Advanced vacuolation indicates propagation of various salmonid alphavirus type 2 isolates in Acholeplasma-infected BF-2 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.

First confirmation of salmonid alphavirus infection in Arctic char Salvelinus alpinus and in Austria.

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.

Characterization of Austrian koi herpesvirus samples based on the ORF40 region.

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.

Phylogenetic analysis of spring viraemia of carp virus isolates from Austria indicates the existence of at least two subgroups within genogroup Id.

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.

Myxosporidia and macrophage centres in chub (Leuciscus cephalus)--quantitative interactions focus on Myxobolus cyprini.

Six myxosporidian species were found in chub (Leuciscus cephalus) originating from Lower Austrian rivers. The frequency of the parasites and their localization was recorded. In all chub, independent of size and origin, Myxobolus cyprini occurred predominantly in the macrophage centres (MCs) of the haematopoietic organs, spleen and kidney. Exclusively in the head kidney of young fish not yet described vermicular plasmodia containing spores of M. cyprini were found. In muscle tissue the prevalence of M. cyprini was comparatively low. Other species of Myxobolus characterized by plasmodial cysts frequently occurred in gills and swimbladder but were rarely detected, and only in small numbers, in the haematopoietic organs. The number of M. cyprini spores and the relative volume of MCs in the haematopoietic organs were estimated in order to examine possible correlations. Significant interrelated changes were found only in juvenile fish up to a size of 15 cm. In bigger fish, the number and size of macrophage aggregates were highly variable and independent of infection intensity and fish size, but the number of spores never exceeded that of the aggregated macrophages. The data suggest that due to an early date of infection M. cyprini is the only species which is closely associated with macrophage aggregation.