reticulum (sER), much of it with a reticulated appearance (rsER) or occurring as rows of vesicles. Lysosomes, degenerating mitochondria and virions in various stages of assembly, and paracrystalline arrays were also present. Long electron-dense inclusions (EDIs) with 15 nm repeating units split terminally and curled to form tubular structures internalising the 15 nm repeating structures. These tubular structures appeared to form the virion capsids. Large parallel arrays of sER sometimes alternated with aligned arrays of crinkled cisternae along which passed a uniformly wide (20 nm) thread-like structure. Strings of small vesicles near open capsids may also have been involved in formation of an inner lipid layer. Granules with a fine fibrillar appearance also occurred in the viromatrix, and from the presence of a halo around mature virions it appeared that the fibrils may form a layer around the capsid. The general features of virogenesis of large icosahedral dsDNA viruses, the large amount of ER, particularly rsER and the EDIs, are features of nucleo-cytoplasmic large DNA viruses, rather than features of 1 genus or family.

Multiple black raised nodular masses were noted on the dorsal surface of an adult male Haller's round ray Urobatis halleri. Biopsy of 2 masses was performed, and histopathology revealed proliferative sheets of melanocytes exhibiting mild anisocytosis and anisokaryosis, supporting a diagnosis of malignant melanoma. Approximately 2 mo following the biopsy procedure, the round ray became acutely anorexic and was found dead in its enclosure. A full necropsy was performed, and tissues were submitted for histopathology. The black raised nodular masses again exhibited histologic features of a melanoma. In addition to the nodular masses present, multiple flat areas of increased pigmentation were also present throughout the course of the case and were not suggestive of neoplasia histologically. The transformation of benign to malignant neoplasia has been well described in other species and may have played a role in the development of multiple tumors in this case.

Flavobacterium columnare is the causative agent of columnaris disease and is responsible for significant economic losses in aquaculture. F. columnare is a Gram-negative bacterium, and 5 genetic types or genomovars have been described based on restriction fragment length polymorphism of the 16S rRNA gene. Previous research has suggested that genomovar II isolates are more virulent than genomovar I isolates to multiple species of fish, including rainbow trout Oncorhynchus mykiss. In addition, improved genotyping methods have shown that some isolates previously classified as genomovar I, and used in challenge experiments, were in fact genomovar III. Our objective was to confirm previous results with respect to genomovar II virulence, and to determine the susceptibility of rainbow trout to other genomovars. The virulence of 8 genomovar I, 4 genomovar II, 3 genomovar II-B, and 5 genomovar III isolates originating from various sources was determined through 3 independent challenges in rainbow trout using an immersion challenge model. Mean cumulative percent mortality (CPM) of ~49% for genomovar I isolates, ~1% for genomovar II, ~5% for the II-B isolates, and ~7% for the III isolates was observed. The inability of genomovar II isolates to produce mortalities in rainbow trout was unanticipated based on previous studies, but may be due to a number of factors including rainbow trout source and water chemistry. The source of fish and/or the presence of sub-optimal environment may influence the susceptibility of rainbow trout to different F. columnare genomovars.

A severe fungal infection affecting the head and lateral line system was diagnosed in 7 captive scalloped hammerhead sharks Sphyrna lewini in an aquarium in Thailand. Extensive and severe necrotizing cellulitis was consistently observed microscopically along the cephalic and lateral line canals in conjunction with positive fungal cultures for Fusarium sp. Molecular phylogenetic analysis was performed from 3 isolates based on the nucleotide sequences containing internally transcribed spacer (ITS) and a portion of 5.8S and 28S rDNA. The fungus was highly homologous (100%) and closely related to F. solani species complex 2 (FSSC 2), which belongs to Clade 3 of the FSSC. Our results illustrate the histopathological findings and expand upon our knowledge of the prevalence of invasive fusariosis in the head and lateral line system of hammerhead sharks.

Surveillance for pathogens of Atlantic herring, including viral hemorrhagic septicemia virus (VHSV), Ichthyophonus hoferi, and hepatic and intestinal coccidians, was conducted from 2012 to 2016 in the NW Atlantic Ocean, New Jersey, USA. Neither VHSV nor I. hoferi was detected in any sample. Goussia clupearum was found in the livers of 40 to 78% of adult herring in varying parasite loads; however, associated pathological changes were negligible. Phylogenetic analysis based on small subunit 18S rRNA gene sequences placed G. clupearum most closely with other extraintestinal liver coccidia from the genus Calyptospora, though the G. clupearum isolates had a unique nucleotide insertion between 604 and 729 bp that did not occur in any other coccidian species. G. clupearum oocysts from Atlantic and Pacific herring were morphologically similar, though differences occurred in oocyst dimensions. Comparison of G. clupearum genetic sequences from Atlantic and Pacific herring revealed 4 nucleotide substitutions and 2 gaps in a 1749 bp region, indicating some divergence in the geographically separate populations. Pacific G. clupearum oocysts were not directly infective, suggesting that a heteroxenous life cycle is likely. Intestinal coccidiosis was described for the first time from juvenile and adult Atlantic herring. A novel intestinal coccidian species was detected based on morphological characteristics of exogenously sporulated oocysts. A unique feature in these oocysts was the presence of 3 long (15.1 ± 5.1 µm, mean ±SD) spiny projections on both ends of the oocyst. The novel morphology of this coccidian led us to tentatively name this parasite G. echinata n. sp.

Koi carp presenting with coelomatic neoplastic lesions are of growing importance in Switzerland and elsewhere in Europe, as clinical diagnosis is usually difficult and prognosis is usually poor because of late recognition. To gain more information about this condition, between 2008 and 2012, 117 koi diagnosed with coelomatic tumours were included in this study. The nature and growth characteristics of these pathologic tissues were documented. Out of 117 koi, 48.9% (n = 57) were female; the ovary was the most often affected organ (35%, n = 41), and in 62.4% (n =73) of cases, the cells of origin were identified as stromal cells of the sex-cord and the tumours were identified as sex-cord stromal tumours. The characteristics of growth and histology showed that the majority of tumours were malignant.

A wild adult male conger Conger conger, captured by a SCUBA diver in the waters of coastal Italy, was sent for laboratory analysis due to the presence of multiple productive ulcerous skin lesions localized in the dorso-lateral body area, caudally to the gill operculum under the dorsal fin. The main mass was sessile, ulcerated and hemorrhaging in appearance and was surrounded by several smaller masses, which originated peripherally from the same mass or were isolated, always with a tendency to ulceration. Histology confirmed that the newly formed tissue originated from derma invading the closer tissues. The tumour consisted of spindle cells, each with an ovoidal nucleus and rarely with evident nucleoli, which were arranged in parallel or storiform patterns and were often surrounding blood-filled spaces discontinuously lined by endothelial cells. Tumour tissue was richly vascularized and no mitoses were seen. The overlying epidermis was ulcerated. Masson's trichrome technique indicated the presence of a small amount of perivascular connective tissue. No excessive glycogen storage, bacteria, virus or fungi were detected by periodic acid-Schiff (PAS)-reaction. Immunohistochemistry showed dot-like or diffuse cytoplasmic positivity against smooth muscle actin and the monoclonal antibody D2-40. CD34 exhibited relevant immunoreactivity at plasma membranes. Growth fraction evaluated using MIB-1 was <1%. Immunoreactions for wide spectrum CK, CK5/6, CK8, CK18, EMA, desmin, myoglobin, S-100, CD20, CD68, GFAP, and NSE were negative. Histopathological and immunohistochemical results supported a diagnosis of angioleiomyoma, a benign tumour of the muscular cellular component of the blood vessels. To our knowledge, this is the first report of such neoplasms in fishes in which monoclonal antibodies work on fish tissues, facilitating a useful immunohistochemical approach for differential diagnosis.

Since the initial isolation of the fathead minnow nidovirus (FHMNV), concerns have been raised regarding the risks it may pose to other fish species. In this study, 7 fish species resident to the Laurentian Great Lakes were challenged intraperitoneally with 2 doses of FHMNV: 102.8 and 104.8 median tissue culture infective dose (TCID(50)) ml(-1). Infected spotfin shiner Cyprinella spiloptera and golden shiner Notemigonus crysoleucas suffered morbidity and mortality during the 40 d observation period, while other species, including creek chub Semotilus atromaculatus, rainbow trout Oncorhynchus mykiss, largemouth bass Micropterus salmoides and walleye Sander vitreus, showed no clinical signs or mortality. FHMNV was re-isolated on the epithelioma papulosum cyprini cell line from the tissues of infected spotfin shiner and golden shiner, which harbored high numbers of viral RNA copies as measured by quantitative loop-mediated isothermal amplification. Infected spotfin shiner and golden shiner exhibited external petechiae, exophthalmia, oedematous kidneys, and liver pallor. Histopathological analysis revealed multifocal areas of necrosis in the kidney, spleen and liver of infected fish. Spotfin shiner and golden shiner were then infected with 2 doses of FHMNV (10(3.5) and 10(3.9) TCID(50) ml(-1)) by immersion to mimic more natural modes of infection. Spotfin shiner experienced 60% mortality at both doses, while golden shiner did not experience mortality nor develop any clinical signs following a 40 d observation period. Overall, piscivorous fish tested in this study do not seem to be at risk for infection, while cyprinids appear to vary in their susceptibility to the strain of FHMNV used in this study.

Histopathological alterations in the heart are often reported in fish as a result of exposure to a variety of chemical compounds. However, researchers presently lack a standardized method for the evaluation of histopathological alterations in the cardiovascular system of fish and the calculation of an 'organ index'. Therefore, we designed a method for a standardized assessment and evaluation of histopathological alterations in the heart of fish. As a model species, we used rainbow trout Oncorhynchus mykiss, but the protocol was also successfully applied to other fish species belonging to different taxonomic orders. To test the protocol, we re-evaluated sections of atenolol-exposed and unexposed rainbow trout obtained in a previous study. The results were in accordance with those previously published, demonstrating the applicability of the protocol. The protocol provides a universal method for the comparative evaluation of histopathological changes in the heart of fish.

Although elasmobranch species are increasingly displayed in public aquaria, knowledge of disease in wild and captive elasmobranchs, as well as the agents involved, remains limited, and descriptions are often incomplete. This report describes papillomatous skin lesions in a juvenile giant guitarfish Rhynchobatus djiddensis associated with intranuclear viral particles. Skin biopsies were collected from multiple, friable, raised, villonodular skin lesions affecting pigmented and non-pigmented skin of the caudal fin and ventrum, respectively. Microscopic examination revealed papillary proliferation of the epidermis, with widespread marked karyomegaly of squamous epithelial cells. In approximately 75% of nuclei, chromatin was marginated by one to multiple, large, amphophilic inclusions. Large numbers of unencapsulated, 75 nm, icosahedral viral particles were observed to form large arrays in affected nuclei using transmission electron microscopy. Based on intranuclear location, particle size and morphology, a consensus nested-PCR for adenovirus polymerase was attempted. However, no adenoviral gene sequence was amplified. The nature of the involved virus remains unknown and an ongoing area of investigation. Lesions regressed completely over a 6 mo period, during which time the animal showed no signs of systemic illness, and there has been no recrudescence for 6 mo following resolution. Two cohorts of similar age and in close contact with the case animal were unaffected.

Spontaneous neoplasia of the intestinal tract in sentinel and moribund zebrafish Danio rerio is common in some zebrafish facilities. We previously classified these tumors as adenocarcinoma, small-cell carcinoma, or carcinoma otherwise unspecified based on histomorphologic characteristics. Based on histological presentation, the primary differential diagnosis for the intestinal carcinomas was tumor of neuroendocrine cells (e.g. carcinoids). To further characterize the phenotype of the neoplastic cells, select tissue sections were stained with a panel of antibodies directed toward human epithelial (cytokeratin wide spectrum screening [WSS], AE1/AE3) or neuroendocrine (S100, chromogranin A) markers. We also investigated antibody specificity by Western blot analysis, using a human cell line and zebrafish tissues. Nine of the intestinal neoplasms (64%) stained for AE1/AE3; 7 (50%) also stained for WSS. None of the intestinal neoplastic cells stained for chromogranin A or S100. Endocrine cells of the pituitary gland and neurons and axons of peripheral nerves and ganglia stained for chromogranin A, whereas perineural and periaxonal cells of peripheral intestinal ganglia, and glial and ependymal cells of the brain stained for S100. Immunohistochemistry for cytokeratins confirmed the majority of intestinal neoplasms in this cohort of zebrafish as carcinomas.

Vibrio anguillarum is the etiological agent of a fatal hemorrhagic disease known as vibriosis that affects a wide range of fish species, causing severe economic losses. Several investigations have been carried out to elucidate the virulence mechanisms of this pathogen and to develop rapid detection techniques and effective disease-prevention strategies. The aim of our study was to evaluate the most effective way to induce mild enteritis in a fish model, in order to allow further applications. The experiments were carried out using 2 methods of administration of V. anguillarum serotype O1 to adult zebrafish Danio rerio: via intraperitoneal injection and via ingestion of infected Artemia nauplii. The results showed that the intraperitoneal administration often caused massive fish death due to severe systemic involvement. In our experiments, the effect of intraperitoneal infection was evident 48 h post infection, with cumulative mortality within 7 d post infection with severe histopathological changes in kidney hematopoietic tissue and in the intestine. In contrast, oral infection via Artemia did not show systemic involvement and only a moderate degree of inflammatory influx of the mucosa, a partial recovery at 12 d post infection, and no mortality. For these reasons, oral infection with live food appears to be the most effective method to induce mild enteritis with a local inflammatory response.

Vibrio tapetis is primarily known as the causative agent for brown ring disease in bivalves, although it has been isolated from cultivated fish during mortalities on farms. Here we describe the first isolation of V. tapetis from wild-caught and subsequently captive-held Dover sole Solea solea. Pathological features consisted of multifocal circular greyish-white skin discolourations evolving into vesicular lesions and subsequent ulcerations on the pigmented side. On the non-pigmented side, multiple circular lesions-white at the center and red at the edges-were evident. Histological examination of the vesicular lesions revealed dermal fluid-filled spaces, collagen tissue necrosis and a mixed inflammatory infiltrate, with large numbers of small rod-shaped bacteria. In the deep skin lesions, loss of scales and dermal connective tissue, with degeneration and fragmentation of the myofibres bordering the ulceration, were noted. Serotyping, DNA-DNA hybridization and REP- and ERIC-PCR techniques showed that the retrieved isolates displayed a profile similar to the representative strain of genotype/serotype O2 which originally was isolated from carpet-shell clam Venerupis decussata and to which isolates obtained from wedge sole Dicologoglossa cuneata were also closely related.

Diseases of aquatic animals have had, and continue to have, a significant impact on aquatic animal health. In Australia, where fisheries and aquaculture are important industries, aquatic species have been subject to serious disease outbreaks, including pilchard herpesvirus, the cause of one of the largest wild fish kills ever recorded. At the same time, there is a consensus that Australia's parasite fauna are largely unknown, and that aquatic animal health information is difficult to access. Managing aquatic animal diseases is challenging because they may be entirely new, their hosts may be new to aquaculture, and specialist expertise and basic diagnostic tools may be lacking or absent. The Neptune project was created in response to these challenges, and it aims to increase awareness of aquatic animal diseases, improve disease management, and promote communication between aquatic animal health professionals in Australia. The project consists of an online database, a digital microscopy platform containing a whole-slide image library, a community space, and online communications technology. The database contains aquatic animal health information from published papers, government reports, and other sources, while the library contains slides of key diseases both endemic and exotic to Australia. These assets make Neptune a powerful resource for researchers, students, and biosecurity officials.

A new species of the genus Thelohanellus Kudo, 1933 (Myxosporea, Bivalvulida) was isolated from the fins of goldfish Carassius auratus auratus (Linnaeus 1758). The fish had been imported from China by an Austrian retailer. Nodules from the margins of the fins contained pyriform myxospores with a singular polar capsule. In valvular view, the spores measured 12.2 µm in length and 6.4 µm in width. In sutural view, the thickness was 2.9 µm. The polar capsule measured 4.2 × 3.1 µm and contained a polar filament with 8 to 9 coils. Histological sections showed plasmodia of 0.2 to 4.0 mm diameter with the earlier developmental stages of the parasite in the periphery and the mature spores closer to the center. In the transmission electron microscope examination, the different developmental stages could be observed. Morphological data, host specificity, tissue tropism, and molecular analysis of the small subunit rDNA identify this parasite as a new species of Thelohanellus, which we have named Thelohanellus hoffmanni sp. nov.

Ichthyophthirius multifiliis is a recurring problem in Australian rainbow trout Oncorhynchus mykiss farms and requires strategically timed, repeat treatments for effective management. Sodium percarbonate (SPC) is permitted for use in Australia, with host safety margins based on the toxicity of acute exposures to hydrogen peroxide (HP), the active product released when SPC is added to water. The effects of exposure to HP released by SPC, of repeated doses and of doses exceeding 100 mg l-1 on rainbow trout are unknown. We exposed juvenile rainbow trout (mean weight: 30.5 ± 9 g) to repeated doses of 50, 150 and 250 mg l-1 SPC for 1 h on Days 1, 2, 7 and 8 of a treatment regime. The effect of SPC was assessed by histological evaluation of structural changes in gill tissue. Survival was 100% in all groups, but some fish exposed to 250 mg l-1 SPC displayed impaired swimming performance, and on Day 9 after the final treatment, oedema was present in 9.8% of lamella, which was significantly higher than the mean occurrence of 1.7, 4.2 and 1.3% in fish treated with 0, 50 and 150 mg l-1 SPC, respectively. These changes resolved within 24 h of the cessation of treatment. We conclude that SPC is safe to use on rainbow trout in doses of ≤150 mg l-1 at 17°C, however caution is advised at doses approaching 250 mg l-1. Water temperature, fish age, fish size and maturity, intensity of parasite infection and stocking density could alter the sensitivity of rainbow trout to SPC treatments.

Fish, like mammals, can be affected by neoplastic proliferations. As yet, there are only a very small number of studies reporting on the occurrence of tumours in koi carp Cyprinus carpio koi and only sporadic reports on the nature of the tumours or on risk factors associated with their development. Between 2008 and 2012, koi with abdominal swelling were examined pathologically: neoplastic lesions were diagnosed and classified histologically. We evaluated possible risk factors for the development of these internal neoplasms in koi carp in Switzerland, using an online 2-part questionnaire sent to fish keepers with koi affected by internal tumours and to fish keepers who had not previously reported any affected koi. Part 1 addressed all participants and focused on general information about koi husbandry and pond technical data; Part 2 addressed participants that had one or several case(s) of koi with internal tumour(s) between 2008 and 2012, and consisted of specific questions about affected koi. A total of 112 internal tumours were reported by the 353 koi keepers participating in the survey. Analysis of the obtained data revealed that tumour occurrence was significantly associated with the location (indoors vs. outdoors) and volume of the pond, frequency of water changes, origin of the koi, number of koi kept in a pond and the use of certain pond disinfectant/medication products. Our results contribute to the identification of possible risk factors, which in turn could help to establish prophylactic measures in order to reduce the occurrence of internal neoplasms in koi.

Proliferative kidney disease (PKD) is an emerging disease threatening wild salmonid populations. In temperature-controlled aquaria, PKD can cause mortality rates of up to 85% in rainbow trout. So far, no data about PKD-related mortality in wild brown trout Salmo trutta fario are available. The aim of this study was to investigate mortality rates and pathology in brown trout kept in a cage within a natural river habitat known to harbor Tetracapsuloides bryosalmonae. Young-of-the-year (YOY) brown trout, free of T. bryosalmonae, were exposed in the River Wutach, in the northeast of Switzerland, during 3 summer months. Samples of wild brown trout caught by electrofishing near the cage location were examined in parallel. The incidence of PKD in cage-exposed animals (69%) was not significantly different to the disease prevalence of wild fish (82 and 80% in the upstream and downstream locations, respectively). The mortality in cage-exposed animals, however, was as low as 15%. At the termination of the exposure experiment, surviving fish showed histological lesions typical for PKD regression, suggesting that many YOY brown trout survive the initial infection. Our results at the River Wutach suggest that PKD in brown trout does not always result in high mortality under natural conditions.

Whirling disease (WD) is an ecologically and economically debilitating disease of rainbow trout Oncorhynchus mykiss caused by the actinosporean spores of the parasite Myxobolus cerebralis. M. cerebralis has a complex, 2-host life cycle alternating between salmonid fish and the oligochaete host Tubifex tubifex. The parasite alternates between 2 spore forms as transmission stages: an actinosporean triactinomyxon spore that is produced in the oligochaete host and a myxosporean spore that develops in the salmonid host. Waterborne triactinomyxon spores released from infected T. tubifex oligochaetes attach to the salmonid host by polar filament extrusion elicited by chemical (nucleoside) and mechanical (thigmotropy) stimuli-a process which is rapidly followed by active penetration of the sporoplasms into the fish epidermis. Upon penetration, sporoplasms multiply and migrate via peripheral nerves and the central nervous system to reach the cartilage where they form trophozoites which undergo further multiplication and subsequent sporogenesis. M. cerebralis myxospores are released into the aquatic environment when infected fish die and autolyse, or when they are consumed and excreted by predators. Myxospores released into the water are ingested by susceptible T. tubifex where they develop intercellularly in the intestine over a period of 3 mo through 4 developmental stages to give rise to mature actinospores. In this article, we review our current understanding of WD-the parasite and its alternate hosts, life cycle and development of the parasite in either host, disease distribution, susceptibility and resistance mechanisms in salmonid host and strategies involved in diagnosis, prevention and control of WD.

Host genetic resistance against disease-causing pathogens can be enhanced through family-based selective breeding. At present, there is an incomplete understanding of how artificial selection of fish alters host physiology and response following pathogen exposure. We previously reported the generation of selectively-bred rainbow trout Oncorhynchus mykiss lines with either increased resistance (ARS-Fp-R) or susceptibility (ARS-Fp-S) to bacterial cold water disease (BCWD). This study (1) determined baseline reference-range intervals for packed cell volume (PCV) and 18 plasma biochemistry analytes, and (2) examined pathophysiological changes following infection between the genetic lines. PCV and biochemistry reference-range intervals did not significantly differ between genetic lines; thus data were pooled into a single reference-range population (n = 85). ARS-Fp-R and ARS-Fp-S line fish were intraperitoneally challenged with Flavobacterium psychrophilum, and plasma was collected on Days 1, 3, 6, and 9 post-challenge. Splenic bacterial load was measured using an F. psychrophilum-specific qPCR assay. In both genetic lines, changes were observed in mean PCV, total protein, albumin, glucose, cholesterol, chloride, and calcium, falling outside the established reference intervals and significantly differing from phosphate-buffered saline challenged fish, on at least 1d post-challenge. Mean PCV, total protein, and calcium significantly differed between ARS-Fp-R and ARS-Fp-S line fish on Day 9 post-infection, with values in the ARS-Fp-S line deviating most from the reference interval. PCV, total protein, cholesterol, and calcium negatively correlated with bacterial load. These findings identify divergent pathophysiological responses between ARS-Fp-R and ARS-Fp-S line fish following laboratory challenge that are likely associated with differential survival.

A series of fungal cases in hatchery-reared juvenile and young adult Siberian sturgeon Acipenser baerii and white sturgeon A. transmontanus occurred at production facilities in Florida and California, USA, respectively. Affected fish exhibited abnormal orientation and/or buoyancy, emaciation, coelomic distension, exophthalmos, cutaneous erythema, and ulcerative skin and eye lesions. Necropsies revealed haemorrhage throughout the coelom, serosanguinous coelomic effusion and organomegaly with nodular or cystic lesions in multiple organs. Fungal hyphae were observed in 27 fish (24 A. baerii and 3 A. transmontanus) via microscopic examination of tissue wet mounts and on slides prepared from colonies grown on culture media. Histopathological examination of these infected tissues revealed extensive infiltration by melanised fungal hyphae that were recovered in culture. Phenotypic characteristics and sequencing of the fungal isolates with the use of the internal transcribed spacer region and 28S rRNA gene confirmed the aetiological agent as Veronaea botryosa. To our knowledge, this is the first documentation of V. botryosa infection in fish, although melanised fungi of the closely related genus Exophiala are well-known pathogens of freshwater and marine fishes.

This report describes a peripheral nerve sheath tumour in 8 European eels Anguilla anguilla L. from a fish farm located in Croatia. The newborn tissue appeared as smooth and soft skin nodules without pronounced colour change. Nodules were dome-shaped with a pale crater and were present on different body areas. In general, nodules were located as series of differently sized protrusions extending along the lateral line on both sides of the fish, as well as sensory canals on the head. Cut sections showed a homogeneous, pale white-grey texture. Histologically, the pathological tissue was located in the dermis, occasionally intruding into the hypodermis, and pushing as a space-occupying mass against the underlying muscle tissue without any evident boundaries. The pressure also caused changes in the overlying epidermis, such as atrophy, spongiosis and erosion. In some areas, the epidermis was 1 cell thick and club and goblet cells had completely disappeared. Ultimately, these changes resulted in shallow ulceration. Tumour tissue was characterized by a scant population of spindle or stellate cells, with oval, hyperchromatic nuclei and pale cytoplasm embedded in a copious myxoid matrix. Cells were arranged in fascicles and whorls, extending in a poorly defined manner among the dermal collagen bundles. Occasionally, adipose cells were also detected, mainly in the central portion of the bulges. Myxoid areas appeared rich in metachromatic and alcianophilic mucous ground substance. Reticular fibres and collagenous connective tissue were scarce. Immunohistochemistry (IHC) using antibodies against S-100 and glial fibrillary acidic protein caused a slight positive reaction in neoplastic dendritic cells. High magnification showed the immunostaining to be cytoplasmic in all tumour cells. IHC with anti-calretinin antibody gave only negative results. Macroscopic, histological, histochemical and immunohistochemical findings were consistent with a diagnosis of multicentric myxoma of the dermal nerve sheaths, a tumour not yet reported in fish.

Sleeping disease is a contagious disease mainly of freshwater farmed rainbow trout, caused by salmonid alphavirus (SAV) Subtype 2. Here we describe the first case in Switzerland. Pathological changes ranged from acute pancreas necrosis to more chronic lesions with complete loss of exocrine pancreas and simultaneous degenerative, inflammatory and regenerative heart and muscle lesions. The partial sequencing of SAV E2 and nsp3 genes placed the Swiss SAV variant within the Subtype 2 clustering together with freshwater isolates from UK and continental Europe. Although mortality stayed low, growth rates were significantly reduced, making the disease economically relevant.

An adult European eel Anguilla anguilla, showing typical signs of the so-called cauliflower disease, was subjected to pathological and molecular virological examinations. Samples taken from internal organs and the polypoid proliferative tissue from the mouth were examined by PCR for the detection of several viruses. Positive results were obtained with a nested PCR targeting the rep gene of circoviruses. Analysis of the partial rep sequence indicated the presence of a putative novel circovirus, but attempts to isolate it remained unsuccessful. The missing part of the genome was acquired by an inverse nested PCR with 2 specific primer pairs, designed from the newly determined rep sequence, followed by genome walking. The circular full genome was found to consist of 1378 nt (GenBank accession no. KC469701). Two oppositely oriented open reading frames (ORFs) were present, of which one was unambiguously identified as a circoviral rep gene. However, the predicted product of the other ORF, though it is a clear positional counterpart of the cap genes, showed no obvious homology to any known circoviral capsid proteins. A stem-loop-like element in the intergenic region between the 5' ends of the ORFs was also found. Phylogenetic calculations indicated that the novel virus belongs to the genus Circovirus in the family Circoviridae. The relative amount of the viral DNA in the organ samples was estimated by quantitative real-time PCR. The results suggested that the examined fish was caught in an active viremic state, although the role of this circovirus in the etiology of the cauliflower diseases could not be ascertained.

The parasitic nematode Philometra floridensis infects the ovary of its only host, the economically important fish species Sciaenops ocellatus, but the factors influencing host susceptibility and potential pathogenic effects are unknown. Here we report new information on these topics from evaluations of infected and uninfected hosts collected from the northeastern Gulf of Mexico. Fish length and age were evaluated vis-à-vis nematode prevalence to check for ontogenetic differences in host susceptibility. To evaluate health and reproductive consequences of infection, we looked for effects in Fulton's condition factor (K) and batch fecundity estimates (BF), and we evaluated ovarian tissue histologically to check for oocyte atresia and other host responses. We observed localized pathological changes in fish ovarian tissue associated with female nematodes, including leucocytic exudates, granulomatous inflammation, and Langhans-type multinucleated giant cells; the hosts, however, appeared to maintain high fecundity and actually exhibited, on average, better health index scores and higher relative fecundity than did uninfected fish. These differences are likely explained by the parasite's tendency to disproportionately infect the largest, actively spawning fish and by the localization of pathogenic changes, which could have masked effects that otherwise would have been reflected in mass-based health indicators. Although we did not detect negative effects on measures of overall health or reproductive output, further research is needed to better elucidate the relationship between these parasites and other factors affecting host reproductive potential, such as egg quality.

Vibrio vulnificus is a potentially zoonotic bacterial pathogen of fish, which can infect humans (causing necrotic fasciitis). We analysed 24 V. vulnificus isolates (from 23 severe eel disease outbreaks in 8 Dutch eel farms during 1996 to 2009, and 1 clinical strain from an eel farmer) for genetic correlation and zoonotic potential. Strains were typed using biotyping and molecular typing by high-throughput multilocus sequence typing (hiMLST) and REP-PCR (Diversilab®). We identified 19 strains of biotype 1 and 5 of biotype 2 (4 from eels, 1 from the eel farmer), that were subdivided into 8 MLST types (ST) according to the international standard method. This is the first report of V. vulnificus biotype 1 outbreaks in Dutch eel farms. Seven of the 8 STs, of unknown zoonotic potential, were newly identified and were deposited in the MLST database. The REP-PCR and the MLST were highly concordant, indicating that the REP-PCR is a useful alternative for MLST. The strains isolated from the farmer and his eels were ST 112, a known potential zoonotic strain. Antimicrobial resistance to cefoxitin was found in most of the V. vulnificus strains, and an increasing resistance to quinolones, trimethoprim + sulphonamide and tetracycline was found over time in strain ST 140. Virulence testing of isolates from diseased eels is recommended, and medical practitioners should be informed about the potential risk of zoonotic infections by V. vulnificus from eels for the prevention of infection especially among high-risk individuals. Additional use of molecular typing methods such as hiMLST and Diversilab® is recommended for epidemiological purposes during V. vulnificus outbreaks.

We examined the occurrence of odontoma in the marine fish Sphyraena jello sourced from 3 different landing centers (Cuddalore, Parangipettai and Nagapattinam) in Tamil Nadu (southeast India). A total of 19783 fishes were examined for odontoma presence, of which 2393 were affected with odontoma. The overall prevalence was 12.1% among the 3 stations. Fish landed at Parangipettai showed the highest peak prevalence of odontoma (16.8%) during the pre-monsoon, followed by Nagapatinam (9.1%) during summer 2011. The tumour lengths in premaxilla, supermaxilla and dentary bone were 1.1-3.6, 1.4-5.9 and 1.2-4.1 cm, respectively, and tumour widths were 0.3-1.9, 0.7-3.1 and 0.5-1.9 cm. Higher prevalence (0.206%) of tongue tumour along with odontoma was observed at Nagapattinam whereas it was lower (0.162%) at Cuddalore. Odontoma histopathology showed dense fibrous tissue with fine teeth roots. TEM analysis showed virus-like particles associated with odontoma. Radiography of the odontoma showed that the tumour masses were bony in nature and tissues were merged with upper and lower jaw.

For several species of fish myxosporeans known at present, the musculature has been designated as the location of intrapiscine development. In the majority of these cases, plasmodia and spores are actually found in the muscle cells, but there are also myxosporeans that select intermuscular connective tissue, fish bones, nerves and capillaries of the musculature as their site of development. During the plasmodial development of Myxobolus, Henneguya and Thelohanellus species in fish inhabiting Hungarian freshwaters, 3 main locations of development inside the muscles were identified. Pseudodispar-type plasmodia, such as M. cyprini, M. musculi and M. pseudodispar, form plasmodia intracellularly in the muscle cells, while the plasmodia of M. pfeifferi, M. sandrae and T. hovorkai develop in the intermuscular connective tissue. A similar development in the connective tissue of the ventricle and the bulbus arteriosus was observed for M. dogieli, a heart parasite found in some cyprinid fishes. The third type of development is represented by M. tauricus, which prefers the collagenous elements of the fin rays, but its plasmodia are commonly found in the muscle attached to the fish bones.

A 7.5 kg common carp Cyprinus carpio presented with prominent localized swelling in the caudal right coelomic area, identified by ultrasound as a fluid filled mass. Fine needle aspirate (FNA) and culture results suggested a sterile seroma. Centesis removed 290 ml of serosanguinous fluid that returned within days. Recheck ultrasonography revealed a solid component within the cavity. Radiography demonstrated irregular lysis and misalignment of vertebrae adjacent to the mass, most suggestive of bacterial osteitis or neoplasia. Treatment with antibiotics followed for 2 mo but failed to resolve the lesion. Repeated radiography and ultrasonography showed progressive enlargement of the mass, with vertebral lysis and invasion characteristic of neoplasia. Ultrasound-guided FNA of the solid component of the mass was non-diagnostic. Euthanasia was elected due to the poor clinical response and primary differential of neoplasia. Post-mortem MRI and CT confirmed a retroperitoneal soft tissue mass, partially surrounded by a fluid-filled cavity, causing vertebral lysis and infiltration of the spinal canal. Expansion of the mass caused severe muscle loss and an associated elevation in creatine kinase (>120000 U l-1). Necropsy results corroborated the MRI and CT findings, revealing a retroperitoneal, multilobular, red and tan mass causing dorsal displacement of the vertebral column, with vertebral lysis, pathologic fracture and invasion of the spinal canal. Histopathologic examination revealed a locally aggressive neoplasm exhibiting multiple patterns of growth, including endothelial lined vascular channels and solid areas formed by more pleomorphic polygonal and spindle cells, consistent with hemangiosarcoma.

Henneguya piaractus and Myxobolus colossomatis (Myxosporea: Myxobolidae) are commonly found in the characid Piaractus mesopotamicus, an important fish farm species in Brazil. This paper describes the prevalence, mean intensity, molecular phylogeny, ultrastructure, and histology of H. piaractus and M. cf. colossomatis found infecting specimens of P. mesopotamicus collected from fish farms in the state of São Paulo, Brazil. A total of 278 fish were collected from 3 fish farms between February 2008 and July 2010. Parasite prevalence and mean intensity varied throughout the study period, and according to location and year. A phylogenetic tree, placing South American species in a global context, showed a clear tendency among myxosporean species to cluster according to host families. Ultrastructural analysis for M. cf. colossomatis showed the plasmodial wall with numerous projections toward host cells and phagocytic activity. Histopathological data showed hyperplasia caused by H. piaractus in highly infected fish. Histological and ultrastructural analysis of H. piaractus showed results similar to those that have previously been reported.

We provide the first description of a virus responsible for a systemic hemorrhagic disease causing high mortality in farmed rice field eels Monopterus albus in China. Typical signs exhibited by the diseased fish were extensive hemorrhages in the skin and viscera and some neurological signs, such as loss of equilibrium and disorganized swimming. Histopathological examination revealed various degrees of necrosis within the spleen and liver. Virus isolation was attempted from visceral tissues of diseased fish by inoculation on 6 fish cell lines. Typical cytopathic effects (CPE) were produced in bluegill fry (BF2) cells, so this cell line was chosen for further isolation and propagation of the virus. Electron microscopy observation showed that the negative stained viral particles had the characteristic bullet shape of rhabdoviruses and an estimated size of 60 × 120 nm. We therefore tentatively refer to this virus as Monopterus albus rhabdovirus (MoARV). Molecular characterization of MoARV, including sequence analysis of the nucleoprotein (N), phosphoprotein (P), and glycoprotein (G) genes, revealed 94.5 to 97.3% amino acid similarity to that of Siniperca chuatsi rhabdovirus. Phylogenetic analysis based on the amino acid sequences of N and G proteins indicated that MoARV should be a member of the genus Vesiculovirus. Koch's postulates were fulfilled by infecting healthy rice field eels with MoARV, which produced an acute infection. RT-PCR analysis demonstrated that MoARV RNA could be detected in both naturally and experimentally infected fish. The data suggest that MoARV was the causative pathogen of the disease.

The aim of the present study was to analyse the occurrence of macroscopically visible ovary alterations in 2 populations of northern pike Esox lucius L. originating from lakes in the Mazurian Lake District (NE Poland). The alterations were characterised by ovary tissue that was morphologically malformed, in part or in whole, and contained immature oocytes, i.e. trophoplastic or previtellogenic oocytes instead of vitellogenic oocytes. These alterations were found only in the ovaries, and no morphological alterations of the testes were noted. Macroscopic and histological analyses were carried out in order to classify the observed alterations in the ovaries. Three types of alterations were identified in which morphological malformations as well as histological investigation of the ovaries were considered. An analysis of the size and age of the fish in relation to the occurrence of alterations as well as of the macroscopic and histological nature of the alteration types was made. The data obtained revealed no lake or age dependency of the observed alterations. Based on the results obtained, we suggest that the presence of endocrine disruptors in the environment or/and genetic factors could be responsible for these kinds of gonad anomalies. However, our results did not allow us to determine the aetiology of the alterations.

Edwardsiella ictaluri, a Gram-negative enteric bacterium, is the known etiological agent of enteric septicemia of catfish. In the last few years, different strains have been implicated as the causative agent of mortality events in cultured fish, including Nile tilapia Oreochromis niloticus L. Due to the emergent nature of edwardsiellosis in non-ictalurid fish, little is known about the dynamics of E. ictaluri infection in tilapia. The purpose of this study was to gain a better understanding of the pathogenesis of edwardsiellosis in tilapia by determining the median lethal and infective doses, tissue targets of infection, rate of bacterial dissemination, and the specific tissue response to E. ictaluri following an immersion challenge with bacterial strains recovered from outbreak events in tilapia. In addition to histopathology assessment, the bacterial burdens in several tissues of infected fish were determined over a 2 wk course of infection using quantitative real-time PCR (qPCR). The collected data suggest the cutaneous and oral routes as the main ports of entry for the organism, which later spreads hematogenously throughout the body. Even though histopathological assessment of infected fish revealed involvement of a wide range of tissues, the severity of the necrotizing and granulomatous lesions in the spleen and head kidney, with concomitant high levels of bacterial DNA in these organs determined by qPCR, identifies them as the main targets of infection.

Proliferative kidney disease (PKD) is a temperature-dependent disease caused by the myxozoan Tetracapsuloides bryosalmonae. It is an emerging threat to wild brown trout Salmo trutta fario populations in Switzerland. Here we examined (1) how PKD prevalence and pathology in young-of-the-year (YOY) brown trout relate to water temperature, (2) whether wild brown trout can completely recover from T. bryosalmonae-induced renal lesions and eliminate T. bryosalmonae over the winter months, and (3) whether this rate and/or extent of the recovery is influenced by concurrent infection. A longitudinal field study on a wild brown trout cohort was conducted over 16 mo. YOY and age 1+ fish were sampled from 7 different field sites with various temperature regimes, and monitored for infection with T. bryosalmonae and the nematode Raphidascaris acus. T. bryosamonae was detectable in brown trout YOY from all sampling sites, with similar renal pathology, independent of water temperature. During winter months, recovery was mainly influenced by the presence or absence of concurrent infection with R. acus larvae. While brown trout without R. acus regenerated completely, concurrently infected brown trout showed incomplete recovery, with chronic renal lesions and incomplete translocation of T. bryosalmonae from the renal interstitium into the tubular lumen. Water temperature seemed to influence complete excretion of T. bryosalmonae, with spores remaining in trout from summer-warm rivers, but absent in trout from summer-cool rivers. In the following summer months, we found PKD infections in 1+ brown trout from all investigated river sites. The pathological lesions indicated a re-infection rather than a proliferation of remaining T. bryosalmonae. However, disease prevalence in 1+ trout was lower than in YOY.

An increasing trend in the prevalence of hyperpigmentation in the common dab Limanda limanda from the North Sea prompted us to investigate the potential role of infectious agents as causes or contributing factors to the condition. Dab representing 3 severity grades of hyperpigmentation were sampled for virology, bacteriology, histopathology and ultrastructure assessments. No cytopathic effect was recorded during virology testing, and bacteriological results showed no differences between normal and hyperpigmented dab. Histopathological assessment showed that the most significant changes occurred in the dermis as a result of chromatophore hyperplasia, namely melanophores and iridophores, alongside loose melanin granules. Dermal lymphocytic infiltration occasionally expanding into the epidermis and the underlying musculature was more frequent in highly pigmented dab than in normal fish, suggesting an active immune response. Ultrastructure studies showed additional disruption of the epithelial layer, with loose melanin granules between cells and a number of single or aggregated melanocytes. Dab representing different grades of hyperpigmentation kept in the laboratory alongside normal fish for a monitoring period of 18 mo showed no changes in their pigment distribution pattern, nor occurrence of new pigment in the normal fish. The current investigation found no association of hyperpigmentation in the common dab with infectious agents; therefore, understanding the cause of the condition remains a challenge which can now more reliably focus on a non-infectious origin hypothesis.

A newly discovered virus, Namao virus, associated with morbidity and mortality, was detected among juvenile lake sturgeon Acipenser fulvescens being propagated by a conservation stocking program for this endangered species in Manitoba, Canada. The outbreaks resulted in cumulative mortalities of 62 to 99.6% among progeny of wild Winnipeg River or Nelson River lake sturgeon and occurred at 2 geographically separate facilities. Namao virus was detected in almost 94% of the moribund or dead lake sturgeon according to a conventional polymerase chain reaction (cPCR) test that is based upon amplification of a 219 bp fragment of the virus major capsid protein (MCP). The virus itself was large (242 to 282 nm) and icosahedral-shaped with 2 capsids and a condensed bar-shaped core. It was found in virus factories within the host cell cytoplasm and displayed a tropism for the integument. Namao virus caused cellular changes characterized by enlarged eosinophilic epithelial cells in the gills and skin. Samples suspected of containing Namao virus did not have cytopathic effects on primary lake sturgeon or established white sturgeon cell lines. However, viral nucleic acid was detected in the former after prolonged incubation periods. Using primers designed from conserved regions of the MCP from NCLDVs, an estimated 95 to 96% of the Namao virus MCP open reading frame was captured. Phylogenetic analysis using the MCP of Namao virus and 27 other NCLDVs suggested that Namao virus and white sturgeon iridovirus share a common evolutionary past and might be members of the family Mimiviridae or a new, as yet unrecognized, virus family.

Juvenile meagre Argyrosomus regius (Asso, 1809) maintained in experimental conditions developed lateral and/or bilateral circular-shaped sarcoma within the opercular cavity. The sarcoma was dense, reddish and its growth from the branchial arch exerted pressure on the operculum forcing it to open. Histologically, the neoplasm exhibited marked proliferation of mesenchymal connective tissue composed largely of fusiform cells, which developed in a solid pattern accompanied by abundant mononuclear cell types. Multifocal areas of discrete necrosis were also observed, compatible with a sarcomatous proliferation. The immunological parameters analysed suggested an inflammatory response. No bacteria were isolated from the hematopoietic organs. However, Vibrio species, components of the normal seawater flora, were isolated from the tumour, which may have had a role in eliciting the immune response. No evidence of viral pathogens was found by electron microscopy. In order to look for cytogenetic alterations often linked to sarcomas, the diploid number and karyotype of this species were determined for the first time. An increase in the aneuploidy level was observed in sarcoma cell metaphase stages compared to other tissues. The aetiology of this tumour remains unknown.

Little is known about the environmental conditions that allow Francisella noatunensis subsp. orientalis, a worldwide emergent bacterial fish pathogen, to colonize and infect wild and cultured fish. We evaluated the effect of temperature and salinity on the infectivity of F. noatunensis subsp. orientalis in Nile tilapia Oreochromis niloticus (L). Immersion challenges of tilapia with F. noatunensis subsp. orientalis at water temperatures of 25 and 30°C in both sea and fresh water were conducted for 14 d. Morbidity and mortality were recorded daily, and at the completion of the study, a quantitative assessment of the splenic bacterial burden was performed in surviving fish. Fish maintained at 25°C developed francisellosis and had considerably higher mortality and splenic bacterial concentrations compared to control fish and fish maintained at 30°C. Moreover, increasing the water temperature from 25 to 30°C prevented the development of clinical signs and mortality in Francisella-challenged fish. In conclusion, temperature significantly influenced the development of francisellosis in tilapia, whereas salinity had no effect. Our findings may be useful in the establishment of improved prophylactic practices and in the management of outbreaks of francisellosis in the aquaculture industry.

Prevalence of the non-native swim bladder nematode Anguillicoloides crassus has recently increased in American eels from estuaries of the North American Atlantic coast, but little is known about parasite prevalence or conditions of previous infection in upstream migrant eels within upper watersheds. This study is the first to confirm presence of A. crassus in the upper Potomac River watershed. We estimated A. crassus prevalence during 3 time periods: September to October 2006 (5/143 eels, 3.5%), August to October 2007 (0/49 eels), and June 2008 (0/50 eels). All eels were sampled from the Millville Dam eel ladder on the lower Shenandoah River, a Potomac River tributary located approximately 285 km upstream of Chesapeake Bay, USA. Of the 5 infected eels, parasite intensity was 1 for each eel, and mean intensity was also 1.0. A swim bladder degenerative index (SDI) was calculated for the 50 eels from the final sampling period, and 38% of those eels (19 of 50) showed signs of previous infection by A. crassus. We also aged 42 of the 50 eels (mean ± SE = 6.7 ± 0.29 yr, range 4 to 11 yr) from the final sampling period. Based on the range of possible SDI scores (0 to 6), severity of previously infected swim bladders was moderate (SDI = 1 or 2). Previously infected eels, however, had a lower length-at-age than that of uninfected eels. Female yellow-phase eels in upper watersheds develop into large highly fecund silver-phase adults; hence, a parasite-induced effect on growth of yellow-phase eels could ultimately reduce reproductive potential.

During a 4 mo epizootic, 100% of 152 lined seahorses Hippocampus erectus in 3 separate groups died while in quarantine following shipment to a public aquarium. Twelve animals with skin depigmentation and ulceration were received by the Aquatic Pathology Service, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA, for diagnostic evaluation. Microscopically, lesions in 11 seahorses included multifocal epithelial necrosis and ulceration associated with 2 to 7 µm diameter, branching, septate fungal hyphae, typically accompanied by deeper infiltration into underlying skeletal muscle. Angioinvasion, with vascular thrombosis and tissue infarction, was a prominent feature in multiple animals. Fungal invasion of one or more internal organs was observed in 4 animals. Hyphae appeared to course freely through tissues and elicited little or no inflammatory response. Fusariosis has been reported sporadically in fish and other aquatic organisms, but identification has often been limited to the genus level based solely on morphologic features. Morphologic characteristics of the fungus isolated from this case were consistent with the Fusarium solani species complex (FSSC), which includes over 50 members that can only be identified definitively using DNA sequence data. A 3-locus typing scheme identified the isolate as a distinct species/haplotype, designated FSSC 12-a, belonging to a specific lineage that appears adapted to aquatic environments and disease in marine animals. Empirical treatment with itraconazole failed to stop mortalities, and subsequent in vitro antifungal susceptibility data explained a lack of clinical efficacy for this agent. Effective treatment in human medicine has similarly been limited by poor susceptibility to several classes of antifungal compounds.