Yersinia ruckeri is a facultative intracellular enterobacterium mostly known as the causative agent of enteric redmouth disease in salmonid fish. In the present study, we applied RNA inhibition to silence twenty pre-selected genes on the genome of a fish cell line (CHSE-214) followed by a gentamicin assay to quantify the effect of silencing on the cells’ susceptibility to infection and found that silencing of 18 out of 20 genes significantly reduced the number of Y. ruckeri recovered. These findings improve our understanding of the infection process by Y. ruckeri and of the interactions between this bacterial pathogen and host cells.

During previous routine inspections of bluegill fry (BF-2) and rainbow trout gonad (RTG-2) cells incubated with organ samples from asymptomatic Arctic char Salvelinus alpinus, brook trout Salvelinus fontinalis, and rainbow trout Oncorhynchus mykiss, a distinctive, reproducible cytopathic effect (CPE) appeared. The striking CPE, involving progressive vacuolation turning into slowly proceeding pyknotic degeneration, was originally attributed exclusively to enhanced growth of Acholeplasma sp. However, at a recent re-examination of re-infected BF-2 cells using electron microscopy (EM), conventional PCR, and quantitative PCR (qPCR), a virus was also detected. Two days post inoculation (dpi), EM revealed characteristic virions inside cytoplasmic vacuoles and next to bacteria outside the cells. The nucleotide sequences of the viral nsP3 gene fragment obtained from supernatants of infected cells were 100% identical and representative for salmonid alphavirus type 2 (SAV 2). The 16S RNA gene (16S rDNA) fragment sequences of the Mollicutes-specific PCR product obtained from SAV-infected as well as virus-free BF-2 control cells were identical with Acholeplasma laidlawii. In addition, qPCR results indicated enhanced propagation of virus and bacteria increasing with vacuolation between 5 and 8 dpi. Advanced vacuolation can be regarded as a CPE of both SAV and A. laidlawii, suggesting a viral impact on the bacterial infection that turns a latent intracellular stage into an apparent degenerative condition.

To date, sleeping disease (SD) caused by salmonid alphavirus 2 (SAV 2) has been reported in freshwater rainbow trout Oncorhynchus mykiss and Atlantic salmon Salmo salar. This study describes for the first time the occurrence of SD in farm-reared Arctic char Salvelinus alpinus and the occurrence of SAV in Austria. Clinical symptoms were indicative of the disease, and the diagnosis was confirmed by histopathology, infectivity in first passages of CHSE-214 cells and PCR. The phylogenetic analysis of the amplified SAV-nonstructural protein-3 (nsP3) fragment revealed the affiliation to the SAV 2 genotype.

Monolayers of different salmon embryo cells were more adherent to cell culture flasks and resistant to bacteria but almost as susceptible to Infectious haematopoietic necrosis virus (IHNV) as epithelial cells. Infection of Chinook salmon embryo ceUs (CHSE-214 cells) with Mycoplasma spp. did not prevent sensitivity to Epizootic haematopoietic necrosis virus (EHNV), Salmon alphavirus (SAV) and distinct Viral haemorrhagic septicaemia virus (VHSV), Infectious pancreatic necrosis virus (IPNV) and Infectious haematopoietic necrosis virus (IHNV) isolates.

IntroductionUp to 2015 cell culture isolation of Infectious Hematopoietic Necrosis Virus (IHNV) was obligatory for the surveillance of the related notifiable fish disease. The EU diagnostic guidelines recommended the use of distinct epithelioid cell lines. The final case of IHN in Austria in 2014 showed an unusual susceptibility of fibroblastoid cells. Reasons for the different affinities of IHNV to various cell lines are suggested.Materials and MethodsIHNV was isolated from farmed rainbow trout. Six cell lines were used for virus cultivation. The impact of adaptation to cell culture on the infectivity of IHNV was assessed by crosswise titration of selected isolates from various cell lines. Infectivity was determined in cell lines grown in normal, glucose-supplemented and diluted culture medium. Enzymatic activities were visualized on API ZYM test strips. The toxicity of mercaptane and the binding of the N-acetylglucosaminespecific lectins Bandeiraea simplicifolia and wheat germ agglutinin (WGA) were inspected microscopically. Mycoplasma were stained with diamidino phenylindole.ResultsThe consequences of IHN infection varied between individual rainbow trout. IHNV has been detected in Austria less often since 1994. For routine diagnostics as well as in annual proficiency tests for comparison of cell line susceptibility IHNV was usually grown only from epithelioid cell lines, including Chinook salmon embryo (CHSE-214). Fibroblastoid cells were refractory to infection. Embryonic salmon cells - combining properties of both cell lines - may mediate unspecific infectivity. The susceptibility of epithelioid cells could be restricted by the addition of D-glucose and elevated in diluted culture medium. IHNV-susceptible fibroblastoid Rainbow trout gonad (RTG-2) cells, infected with Mycoplasma, may regain some resistance in diluted medium. Fibroblastoid cells displayed limited enzymatic activity and a higher resistance to mercaptane. The WGA-binding pattern of IHNV-infected RTG-2 cells resembled virus-specific immunofluorescence and the distribution of DAPI-stained Mycoplasma spp.ConclusionVarying IHNV susceptibilities reflect quantitative differences in enzymatic activities and contents of N-acetylglucosamine. High mercaptane tolerance of fibroblastoid cells with lower sugar demands may indicate a distinct glycoprotein with abundant sulfur bridges, which normally restricts a progressive virus infection, and massive bacterial sugar degradation. Immediate vicinity of Mycoplasma on RTG-2 cells that bind WGA and IHNV antibodies suggests that IHNV susceptibility is mediated by microbes. An osmotic imbalance my result from Mycoplasma breaking disulfide bonds and making sugar binding sites available and might lead to IHNV susceptibility and pathogenesis.

During a fish health inspection in the Viennese waterway 'Old Danube', a virus was isolated exclusively from white bream Blicca bjoerkna (L.) (formerly Abramis bjoerkna L.), one of the most abundant cyprinids present and not known as a host species for this virus. The virus preferentially replicated in cultures of the epithelioma papulosum cyprini cell line where focal plaques of infection developed slowly. Examination of infected cell cultures by electron microscopy revealed non-enveloped 60 to 70 nm icosahedral virions that had characteristic multiple segregated protrusions of their outer capsid. A partial RNA-dependent RNA polymerase gene sequence was obtained and a BLAST search indicated 76% identity to golden shiner reovirus and grass carp reovirus. These results suggested that the virus belonged to the genus Aquareovirus (Family Reoviridae). Phylogenetic analysis placed the isolated virus within a clade of the species Aquareovirus C species. Accordingly, the virus was tentatively designated as white bream reovirus (WBRV) strain A-127/06 within the species Aquareovirus C.

Spring viraemia of carp (SVC) is a viral disease that mainly affects carp Cyprinus carpio and other cyprinid fish, causing severe economic losses. Rapid detection and identification of spring viraemia of carp virus (SVCV) is crucial for effective disease management. Recent advances in nanoscience are having a significant impact on many scientific fields, especially biodiagnostics, where a number of nanoparticle-based assays have been introduced for biomolecular detection. Single- and double-stranded oligonucleotides can be adsorbed on gold nanoparticles (AuNPs) in colloidal solution under certain conditions. We exploited this phenomenon to develop a specific hybridization assay for direct detection of SVCV-RNA without prior amplification. The result of the hybridization process could be detected visually within 1 min when the colour of the reaction mixture changed from red to blue (positive reaction) or remains red (negative). The lower detection limit of the assay was estimated to be 10-3 TCID50 ml-1 SVCV-RNA, and it has the feasibility to detect the target virus-RNA in clinical specimens without previous amplification. In order to obtain an indication of the assay's performance on clinical samples we compared the optimized assay with nested RT-PCR in detection of SVCV-RNA in infected fish samples. The concordance of the 2 methods was defined as 100% when compared to nested RT-PCR positive and negative samples. The SVC-AuNPs assay requires only 15 min, eliminates the need for thermal cycling or detection instruments and is a specific and rapid tool for detection of SVCV-RNA directly from clinical samples.

Using a PCR that amplifies a region of the thymidine kinase (TK) gene, an epidemic spread of koi herpesvirus (KHV) was determined in koi carps in Austria in 2007. A total of 15 virus samples from different locations in Austria were analyzed to determine their genetic relatedness following PCR and nucleic acid sequencing of the open reading frame 40 (ORF40) region of the KHV genome. ORF40-specific PCR amplification products that were obtained from tissue samples shared 100% nucleotide sequence identity with the published sequence of the Japanese strain of KHV. The ORF40 sequence of one isolate from the UK that was included in the present study was 100% identical with the published sequence of an Israeli strain of KHV. This is the first study that used a larger number of samples and a PCR method, which allowed distinguishing all 3 strains of KHV. The present investigation provides information on the epidemiology of KHV infections in Europe and describes a useful molecular tool for epidemiological studies.

Genetic relationships between 22 spring viraemia of carp virus (SVCV) isolates from Austria collected between 1994 and 2007 were determined based on the partial nucleotide sequence of the glycoprotein gene (G gene). Phylogenetic analyses located all Austrian isolates except one in genogroup Id. One isolate collected in 2007 was placed within the SVCV Ia genogroup. More importantly, the study also revealed 3 distinct clusters within genogroup Id, designated Id1, Id2 and Id3. Existence of subgroups Id2 and Id3 within the genogroup Id was supported by high bootstrap values. The genetic clustering could neither be linked to host species nor to geographic localization of fish farms. Furthermore, no clear link could be established between the pathological lesions and phylogenetic relationship. However, time-dependent division of the isolates was observed. Viruses from the Id1 cluster were mainly sampled in Austria in the 1990s and up until 2003, whereas all viruses from the Id2 subgroup were isolated after 2003.

29 Infectious hematopoietic necrosis virus (IHNV) is one of the most important pathogens of 30 salmonid fish. In this study a comprehensive phylogenetic analysis of the genetic evolution 31 and variety of Austrian IHNV strains as well as selected strains ensuring worldwide coverage 32 is presented. The phylogenetic investigation is based on sequences comprising the “mid G” 33 region of the G gene, and it includes all to-date available IHNV sequences of the G gene with 34 a length of at least 615 bp. Austrian IHN viruses are located together with other European 35 IHNV isolates in two clusters of genogroup M (M-Eur1 and M-Eur2), and are clearly 36 separated from American and Asian lineages. The genetic clustering, however, could not be 37 linked to certain clinical symptoms or significant differences in the mortality rates. 38

ABSTRACT Infectious hematopoietic necrosis virus (IHNV) is one of the most important pathogens of salmonid fish. In this study a comprehensive phylogenetic analysis of the genetic evolution and variety of Austrian IHNV strains, as well as selected strains ensuring worldwide coverage, is presented. The phylogenetic investigation is based on sequences comprising the “mid-G” region of the G gene, and it includes all currently available IHNV sequences of the G gene with a length of at least 615 bp. Austrian IHNVs are located—together with other European IHNV isolates—in two clusters of genogroup M (M-Eur1 and M-Eur2) and are clearly separated from American and Asian lineages. The genetic clustering, however, could not be linked to certain clinical symptoms or significant differences in the mortality rates.

Between April and November 2003, parasitological examinations of the nase Chondrostoma nasus L. and the chub Leuciscus cephalus L. from the neighbouring Melk and Pielach rivers in Lower Austria were conducted. Amongst various gill parasites, Lamproglena pulchella Nordmann 1832 was detected on both fish species, which was the first record of this parasitic crustacean in Austria. Physico-chemical examinations of water samples of the two rivers were carried out during the same period. The results indicate that general water parameters in the Melk were subjected to more vigorous changes than in the Pielach. Critical temperature levels and ammonia concentration as well as drastic changes in the ionic composition occurred more frequently in the Melk River. The observed distribution of L. pulchella indicates its sensitivity to such stress factors: there was no evidence of the parasite in the Melk River until late November and it only then occurred on the gills of L. cephalus with a prevalence of 20% and a mean intensity of 2. In the Pielach River, infestation on chub had already occurred in June with a prevalence of 40% and a mean intensity of 3, rising to 60% and 7 in November; 45% of the nase was also infested in November at a mean intensity of 3.

Of 150 wild stock chub, Leuciscus cephalus L, captured in Lower Austrian watercourses, 112 revealed disc like plasmodia of Myxobolus cycloides Gurley, 1893 on the caudal chamber of the swim bladder. Other cyprinid species from the same waters lacked M. cycloides or other myxosporeans in this specific localisation, In chub, the intensity of infection (number of discs on the swim bladder) showed a logarithmic, age-dependent increase, The plasmodia of M cycloides were situated in the connective tissue-mainly along blood vessels-and exhibited a delicate envelope of host tissue, thus forming a characteristic myxosporean cyst. Occasionally single trophozoites seemed to merge. A general process of fibroblast proliferation leading to encapsulation and degradation of the parasite was observed, This process was initiated by the formation of small multiple encapsulations within the spore containing trophozoid, before thickening of the outer cyst wall occurred. The general noninflammatory course of the M. cycloides infection, and the obvious good health of the investigated chub suggest that this myxosporean in its host specific localisation cannot be regarded as a serious pathogen-on the contrary: parasite multiplication and degradation seemed to occur in a well-defined equilibrium controlled by the host fish.