Carp edema virus infection associated gill pathobiome: A case report.

Understanding disease aetiology and pathologic mechanisms is essential for fish health evaluation. Carp edema virus (CEV) is the causative agent of a disease (CEVD) responsible for high mortality rates in both wild and cultured common carp Cyprinus carpio. Inspection of two carp specimens from a pond with high fish mortality revealed CEV infection in both the host and its ectoparasite (Argulus foliaceus). In addition to flavobacteria, well known to be associated with gill lesions, we found that free-living eukaryotes (amoebae and ciliates) and a temporary parasite (Ichthyobodo spp.) colonizing the gills may also contribute to alterations in gill structure and/or function, either directly, through firm (Ichthyobodo) or weak (amoebae) attachment of trophozoites to the gill epithelium, or indirectly, through carriage of pathogenic bacteria. Bacterial assemblages rich in families and genera, with predominance of Cetobacterium spp. in low-intensity alteration of the gill tissue and of Flavobacterium spp. in gills with extensive necrotic lesions, were detected in gills and within the cytoplasm of associated amoebae using high-throughput sequencing. Quantitative PCR indicated F. swingsii as the prevailing flavobacterial species within amoebae from less affected gills and F. psychrophilum within amoebae from extensively affected gills. This case study suggests that eukaryotic organisms as part of the gill pathobiome may also contribute to irreversible gill lesions seen in CEVD. Emphasizing the complexity of mutual relationships between bacterial assemblages and eukaryotic co-pathogens, further studies regarding factors that trigger pathology and influence severity in the CEV-positive carp are needed.

Starter feed for carnivorous species as a practical replacement of bloodworms for a vertebrate model organism in ageing, the turquoise killifish Nothobranchius furzeri.

The absence of a controlled diet is unfortunate in a promising model organism for ageing, the turquoise killifish (Nothobranchius furzeri Jubb, 1971). Currently captive N. furzeri are fed bloodworms but it is not known whether this is an optimal diet. Replacing bloodworms with a practical dry feed would reduce diet variability. In the present study, we estimated the nutritional value of the diet ingested by wild fish and determined the fish-body amino acid profile as a proxy for their nutritional requirements. We compared the performance of fish fed four commercial feeds containing 46%-64% protein to that achieved with bloodworms and that of wild fish. Wild fish target a high-protein (60%) diet and this is supported by their superior performance on high-protein diets in captivity. In contrast, feeds for omnivores led to slower growth, lower fecundity and unnatural liver size. In comparison to wild fish, a bloodworm diet led to lower body condition, overfeeding and male liver enlargement. Out of the four dry feeds tested, the fish fed Aller matched wild fish in body condition and liver size, and was comparable to bloodworms in terms of growth and fecundity. A starter feed for carnivorous species appears to be a practical replacement for bloodworms for N. furzeri. The use of dry feeds improved performance in comparison to bloodworms and thus may contribute to reducing response variability and improving research reproducibility in N. furzeri research.

Histopathology of laboratory-reared Nothobranchius fishes: Mycobacterial infections versus neoplastic lesions.

Short-lived killifishes of the genus Nothobranchius Peters, 1868 (Cyprinodontiformes) are considered promising model organisms for biomedical research on ageing and tumorigenesis. We conducted histopathological analysis of 411 adult individuals from three Nothobranchius species to study details on spontaneous age-related neoplastic lesions. Light microscopy based on H&E and toluidine blue-stained sections revealed (a) non-proliferative liver changes with pronounced vacuolation of hepatocytes; (b) proliferation of kidney haemopoietic tissue contributing to excretory system damage; (c) proliferation of splenic mononuclear haemoblasts accompanied by reduced erythropoiesis; (d) proliferation of mononuclear cell aggregates in the liver parenchyma; and (e) rare occurrence of hepatocellular adenomas. Ziehl-Neelsen (ZN) staining revealed that the proliferative lesions are a host defence response to mycobacterial infections manifested by activation of the mononuclear phagocytic system and atypical granulomatous inflammatory reaction. 16S rRNA analysis identified three species of Mycobacterium in our samples. Our findings turn attention to lesions which mimic neoplasms by their gross appearance and question the light microscopic interpretation of lesions unless differential ZN staining is included. Beyond the limitations of our morphological approach, the intensity of mycobacterial infections is a challenging opportunity for research into the molecular-genetic background of the mononuclear phagocytic system reaction in Nothobranchius killifish.

Spontaneous adenocarcinoma of the gas gland in Nothobranchius fishes.

Nothobranchius fishes (Cyprinodontiformes), known for their genetically encoded extremely compressed lifespan, are considered an excellent vertebrate model for the research of aging. Unlike the rapid accumulation of data concerning their biology, ecology and genome, knowledge of their age-related diseases, including tumours, is still very limited. This Note reports spontaneous neoplastic lesions in the swim bladder gas glands of Nothobranchius furzeri, N. kadleci and N. orthonotus. Based on light and transmission electron microscopy, the neoplastic proliferation of gas gland cells was classified as adenocarcinoma. There was a concurrent proliferation of haemopoietic cells in the kidney interstitium in all individuals diagnosed with this type of primary neoplasia.

Sappinia sp. (Amoebozoa: Thecamoebida) and Rosculus sp. (SAR: Cercozoa) Isolated From King Penguin Guano Collected in the Subantarctic (South Georgia, Salisbury Plain) and their Coexistence in Culture.

Two amoeboid organisms of the genera Sappinia Dangeard, 1896 and Rosculus Hawes, 1963 were identified in a sample containing king penguin guano. This sample, collected in the Subantarctic, enlarges the list of fecal habitats known for the presence of coprophilic amoebae. The two organisms were co-isolated and subcultured for over 6 mo, with continuous efforts being invested to separate each one from the mixed culture. In the mixed culture, Rosculus cells were fast growing, tolerated changes in culturing conditions, formed cysts, and evidently were attracted by Sappinia trophozoites. The separation of the Rosculus strain was accomplished, whereas the Sappinia strain remained intermixed with inseparable Rosculus cells. Sappinia cell populations were sensitive to changes in culturing conditions; they improved with reduction of Rosculus cells in the mixed culture. Thick-walled cysts, reportedly formed by Sappinia species, were not seen. The ultrastructure of both organisms was congruent with the currently accepted generic characteristics; however, some details were remarkable at the species level. Combined with the results of phylogenetic analyses, our findings indicate that the ultrastructure of the glycocalyx and the presence/absence of the Golgi apparatus in differential diagnoses of Sappinia species require a critical re-evaluation.

Neovahlkampfia nana n. sp. Reinforcing an Underrepresented Subclade of Tetramitia, Heterolobosea.

The study provides robust genetic evidence that a newly isolated naked ameba with morphological and ultrastructural features indicative of Heterolobosea is a new species. Neovahlkampfia nana n. sp. associates with the yet underrepresented subclade of Tetramitia I. Considerable differences found in 18S rRNA gene sequences of individual molecular clones derived from DNA of five clonal cultures, using a low fidelity DNA polymerase, raised the issue of intragenomic sequence variation, a phenomenon that has not been previously studied in Heterolobosea. However, as proved using a higher fidelity DNA polymerase, the sequence variability observed was introduced by PCR mediated by the low fidelity polymerase and fixed by molecular cloning. This points to the potentially dubious validity of some current nominal species of Heterolobosea that differ from one another in just one or two base positions.

Copromyxa laresi n. sp. (Amoebozoa: Tubulinea) and Transfer of Cashia limacoides (Page, 1967) to Copromyxa Zopf, 1885.

Five amoeboid organisms of different origin (isolated from fish organs, soil and digestive tract of earthworm) that shared light microscopical and ultrastructural features including type and arrangement of mitochondrial cristae were subjected to phylogenetic analyses based on sequences of SSU rDNA and protein coding genes (actin, cytochrome oxidase I, and eukaryotic elongation factor 2). The reconstruction of multigene phylogeny of the strains studied (i) revealed that they belong to the same single-genus Copromyxa clade; (ii) strongly supported position of Copromyxa cantabrigiensis (syn. Hartmannella cantabrigiensis) within the genus; (iii) together with comparisons of light and electron microscopy data justified reclassification of Cashia limacoides (syn. Vexillifera expectata) to Copromyxa limacoides n. comb., and (iv) justified description of a new species, Copromyxa laresi n. sp.

Pathogenic endoparasites of the spotted seatrout, Cynoscion nebulosus: patterns of infection in estuaries of South Carolina, USA.

Six types of pathogenic endoparasites in an economically important fish, spotted seatrout Cynoscion nebulosus, were studied in order to test whether prevalence of infection and assemblage richness varied with season, host sex, host size, or host age. Fish were collected from South Carolina estuaries, USA, over 12 months (n = 216; total lengths 15-663 mm). They were screened histologically for presence of Henneguya cynoscioni (Myxozoa) and Cardicola spp. (Digenea) in the heart, Kudoa inornata (Myxozoa) in the skeletal muscle, Sinuolinea dimorpha (Myxozoa) in the urinary system, Ichthyophonus sp. (Mesomycetozoea) in the kidney, and an unidentified microsporidian in the liver. Prevalence of infection was 29.8, 38.6, 47.2, 41.2, 13.6, and 2.8%, respectively. All factors had significant, but varying effects on the parasites. Parasite infections were more prevalent in winter than other seasons for Cardicola spp. and H. cynoscioni, more prevalent in winter and spring for Ichthyophonus sp., and more prevalent in male fish than female fish for K. inornata, S. dimorpha, and Ichthyophonus. Prevalence of infection by the three myxosporeans and Cardicola spp. increased with fish length, whereas prevalence of Ichthyophonus increased with length among young fish, but decreased with length among older fish. None of the factors affected the liver microsporidian, although statistical power was low due to its rareness. Assemblage richness varied between 0 and 5, was greater during winter and in male fish, and increased with fish length and fish age. Our results demonstrate that spotted seatrout are commonly co-infected by multiple pathogenic endoparasites, suggesting these parasites likely play an import role in controlling fish population numbers.

Vermistella arctica n. sp. Nominates the Genus Vermistella as a Candidate for Taxon with Bipolar Distribution.

A new amoebozoan species, Vermistella arctica n. sp., is described from marine habitats in the central part of Svalbard archipelago. This is the first report on Arctic amoebae belonging to the genus Vermistella Moran and Anderson, 2007, the type species of which was described from the opposite pole of the planet. Psychrophily proved in the new strains qualifies the genus Vermistella as a bipolar taxon. Molecular phylogenetic analyses based on 18S rDNA and actin sequences did not show any affinity of the genus Vermistella to Stygamoeba regulata ATCC(®) 50892(™) strain. A close phylogenetic relationship was found between Vermistella spp. and a sequence originating from an environmental sample from Cariaco basin, the largest marine permanently anoxic system in the world. Possible mechanisms of bipolar distribution are discussed.

Clinical and Laboratory Parameters of Carp Edema Virus Disease: A Case Report.

In the present study, we describe a natural outbreak of carp edema virus disease (CEVD) in koi carp, concentrating on clinical manifestation, gross and microscopic pathology, immunological parameters, viral diagnostics, and phylogenetic analysis. Examination of white blood cell parameters showed increased monocyte and decreased lymphocyte counts in CEV-affected fish compared to healthy control fish. Regarding immune system functioning, the present work shows, for the first time, enhanced phagocytic activity in CEV-affected fish. Respiratory burst of phagocytes was strongly increased in diseased fish, the increase being attributed to an increased phagocyte count rather than enhancement of their metabolic activity. The present work also newly shows histopathological changes in the pancreatic tissue of diseased koi.

From fish to cells: Establishment of continuous cell lines from embryos of annual killifish Nothobranchius furzeri and N. kadleci.

There is a growing need of alternative experimental models that avoid or minimize the use of animals due to ethical, economical, and scientific reasons. Surprisingly, the stable embryonic cell lines representing Nothobranchius spp., emerging vertebrate models in aging research, regenerative medicine, ecotoxicology, or genomics, have been not derived so far. This paper reports establishment and deep characterization of ten continuous cell lines from annual killifish embryos of N. furzeri and N. kadleci. The established cell lines exhibited mostly fibroblast- and epithelial-like morphology and steady growth rates with cell doubling time ranging from 27 to 40 h. All cell lines retained very similar characteristics even after continuous subcultivation (more than 100 passages) and extended storage in liquid nitrogen (∼3 years). The cytogenetic analysis of the cell lines revealed a diploid chromosome number mostly equal to 38 elements (i.e., the native chromosome count for both killifish species), with minor but diverse line/passage-specific karyotype changes compared to the patterns observed in non-cultured N. furzeri and N. kadleci somatic cells. Based on transcriptional analysis of marker genes, the cell lines displayed features of an undifferentiated state without signs of senescence even in advanced passages. We confirmed that the cell lines are transfectable and can form viable 3-D spheroids. The applicability of the cell lines for (eco)toxicological surveys was confirmed by assessing the effect of cytotoxic and growth inhibitory agents. Properties of established Nothobranchius embryonic cell lines open new possibilities for the application of this model in various fields of life sciences including molecular mechanisms of aging, karyotype (in)stability or differences in lifespan.

New species of Myxosporea from frogs and resurrection of the genus Cystodiscus Lutz, 1889 for species with myxospores in gallbladders of amphibians.

Two new myxosporean species in the gallbladders of frogs have recently spread across eastern Australia and cause disease. Cystodiscus axonis sp. n. and Cystodiscus australis sp. n. are species of Myxosporea (Myxozoa) identified from a range of Australian frogs and tadpoles including the introduced Cane toad (Rhinella marina). The new species are defined by their distinct genetic lineage, myxospore morphology and ultrastructure of the pre-sporogonic development. Spores of both species are produced in the gallbladder. Spores of C. axonis sp. n. possess distinct filiform polar appendages (FPA). The pre-sporogonic development of C. axonis sp. n. is within myelinated axons in the central nervous system of hosts, as well as bile ducts of tadpoles. Pre-sporogonic and sporogonic development of C. australis sp. n. is confined to tadpole bile ducts and myxospores of C. australis sp. n. are devoid of FPA. The genus Cystodiscus Lutz, 1889 introduced for Cystodiscus immersus Lutz, 1889 is emended to accompany myxosporean parasites affecting amphibians previously classified in the genus Myxidium sensu lato. A synopsis of described species within Cystodiscus is provided.

Good Practices for Histological Analysis of the Annual Killifish Nothobranchius furzeri (Nothobranchiidae).

Paraffin histology is one of the most important and commonly used laboratory techniques enabling the study of the microscopic structure of animal and plant tissues. This technique uses paraffin wax, which in liquid form impregnates fixed and dehydrated tissues and allows the preparation of thin sections when solidified in blocks. This protocol on good practices in paraffin histology of Nothobranchius furzeri (Nothobranchiidae) summarizes the authors' current experience in terms of technique, evaluation, and interpretation of sectioned tissues. The steps that precede paraffin block preparation are also presented as they play a key role in maximizing the quality of examined sections. The paraffin technique as described only requires basic laboratory conditions to produce good-quality results. The description of staining methods is limited to Mayer's hematoxylin and eosin (H&E), the routinely used histological dye staining cell nuclei in blue-black (hematein) and cell cytoplasm and connective tissue fibers in shades of pink-red (eosin). Killifish specialists are encouraged to engage in the study of histology and histopathology, taking advantage of interdisciplinary cooperation.

Encephalitozoon cuniculi genotype I as a causative agent of brain abscess in an immunocompetent patient.

A brain abscess caused by Encephalitozoon cuniculi genotype I together with Streptococcus intermedius occurred in a patient without major immunocompromise and with diabetes. The distinguishing clinical signs were hemiparesis and epilepsy. The microsporidium was observed in the abscess aspirate, and its specific DNA was also detected in stool and urine. The patient was successfully treated with albendazole and mebendazole.

Neoparamoeba branchiphila infections in moribund sea urchins Diadema aff. antillarum in Tenerife, Canary Islands, Spain.

A total of 109 sea urchins from 3 species collected in 2 localities off the coast of Tenerife Island, Spain, were examined for the presence of free-living amoebae in their coelomic fluid. Amoeba trophozoites were isolated exclusively from moribund individuals of long-spined sea urchins Diadema aff. antillarum (Philippi) (Echinoidea, Echinodermata) that manifested lesions related to sea urchin bald disease on their tests (16 out of 56 examined). No amoebae were detected in Arbacia lixula (L.) and Paracentrotus lividus (Lamarck). From the former sea urchin species, 8 strains, established from 10 primary isolates, were identified as Neoparamoeba branchiphila Dyková et al., 2005 using morphological and molecular methods. Results of this study (limited to the screening for free-living amoebae) together with data on agents of sea urchin mortalities reported to date justify the hypothesis that free-living amoebae play an opportunistic role in D. aff. antillarum mortality. The enlargement of the dataset of SSU rDNA sequences brought new insight into the phylogeny of Neoparamoeba species.

Xenoma-like formations induced by Soricimyxum fegati (Myxosporea) in three species of shrews (Soricomorpha: Soricidae), including records of new hosts.

In South Bohemia, Czech Republic, 178 shrews, including 98 common shrews, Sorex araneus L., 70 pygmy shrews, Sorex minutus L., and 10 lesser white-toothed shrews, Crocidura suaveolens (Pallas), were examined for Soricimyxum fegati Prunescu, Prunescu, Pucek et Lom, 2007 infections, using squash preparations of unfixed tissues, histological sections and molecular methods. The infection was found in 51 (52%) S. araneus, 14 (20%) S. minutus and 1 (10%) C. suaveolens. The records of the latter two species extend host range of S. fegati. Lesions associated with S. fegati infections in the liver, the organ of specific localisation of the parasite, were found to be induced by proliferative stages migrating toward lumina of bile ducts. In other organs of these three host species, xenoma-like formations (XLFs) were found that severely injured blood vessels. XLFs contained presporogonic stages of S. fegati, whose species identity was evidenced using molecular methods.

Henneguya cynoscioni sp. n. (Myxosporea: Bivalvulida), an agent of severe cardiac lesions in the spotted seatrout, Cynoscion nebulosus (Teleostei: Sciaenidae).

A new myxosporean species, Henneguya cynoscioni sp. n., is described from the spotted seatrout, Cynoscion nebulosus (Cuvier) (Sciaenidae) as a causative agent of cardiac henneguyosis. This new myxosporean species is characterized by the morphology of spores and the sequence of SSU rDNA. Examination of 227 spotted seatrout from four South Carolina estuaries in 2008-2010 revealed a 33.5% total prevalence of H. cynoscioni. Henneguya cynoscioni produces lesions in the bulbus arteriosus, its specific site of infection. The severity of lesions and their impact on the bulbus arteriosus is proportional to the number of plasmodial stages developing in this segment of the heart, being most pronounced in host reaction directed against spores liberated from plasmodia.

Strains of Uronema marinum (Scuticociliatia) co-isolated with amoebae of the genus Neoparamoeba.

Mixed infections of histophagous ciliates and Neoparamoeba spp. Page, 1987 were diagnosed in gill tissue of farmed turbot Psetta maxima (synonym: Scophthalmus maximus) and Atlantic salmon Salmo salar during a study of amoebic gill disease. Ciliates co-isolated from lesions grossly visible on gills of both fish hosts and from 2 species of red algae Lithophyllum racemus and Palmaria palmata were characterized morphologically and by using molecular markers. Sequences of small subunit (SSU) rDNA were determined for 6 strains of ciliates isolated from hosts collected in geographically distant localities. Of these, sequences of 5 strains revealed a surprisingly high level of similarity and identified the corresponding strains with Uronema marinum Lynn et Small, 1997. Thus, the set of environmental sequences of U. marinum available in the GenBank database to date was supplemented with the first sequences of potentially histophagous strains. On the basis of SSU rDNA, the 6th strain, also isolated from affected fish gills, was identified as Aristerostoma sp.

New data on aetiology of nodular gill disease in rainbow trout, Oncorhynchus mykiss.

We studied amoebae associated with nodular gill disease (NGD) outbreaks in rainbow trout Oncorhynchus mykiss (Walbaum) in fish farms in South-Western Germany. Gills of 12 diseased rainbow trout were examined in fresh, by isolation attempts, histologically and using in situ hybridisation (ISH). A total of nine amoeba strains of the genera Acanthamoeba (1), Hartmannella (2), Naegleria (1), Protacanthamoeba (1) and Vannella (4) were isolated and determined using light microscopical, ultrastructural and molecular methods. Specific molecular probes designed from the SSU rDNA sequences of individual amoeba strains were used for non-radioactive ISH in histological sections. Association of Naegleria sp. with NGD and a direct ISH proof of Naegleria trophozoites attached to hyperplastic gill epithelium are novel findings, expanding the number of possible agents of NGD and supporting the hypothesis on multicausal aetiology of this disease.

Good performance of turquoise killifish (Nothobranchius furzeri) on pelleted diet as a step towards husbandry standardization.

Dietary alteration is one of the most universally effective aging interventions, making its standardization a fundamental need for model organisms in aging. In this dietetic study we address the current lack of standardized formulated diet for turquoise killifish Nothobranchius furzeri - a promising model organism. We first demonstrated that N. furzeri can be fully weaned at the onset of puberty onto a commercially available pelleted diet as the sole nutrition when kept in social tanks. We then compared nine somatic and six reproductive parameters between fish fed a typical laboratory diet - frozen chironomid larvae (bloodworms) and fish weaned from bloodworms to BioMar pellets. Both dietary groups had comparable somatic and reproductive performance. There was no difference between diet groups in adult body size, specific growth rate, condition or extent of hepatocellular vacuolation. Fish fed a pelleted diet had higher juvenile body mass and more visceral fat. Pellet-fed males had lower liver mass and possessed a lipid type of hepatocellular vacuolation instead of the prevailing glycogen-like vacuolation in the bloodworm-fed group. No considerable effect was found on reproductive parameters. The negligible differences between dietary groups and good acceptance of pellets indicate their suitability as a useful starting point for the development of standardized diet for Nothobranchius furzeri.