The myxozoan Tetracapsuloides bryosalmonae is the causative agent of Proliferative Kidney Disease (PKD) targeting primarily the kidney of infected fish where it causes a chronic lymphoid immunopathology. Although known to be associated with suppression of some cellular aspects of innate immunity and a prominent lymphocytic hyperplasia, there remains a considerable knowledge gap in our understanding of the underlying immune mechanisms driving PKD pathogenesis. To provide further insights, the expression profiles of a panel of innate / inflammatory and adaptive immune molecules were examined in rainbow trout Oncorhynchus mykiss following a natural exposure to the parasite. Relative to controls, fish with early to advanced stages of kidney pathology exhibited up-regulation of the inflammatory cytokines interleukin (IL)-6 and IL-11, although remaining refractory towards genes indicative of macrophage activity. Antimicrobial peptides (AMPs) and anti-inflammatory markers, including cathelicidin (CATH) and IL-10 were markedly up-regulated during clinical disease. Up-regulation of adaptive immune molecules, including cell markers and antibody genes reflect the lymphocytic dominance of this disease and the likely importance of lymphocyte subsets in PKD pathogenesis. Up-regulation of T helper (TH) cell-like response genes and transcription factors implies that T. bryosalmonae may elicit a complex interplay between TH cell subsets. This work, for the first time in the study of fish-myxozoan interactions, suggests that PKD pathogenesis is shaped by an anti-inflammatory phenotype, a profound B cell / antibody response and dysregulated TH cell-like activities. A better understanding of the functional roles of fish immune cells and molecules in PKD pathogenesis may facilitate future development of control measures against this disease.

Koi sleepy disease (KSD), also known as carp edema virus (CEV), was first reported from juvenile colour carp in Japan in the 1970s. Recently, this pox virus was detected in several European countries, including Germany, France and the Netherlands. In England, in addition to colour carp, outbreaks in common carp are reported. KSD/CEV is an emerging infectious disease characterized by a typical sleepy behaviour, enophthalmia, generalized oedematous condition and gill necrosis, leading to hypoxia. High mortality, of up to 80-100%, is seen in juvenile koi collected from infected ponds. In Austria, this disease had not been detected until now. In spring 2014, diagnostic work revealed the disease in two unrelated cases. In one instance, a pond with adult koi was affected; in the other, the disease was diagnosed in adult common carp recently imported from the Czech Republic. A survey was carried out on recent cases (2013/2014), chosen from those with similar anamnestic and physical examination findings, revealing a total of 5/22 cases positive for KSD/CEV. In this study, two paradigmatic cases are presented in detail. Results together with molecular evidence shaped the pattern of the first diagnosis of KSD/CEV in fish from Austrian ponds. In the light of the positive cases detected from archived material, and the spread of the disease through live stock, imported from a neighbouring country, the need for epidemiological investigations in Austria and surrounding countries is emphasized.

Fish type I IFNs are classified into two groups with two (group I) or four (group II) cysteines in the mature peptide and can be further divided into four subgroups, termed IFN-a, -b, -c, and -d. Salmonids possess all four subgroups, whereas other teleost species have one or more but not all groups. In this study, we have discovered two further subgroups (IFN-e and -f) in rainbow trout Oncorhynchus mykiss and analyzed the expression of all six subgroups in rainbow trout and brown trout Salmo trutta. In rainbow trout RTG-2 and RTS-11 cells, polyinosinic-polycytidylic acid stimulation resulted in early activation of IFN-d, whereas the IFN-e subgroup containing the highest number of members showed weak induction. In contrast with the cell lines, remarkable induction of IFN-a, -b, and -c was detected in primary head kidney leukocytes after polyinosinic-polycytidylic acid treatment, whereas a moderate increase of IFNs was observed after stimulation with resiquimod. Infection of brown trout with hemorrhagic septicemia virus resulted in early induction of IFN-d, -e, and -f and a marked increase of IFN-b and IFN-c expression in kidney and spleen. IFN transcripts were found to be strongly correlated with the viral burden and with marker genes of the IFN antiviral cascade. The results demonstrate that the IFN system of salmonids is far more complex than previously realized, and in-depth research is required to fully understand its regulation and function.

The intracellular suppressors of cytokine signaling (SOCS) family members, including CISH and SOCS1 to 7 in mammals, are important regulators of cytokine signaling pathways. So far, the orthologues of all the eight mammalian SOCS members have been identified in fish, with several of them having multiple copies. Whilst fish CISH, SOCS3, and SOCS5 paralogues are possibly the result of the fish-specific whole genome duplication event, gene duplication or lineage-specific genome duplication may also contribute to some paralogues, as with the three trout SOCS2s and three zebrafish SOCS5s. Fish SOCS genes are broadly expressed and also show species-specific expression patterns. They can be upregulated by cytokines, such as IFN-γ, TNF-α, IL-1β, IL-6, and IL-21, by immune stimulants such as LPS, poly I:C, and PMA, as well as by viral, bacterial, and parasitic infections in member- and species-dependent manners. Initial functional studies demonstrate conserved mechanisms of fish SOCS action via JAK/STAT pathways.

The recently discovered Tilapia parvovirus (TiPV) was the first Parvovirus confirmed to infect fish, causing mortality outbreaks in farmed adult Nile tilapia in China. Severe mortality outbreaks caused by Tilapia tilapinevirus (TiLV) to farmed tilapia in Thailand revealed the concomitant occurrence of TiPV. Out of ten fish farms screened, TiPV was detected in one site rearing juvenile red hybrid tilapia. Clinical signs included abnormal swimming, scale protrusion, skin and muscle haemorrhaging, exophthalmia and generalized anaemia. Histological findings showed extensive infiltration of lymphocytes, with increased melanomacrophage centres in the anterior kidney and spleen, erythrocyte depletion in the spleen and hepatic syncytial cells. Both TiLV and TiPV were systemically distributed in the body of moribund fish. The analysis of the near-complete TiPV genome isolated from Thailand revealed 98.74% sequence identity to the formerly isolated from China, together with a highly conserved and comparable genomic organization and with a 3 nucleotides deletion in the 5-UTR.The viral genome structure was highly conserved for each of its components, with nucleotide and amino acid identity ranging from 100% for ORF1 to 97% for ORF2, and with conserved HuH and Walker loop motifs within NS1. Taken together, our results document the first detection of TiPV outside China, thus for the first time in Thailand. Moreover, TiPV was detected for the first time during a natural occurrence in farmed red hybrid tilapia and involved in co-infection pattern with TiLV. Diagnostic investigations during tilapia disease outbreaks should include the screening for TiPV.Further studies are needed to elucidate TiPV genomic variance, pathobiology, including focussing on the outcomes of TiLV-TiPV co-infection patterns, necessary to enable risk assessment for the worldwide spreading of TiPV and to design adequate control measures against these emerging viruses in tilapia.

Tilapia lake virus (TiLV) causes an emerging viral disease associated with high mortality and economic damage in tilapia farming around the world. The use of probiotics in aquaculture has been suggested as an alternative to antibiotics and drugs to reduce the negative impact of bacterial and viral infections. In this study, we investigate the effect of probiotic Bacillus spp. supplementation on mortality, viral load, and expression of immune-related genes in red hybrid tilapia (Oreochromis spp.) upon TiLV infection. Fish were divided into three groups, and fed with: control diet, 0.5% probiotics-supplemented diet, and 1% probiotics-supplemented diet. After 21 days of experimental feeding, the three groups were infected with TiLV and monitored for mortality and growth performances, while organs were sampled at different time points to measure viral load and the transcription modulation of immune response markers. No significant difference was found among the groups in terms of weight gain (WG), average daily gain (ADG), feed efficiency (FE), or feed conversion ratio (FCR). A lower cumulative mortality was retrieved from fish fed 0.5% and 1% probiotics (25% and 24%, respectively), compared to the control group (32%). Moreover, fish fed with 1% probiotic diet had a significantly lower viral load, than those fed with 0.5% probiotic and control diet at 5, 6, 9, and 12 days post infection-challenge (dpc). The expression patterns of immune-related genes, including il-8 (also known as CXCL8), ifn-γ, irf-3, mx, rsad-2 (also known as VIPERIN) showed significant upregulation upon probiotic treatment during the peak of TiLV pathogenesis (between 9 and 12 dpc) and during most of the study period in fish fed with 1% probiotics-supplemented diet. Taken together, these findings indicate that dietary supplementation using Bacillus spp. probiotics may have beneficial effects to strengthen tilapia immunity and resistance against TiLV infections. Therefore, probiotic treatments may be preventively administered to reduce losses caused by this emerging viral infection in tilapia aquaculture.

Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish rhabdovirus found in discrete locales throughout the Northern Hemisphere. VHSV infection of fish cells leads to upregulation of the host's virus detection response, but the virus quickly suppresses interferon (IFN) production and antiviral gene expression. By systematically screening each of the six VHSV structural and nonstructural genes, we identified matrix protein (M) as the virus' most potent antihost protein. Only M of VHSV genotype IV sublineage b (VHSV-IVb) suppressed mitochondrial antiviral signaling protein (MAVS) and type I IFN-induced gene expression in a dose-dependent manner. M also suppressed the constitutively active simian virus 40 (SV40) promoter and globally decreased cellular RNA levels. Chromatin immunoprecipitation (ChIP) studies illustrated that M inhibited RNA polymerase II (RNAP II) recruitment to gene promoters and decreased RNAP II C-terminal domain (CTD) Ser2 phosphorylation during VHSV infection. However, transcription directed by RNAP I to III was suppressed by M. To identify regions of functional importance, M proteins from a variety of VHSV strains were tested in cell-based transcriptional inhibition assays. M of a particular VHSV-Ia strain, F1, was significantly less potent than IVb M at inhibiting SV40/ luciferase (Luc) expression yet differed by just 4 amino acids. Mutation of D62 to alanine alone, or in combination with an E181-to-alanine mutation (D62A E181A), dramatically reduced the ability of IVb M to suppress host transcription. Introducing either M D62A or D62A E181A mutations into VHSV-IVb via reverse genetics resulted in viruses that replicated efficiently but exhibited less cytotoxicity and reduced antitranscriptional activities, implicating M as a primary regulator of cytopathicity and host transcriptional suppression.IMPORTANCE Viruses must suppress host antiviral responses to replicate and spread between hosts. In these studies, we identified the matrix protein of the deadly fish novirhabdovirus VHSV as a critical mediator of host suppression during infection. Our studies indicated that M alone could block cellular gene expression at very low expression levels. We identified several subtle mutations in M that were less potent at suppressing host transcription. When these mutations were engineered back into recombinant viruses, the resulting viruses replicated well but elicited less toxicity in infected cells and activated host innate immune responses more robustly. These data demonstrated that VHSV M plays an important role in mediating both virusinduced cell toxicity and viral replication. Our data suggest that its roles in these two processes can be separated to design effective attenuated viruses for vaccine candidates.

Proliferative Kidney Disease (PKD) was diagnosed in juvenile autochthonous brown trout Salmo trutta for the first time in Austria during summer 2014. Cytology showed Tetracapsuloides bryosalmonae sporoblasts, and histology revealed sporogonic (coelozoic) and extrasporogonic (histozoic) stages. Analysis of malacosporean ribosomal small subunit revealed that this strain is closely related to European isolates, although its source is unknown. Infection and high pathogenicity were reproduced upon a pre-restocking test with specific pathogen free (SPF) juvenile trout, resulting in 100% mortality between 28 and 46 d post exposure (dpe), with high ectoparasitosis. Fish showed grade 2 of the Kidney Swelling Index and grade 3 of the PKD histological assessment. T. bryosalmonae enzootic waters were demonstrated in further locations along the River Kamp, with infected bryozoans retrieved up to 6 km upstream of the farm with the PKD outbreak. Fredericella sultana colonies collected from these locations were cultivated in laboratory conditions. Released malacospores successfully induced PKD, and contextually Black Trout Syndrome (BTS), in SPF brown trout. In the absence of co-infections mortality occurred between 59 and 98 dpe, with kidneys enlarged up to 6.74% of total body weight (normal 1.23%). This study confirms the first isolation of a pathogenic myxozoan from an Austrian river tributary of the Danube, where its 2-host life cycle is fully occurring. Its immunosuppressant action could link PKD as a key factor in the multifactorial aetiology of BTS. This T. bryosalmonae isolation provides an impetus to undertake further multi-disciplinary research, aiming to assess the impact of PKD and BTS spreading to central European regions.