Multipathogen infections and multifactorial pathogenesis involved in noble pen shell (Pinna nobilis) mass mortality events: Background and current pathologic approaches.

Disease outbreaks in several ecologically or commercially important invertebrate marine species have been reported in recent years all over the world. Mass mortality events (MMEs) have affected the noble pen shell (Pinna nobilis), causing its near extinction. Our knowledge of the dynamics of diseases affecting this species is still unclear. Early studies investigating the causative etiological agent focused on a novel protozoan parasite, Haplosporidium pinnae, although further investigations suggested that concurrent polymicrobial infections could have been pivotal in some MMEs, even in the absence of H. pinnae. Indeed, moribund specimens collected during MMEs in Italy, Greece, and Spain demonstrated the presence of a bacteria from within the Mycobacterium simiae complex and, in some cases, species similar to Vibrio mediterranei. The diagnostic processes used for investigation of MMEs are still not standardized and require the expertise of veterinary and para-veterinary pathologists, who could simultaneously evaluate a variety of factors, from clinical signs to environmental conditions. Here, we review the available literature on mortality events in P. nobilis and discuss approaches to define MMEs in P. nobilis. The proposed consensus approach should form the basis for establishing a foundation for future studies aimed at preserving populations in the wild.

The efficacy of new oral vaccine feeds against Salmonid novirhabdovirus in rainbow trout.

Salmonid novirhabdovirus (IHNV) causes infectious haematopoietic necrosis (IHN) in salmonid species. Despite an injectable plasmid-based DNA vaccine of the glycoprotein (G) gene is effective, there are no oral vaccines for mass vaccination of rainbow trout (Oncorhynchus mykiss) fry. Recombinant baculoviruses were generated, used in cabbage looper (Trichoplusia ni) insect larvae to produce IHNV G and IHNV G-C5a proteins. Western blotting and chemiluminescence assays confirmed the expression of recombinant proteins, which were added to the fish feeding and top-coated with unflavored gelatin binder. Commercial rainbow trout were fed with experimental diets containing either IHNV G or IHNV G-C5a proteins for 2 weeks, and boosted 4 weeks after. Four weeks post-booster, fish were challenged with IHNV by immersion. Survival upon the infection challenge was evaluated. Spleen were sampled at 7 and 14 days post infection (dpi). Non-vaccinated and IHNV G fed trout reached a mortality of 91.7 and 97.6%, and 70.9 and 88.4%, respectively at 8 and 15 dpi. The IHNV G-C5a fed group exhibited a reduced mortality of 51.2% at 8 dpi, reaching 81.7% at 15 dpi, suggesting some level of antiviral protection. The individual viral load was measured by RT-qPCR detection of IHNV N gene, showing no significant difference across experimental groups. The transcription modulation of selected immune response markers was evaluated across experimental groups, including Type I IFN-a, Mx-1, CD4, and IgM. Further study is needed to assess how new oral vaccines may become effective to mitigate IHNV pathogenesis in juvenile trout by modulating the host immune response to protect towards IHNV exposure.

Uncovering the first occurrence of Tilapia parvovirus in Thailand in tilapia during co-infection with Tilapia tilapinevirus.

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.

Genomic and immunogenic changes of Piscine novirhabdovirus (Viral Hemorrhagic Septicemia Virus) over its evolutionary history in the Laurentian Great Lakes.

A unique and highly virulent subgenogroup (-IVb) of Piscine novirhabdovirus, also known as Viral Hemorrhagic Septicemia Virus (VHSV), suddenly appeared in the Laurentian Great Lakes, causing large mortality outbreaks in 2005 and 2006, and affecting >32 freshwater fish species. Periods of apparent dormancy have punctuated smaller and more geographically-restricted outbreaks in 2007, 2008, and 2017. In this study, we conduct the largest whole genome sequencing analysis of VHSV-IVb to date, evaluating its evolutionary changes from 48 isolates in relation to immunogenicity in cell culture. Our investigation compares genomic and genetic variation, selection, and rates of sequence changes in VHSV-IVb, in relation to other VHSV genogroups (VHSV-I, VHSV-II, VHSV-III, and VHSV-IVa) and with other Novirhabdoviruses. Results show that the VHSV-IVb isolates we sequenced contain 253 SNPs (2.3% of the total 11,158 nucleotides) across their entire genomes, with 85 (33.6%) of them being non-synonymous. The most substitutions occurred in the non-coding region (NCDS; 4.3%), followed by the Nv- (3.8%), and M- (2.8%) genes. Proportionally more M-gene substitutions encoded amino acid changes (52.9%), followed by the Nv- (50.0%), G- (48.6%), N- (35.7%) and L- (23.1%) genes. Among VHSV genogroups and subgenogroups, VHSV-IVa from the northeastern Pacific Ocean has shown the fastest substitution rate (2.01x10-3), followed by VHSV-IVb (6.64x10-5) and by the VHSV-I, -II and-III genogroups from Europe (4.09x10-5). A 2016 gizzard shad (Dorosoma cepedianum) from Lake Erie possessed the most divergent VHSV-IVb sequence. The in vitro immunogenicity analysis of that sample displayed reduced virulence (as did the other samples from 2016), in comparison to the original VHSV-IVb isolate (which had been traced back to 2003, as an origin date). The 2016 isolates that we tested induced milder impacts on fish host cell innate antiviral responses, suggesting altered phenotypic effects. In conclusion, our overall findings indicate that VHSV-IVb has undergone continued sequence change and a trend to lower virulence over its evolutionary history (2003 through present-day), which may facilitate its long-term persistence in fish host populations.

Probiotics Modulate Tilapia Resistance and Immune Response against Tilapia Lake Virus Infection.

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.

Comparative effects of Novirhabdovirus genes on modulating constitutive transcription and innate antiviral responses, in different teleost host cell types.

BACKGROUND: Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are highly contagious, pathogenic Novirhabdoviruses affecting fish and are thusly notifiable diseases with the World Organization for Animal Health. This study assessed the relative capacities of IHNV and VHSV genes to modulate host general transcription and explores the abilities of specific IHNV genes to interfere with the interferon pathway in heterogenous teleost cell-lines. METHODS: Optimized protocols allowed for efficient transient transfections in EPC, BF-2, RTG-2 and RTgill-W1 cell lines of plasmids encoding IHNV (M genogroup) and VHSV (-IVb genotype) genes, including N, P, M, G and NV. Their impact on general cellular transcription was measured 48 hours post transfection (hpt) with luciferase constructs driven by a modified ß-Actin promoter (pCAG). Their modulation of the innate antiviral immune response was characterized 72 hpt, using luciferase constructs measuring rainbow trout Type I IFN or MX-1 promoter augmentation, upon MAVS co-transfection. RESULTS: M was generally confirmed as the strongest constitutive transcriptional suppressor while IHNV P, but not VHSV P, augmented constitutive transcription in fibroblastic cell types. Cell-specific effects were observed for viral G gene, with VHSV G exhibiting suppression of basal transcription in EPC and BF-2 but not in trout cells; while IHNV G was stimulatory in RTG-2, but inhibitory in RTgill-W1. NV consistently stimulated constitutive transcription, with higher augmentation patterns seen in fibroblastic compared to epithelial cells, and for IHNV NV compared to VHSV NV. The innate antiviral immune response, focusing on the IFN pathway, was silenced by IHNV M in all cell lines tested. IHNV N showed a dose-dependent suppression of type I IFN, but with minor effects on MX-1. IHNV P and G played minor IFN-inhibitory roles, consistent and dose-dependent only for G in rainbow trout cells. IHNV NV mediated a consistent stimulatory effect on either Type I IFN or MX-1, but much less pronounced in RTgill-W1. CONCLUSIONS: This study extends our understanding of Novirhabdoviruses-host interaction, showing differential innate immune responses in heterogenous cell types. Viral regulators of innate immune signaling are identified, either as dose-dependent suppressors (such as M and N) or stimulators (mainly NV), indicating novel targets for the design of more efficient vaccination strategies.

Proliferative kidney disease in Alaskan salmonids with evidence that pathogenic myxozoans may be emerging north.

Proliferative kidney disease (PKD) of salmonids, a chronic immunopathology caused by the myxozoan parasite Tetracapsuloides bryosalmonae, is exacerbated by increased water temperatures. PKD causes economic concerns to trout farmers and contributes to the decline of wild salmonid populations in North America and Europe. The parasite occurs as far north as Norway and Iceland in Europe and was confirmed from California to southern British Columbia in the American continent. In mid-September 2011 adult chum salmon (Oncorhynchus keta) were sampled from Kantishna River, a tributary to Yukon River in Alaska. Clinical PKD was diagnosed based on the macroscopic appearance of mottled kidneys that were uniformly swollen and by the detection of tumultuous histozoic extrasporogonic and coelozoic sporogonic stages of T. bryosalmonae in renal tissue by histopathology. Archived samples provided the molecular confirmation and local strain identification, representing the first confirmed case of PKD in wild adult chum salmon, also co-infected with Parvicapsula minibicornis that represents another novel myxozoan detection in Alaska. Our investigation was extended to another case from August/September 1997, with mortality following furunculosis and ectoparasite co-infections, in sockeye salmon (Oncorhynchus nerka) pre-smolts net-pen reared in English Bay Lakes, Alaska. Immunohistochemistry on archived histological preparations confirmed T. bryosalmonae sporogonic and extrasporogonic stages, indicating a severe to resolving PKD, with concomitant Chloromyxum spp. infection. Those cases provide the first documentation that this parasite is present in Alaska and causes PKD in wild and cultured salmonids in the region. The known geographic range of T. bryosalmonae can be extended to ~267 km south of the Arctic Circle, representing the northernmost detection in America. Given the vast size of Alaska and small resident population, it is likely that T. bryosalmonae remained undetected, but more recently became evident due to the clinical manifestation of PKD, possibly linked to increasing water temperatures reported at the sample locations.

Immune response modulation upon sequential heterogeneous co-infection with Tetracapsuloides bryosalmonae and VHSV in brown trout (Salmo trutta).

Simultaneous and sequential infections often occur in wild and farming environments. Despite growing awareness, co-infection studies are still very limited, mainly to a few well-established human models. European salmonids are susceptible to both Proliferative Kidney Disease (PKD), an endemic emergent disease caused by the myxozoan parasite Tetracapsuloides bryosalmonae, and Viral Haemorrhagic Septicaemia (VHS), an OIE notifiable listed disease caused by the Piscine Novirhabdovirus. No information is available as to how their immune system reacts when interacting with heterogeneous infections. A chronic (PKD) + acute (VHS) sequential co-infection model was established to assess if the responses elicited in co-infected fish are modulated, when compared to fish with single infections. Macro- and microscopic lesions were assessed after the challenge, and infection status confirmed by RT-qPCR analysis, enabling the identification of singly-infected and co-infected fish. A typical histophlogosis associated with histozoic extrasporogonic T. bryosalmonae was detected together with acute inflammation, haemorrhaging and necrosis due to the viral infection. The host immune response was measured in terms of key marker genes expression in kidney tissues. During T. bryosalmonae/VHSV-Ia co-infection, modulation of pro-inflammatory and antimicrobial peptide genes was strongly influenced by the viral infection, with a protracted inflammatory status, perhaps representing a negative side effect in these fish. Earlier activation of the cellular and humoral responses was detected in co-infected fish, with a more pronounced upregulation of Th1 and antiviral marker genes. These results reveal that some brown trout immune responses are enhanced or prolonged during PKD/VHS co-infection, relative to single infection.

Differential modulation of host immune genes in the kidney and cranium of the rainbow trout (Oncorhynchus mykiss) in response to Tetracapsuloides bryosalmonae and Myxobolus cerebralis co-infections.

BACKGROUND: Most of the studies on fish diseases focus on single infections, although in nature co-infections occur more often. The two freshwater myxozoan parasites of salmonids, having high economic and ecologic relevance are Tetracapsuloides bryosalmonae (Malacosporea), the etiological agent of proliferative kidney disease, and Myxobolus cerebralis (Myxosporea), the etiological agent of whirling disease. The present study aims to investigate immune modulation in rainbow trouts (Oncorhynchus mykiss) during single and co-infections by these parasites. METHODS: Fish were initially infected with T. bryosalmonae (one group) and M. cerebralis (another group) separately. At 30 days post-exposure (dpe), both the single species infected groups were co-infected, respectively, with the other parasite. Posterior kidney and cartilage cranium samples were collected at 30, 60, 90 and 120 dpe and RT-qPCR was performed on them to assess the transcription of suppressors of cytokine signaling (SOCS) -1 and -3, Janus kinase-1 (JAK-1) and signal transducer and activator of transcription-3 (STAT-3) genes. RESULTS: Kidney samples from the T. bryosalmonae-infected group showed upregulation of all immune genes tested between 60-120 dpe. Crania from the single M. cerebralis-infected group and the M. cerebralis and T. bryosalmonae co-infected group exhibited upregulation of SOCS-1 and JAK-1 between 60-120 dpe and SOCS-3 at 120 dpe. However, only in the single M. cerebralis-infected group, was a statistically significant expression of STAT-3 observed at 30 and 60 dpe. CONCLUSIONS: The results of this study indicate that both T. bryosalmonae and M. cerebralis induce overexpression of SOCS-1 and SOCS-3 genes and modulate the host immune response during the development of parasite to cause immunosuppression.

The impact of Tetracapsuloides bryosalmonae and Myxobolus cerebralis co-infections on pathology in rainbow trout.

BACKGROUND: Myxozoan parasites pose emerging health issues for wild and farmed salmonid fish. Rainbow trout (Oncorhynchus mykiss) is a particularly susceptible species to Tetracapsuloides bryosalmonae (Malacosporea), the etiological agent of Proliferative Kidney Disease (PKD), and to Myxobolus cerebralis (Myxosporea), the etiological agent of Whirling Disease (WD). The objective of this study was to investigate the impact of myxozoan co-infections on the pathogenesis of PKD and WD in the rainbow trout. METHODS: Two groups of rainbow trout (96 fish each) were primarily infected with T. bryosalmonae and triactinomyxons of M. cerebralis; after 30 days half of the fish in each group were co-infected with these parasites vice versa and remaining half was continued as single infection. Mortalities and clinical signs were recorded at different time points. Histopathology and immunohistochemistry were performed to assess the extent of each infection and estimate the parasite burden between groups. RESULTS: Fish firstly infected with M. cerebralis and co-infected with T. bryosalmonae exhibited exacerbated pathological changes of both parasitic diseases and elicited a higher mortality rate. A higher kidney swelling index (grade 4) appeared together with more severe cartilage destruction and displacement, when compared to the pathological changes in fish upon single infections with T. bryosalmonae or M. cerebralis. Conversely, fish firstly infected with T. bryosalmonae and co-infected with M. cerebralis also exhibited typical pathological changes of both parasitic diseases, but with a lower mortality rate, similar as caused by the single T. bryosalmonae or M. cerebralis infection. WD clinical signs were milder, without skeletal deformities, while kidney swelling index was similar to single infection with T. bryosalmonae (grade 2 to 3). CONCLUSIONS: In this study, a co-infection with myxozoan parasites was for the first time successfully achieved in the laboratory under controlled conditions. The impact of co-infections in concurrent myxozoan infections mainly depends on the primary pathogen infecting the host, which could alter the outcomes of the secondary pathogen infection. The primary M. cerebralis infection followed by T. bryosalmonae had a much more serious impact and elicited a synergistic interaction. Contrasting results were instead seen in rainbow trout primarily infected with T. bryosalmonae and then co-infected with M. cerebralis.

Role of Viral Hemorrhagic Septicemia Virus Matrix (M) Protein in Suppressing Host Transcription.

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 virus-induced 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.

Migrating zooids allow the dispersal of Fredericella sultana (Bryozoa) to escape from unfavourable conditions and further spreading of Tetracapsuloides bryosalmonae.

Fredericella sultana (Bryozoa: Phylactolaemata) is a primary host in the two-host life cycle of the myxozoan parasite Tetracapsuloides bryosalmonae, the etiological agent of Proliferative Kidney Disease (PKD) in salmonids. Overtly infected F. sultana colonies were collected from River Kamp (Lower Austria), following the first PKD outbreak affecting autochthonous brown trout (Salmo trutta) in Austria. Zooids cultured under unfavourable conditions, e.g. hypertrophication or sudden temperature changes, disconnected their funiculus from the bottom of the body wall, contracted their retractor muscle and packed all organs into a pear-shaped capsule. Migrating zooids dislocated from larger dying branches by separating from the degenerating zooecial tube. After attaching to a new substrate, a new colony could grow rapidly, similar to newly hatched zooids from statoblasts. This is the first observation of an adaptive dispersal mechanism undertaken by adult viable bryozoan zooids to escape from colony deterioration upon adverse summer-like conditions. The evidence of migrating zooids for F. sultana colonization of new habitats increases their intrinsic capacity of spreading infective T. bryosalmonae malacospores.

First Proliferative Kidney Disease outbreak in Austria, linking to the aetiology of Black Trout Syndrome threatening autochthonous trout populations.

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.

Comparative study of CXC chemokines modulation in brown trout (Salmo trutta) following infection with a bacterial or viral pathogen.

Chemokine modulation in response to pathogens still needs to be fully characterised in fish, in view of the recently described novel chemokines present. This paper reports the first comparative study of CXC chemokine genes transcription in salmonids (brown trout), with a particular focus on the fish specific CXC chemokines (CXCL_F). Adopting new primer sets, optimised to specifically target mRNA, a RT-qPCR gene screening was carried out. Constitutive gene expression was assessed first in six tissues from SPF brown trout. Transcription modulation was next investigated in kidney and spleen during septicaemic infection induced by a RNA virus (Viral Haemorrhagic Septicaemia virus, genotype Ia) or by a Gram negative bacterium (Yersinia ruckeri, ser. O1/biot. 2). From each target organ specific pathogen burden, measured detecting VHSV-glycoprotein or Y. ruckeri 16S rRNA, and IFN-γ gene expression were analysed for their correlation to chemokine transcription. Both pathogens modulated CXC chemokine gene transcript levels, with marked up-regulation seen in some cases, and with both temporal and tissue specific effects apparent. For example, Y. ruckeri strongly induced chemokine transcription in spleen within 24h, whilst VHS generally induced the largest increases at 3d.p.i. in both tissues. This study gives clues to the role of the novel CXC chemokines, in comparison to the other known CXC chemokines in salmonids.

Viral and bacterial septicaemic infections modulate the expression of PACAP splicing variants and VIP/PACAP receptors in brown trout immune organs.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) and PACAP-Related Peptide (PRP) are structurally similar peptides encoded in the same transcripts. Their transcription has been detected not only in the brain but also in a wide range of peripheral tissues, even including organs of the immune system. PACAP exerts pleiotropic activities through G-protein coupled membrane receptors: the PACAP-specific PAC-1 and the VPAC-1 and VPAC-2 receptors that exhibit similar affinities for the Vasoactive Intestinal Peptide (VIP) and PACAP. Recent findings added PACAP and its receptors to the growing list of mediators that allow cross-talk between the nervous, endocrine and immune systems in fish. In this study the expression of genes encoding for PACAP and PRP, as well as VIP/PACAP receptors was studied in laboratory-reared brown trout (Salmo trutta) after septicaemic infections. Respectively Viral Haemorrhagic Septicaemia Virus (VHSV-Ia) or the Gram-negative bacterium Yersinia ruckeri (ser. O1 - biot. 2) were used in infection challenges. Kidney and spleen, the teleost main lymphopoietic organs, were sampled during the first two weeks post-infection. RT-qPCR analysis assessed specific pathogens burden and gene expression levels. PACAP and PRP transcription in each organ was positively correlated to the respective pathogen burden, assessed targeting the VHSV-glycoprotein or Y. ruckeri 16S rRNA. Results showed as the transcription of PACAP splicing variants and VIP/PACAP receptors is modulated in these organs during an acute viral and bacterial septicaemic infections in brown trout. These gene expression results provide clues as to how the PACAP system is modulated in fish, confirming an involvement during active immune responses elicited by both viral and bacterial aetiological agents. However, further experimental evidence is still required to fully elucidate and characterize the role of PACAP and PRP for an efficient immune response against pathogens.

Salmonids have an extraordinary complex type I IFN system: characterization of the IFN locus in rainbow trout oncorhynchus mykiss reveals two novel IFN subgroups.

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.

Immune gene expression profiling of Proliferative Kidney Disease in rainbow trout Oncorhynchus mykiss reveals a dominance of anti-inflammatory, antibody and T helper cell-like activities.

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.

Fish Suppressors of Cytokine Signaling (SOCS): Gene Discovery, Modulation of Expression 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-1ß, 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.