The nedd-8 activating enzyme gene underlies genetic resistance to infectious pancreatic necrosis virus in Atlantic salmon.

Genetic resistance to infectious pancreatic necrosis virus (IPNV) in Atlantic salmon is a rare example of a trait where a single locus (QTL) explains almost all of the genetic variation. Genetic marker tests based on this QTL on salmon chromosome 26 have been widely applied in selective breeding to markedly reduce the incidence of the disease. In the current study, whole genome sequencing and functional annotation approaches were applied to characterise genes and variants in the QTL region. This was complemented by an analysis of differential expression between salmon fry of homozygous resistant and homozygous susceptible genotypes challenged with IPNV. These analyses pointed to the NEDD-8 activating enzyme 1 (nae1) gene as a putative functional candidate underlying the QTL effect. The role of nae1 in IPN resistance was further assessed via CRISPR-Cas9 knockout of the nae1 gene and chemical inhibition of the nae1 protein activity in Atlantic salmon cell lines, both of which resulted in highly significant reduction in productive IPNV replication. In contrast, CRISPR-Cas9 knockout of a candidate gene previously purported to be a cellular receptor for the virus (cdh1) did not have a major impact on productive IPNV replication. These results suggest that nae1 is the causative gene underlying the major QTL affecting resistance to IPNV in salmon, provide further evidence for the critical role of neddylation in host-pathogen interactions, and highlight the value in combining high-throughput genomics approaches with targeted genome editing to understand the genetic basis of disease resistance.

Survival of viral haemorrhagic septicaemia virus and infectious haematopoietic necrosis virus in the environment and dried on stainless steel.

Viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV) are important viral pathogens posing a serious threat to salmonid fish. Survival of two isolates of IHNV and one of VHSV was assessed at temperatures ranging from 4 to 25°C: (a) after drying on stainless steel, (b) in cell culture medium, (c) in filtered river water, (d) in unfiltered river water, and (e) survival, adsorption and desorption in river sediment and five typical soil types. The viruses survived 1 hr to > 84 days depending on the conditions. Survival was inversely related to temperature and organic and inorganic content. Both viruses remained infectious after being dried on stainless steel for several weeks highlighting the risk of mechanical transmission and persistence in a dry environment. Both adsorbed to the soils from the river water inoculum, with titres between 5.56x104 and 2.58x108 TCID50 /ml after 1 hr. Clay soils adsorbed the least virus but had the greatest decrease in the river water inoculum (undetectable in ≤ 1 hr), and there was no desorption. Virus desorbed from the other soils into the surrounding water at different rates dependant on soil type (longest desorption was from chalk loam and sandy soil-detected at 28 days). When desorption was no longer detectable, virus persisted, adsorbed to the soil and remained infectious (the longest adsorption was detected in clay loam for ≥ 49 days, but all the viruses adsorbed to soils were likely to have survived longer than that detected, based on their rate of decay). The long survival of the viruses, particularly at cooler temperatures, highlights the risk of survival in the environment and waterborne spread. The data presented here are highly relevant for assessing risk of pathogen introduction via fomites (stainless steel) and for deciding on best control measures in the context of disease outbreaks.

First detection of infectious spleen and kidney necrosis virus (ISKNV) associated with massive mortalities in farmed tilapia in Africa.

In late 2018, unusual patterns of very high mortality (>50% production) were reported in intensive tilapia cage culture systems across Lake Volta in Ghana. Samples of fish and fry were collected and analysed from two affected farms between October 2018 and February 2019. Affected fish showed darkening, erratic swimming and abdominal distension with associated ascites. Histopathological observations of tissues taken from moribund fish at different farms revealed lesions indicative of viral infection. These included haematopoietic cell nuclear and cytoplasmic pleomorphism with marginalization of chromatin and fine granulation. Transmission electron microscopy showed cells containing conspicuous virions with typical iridovirus morphology, that is enveloped, with icosahedral and/or polyhedral geometries and with a diameter c.160 nm. PCR confirmation and DNA sequencing identified the virions as infectious spleen and kidney necrosis virus (ISKNV). Samples of fry and older animals were all strongly positive for the presence of the virus by qPCR. All samples tested negative for TiLV and nodavirus by qPCR. All samples collected from farms prior to the mortality event were negative for ISKNV. Follow-up testing of fish and fry sampled from 5 additional sites in July 2019 showed all farms had fish that were PCR-positive for ISKNV, whether there was active disease on the farm or not, demonstrating the disease was endemic to farms all over Lake Volta by that point. The results suggest that ISKNV was the cause of disease on the investigated farms and likely had a primary role in the mortality events. A common observation of coinfections with Streptococcus agalactiae and other tilapia bacterial pathogens further suggests that these may interact to cause severe pathology, particularly in larger fish. Results demonstrate that there are a range of potential threats to the sustainability of tilapia aquaculture that need to be guarded against.

In vitro gill cell monolayer successfully reproduces in vivo Atlantic salmon host responses to Neoparamoeba perurans infection.

An in vitro model to study the host response to Neoparamoeba perurans, the causative agent of amoebic gill disease (AGD), was evaluated. The rainbow trout gill derived cell line, RTgill-W1, was seeded onto permeable cell culture supports and maintained asymmetrically with apical seawater. Cells were inoculated with either a passage attenuated or a recent wild clone of N. perurans. Amoebae, loaded with phagocytosed fluorescent beads, were observed associated with host cells within 20 min post inoculation (pi). By 6 h small foci of cytopathic effect appeared and at 72 h cytolysis was observed, with total disruption of the cell monolayer at 96 h pi. Due to cell monolayer disruption, the platform could not support proliferation of amoebae, which showed a 3-log reduction in parasite 18S rRNA mRNA after 72 h (106 copies at 1 h to 103 at 72 h pi). SEM observations showed amoebae-like cells with either short pseudopodia and a malleiform shape, or, long pseudopodia embedded within the gill cells and erosion of the cell monolayer. To study the host immune response, inoculated gill cells were harvested from triplicate inserts at 0, 1, 3, 6, 24 and 48 h pi, and expression of 12 genes involved in the Atlantic salmon response to AGD was compared between infected and uninfected cells and between amoebic clones. Both clones induced similar host inmate immune responses, with the up-regulation of proinflammatory cytokine IL1ß, complement C3 and cell receptor MHC-1. The Th2 pathway was up-regulated, with increased gene expression of the transcription factor GATA3, and Th2 cytokines IL10, IL6 and IL4/13A. PCNA and AG-2 were also up-regulated. The wild clone induced significantly higher up-regulation of IL1ß, MHC-1, PCNA, lysozyme and IL10 than the attenuated clone for at least some exposure times, but AG-2 gene expression was higher in cells inoculated with the attenuated one. A principal component analysis showed that AG-2 and IL10 were key genes in the in vitro host response to N. perurans. This in vitro model has proved to be a promising tool to study host responses to amoebae and may therefore reduce the requirement for in vivo studies when evaluating alternative therapeutants to AGD control.

The skin immune response of rainbow trout, Oncorhynchus mykiss (Walbaum), associated with puffy skin disease (PSD).

Puffy skin disease (PSD) is an emerging skin condition which affects rainbow trout, Oncorhynchus mykiss (Walbaum). The transmission pattern of PSD suggests an infectious aetiology, however, the actual causative infectious agent(s) remain(s) unknown. In the present study, the rainbow trout epidermal immune response to PSD was characterised. Skin samples from infected fish were analysed and classified as mild, moderate or severe PSD by gross pathology and histological assessment. The level of expression of 26 immune-associated genes including cytokines, immunoglobulins and cell markers were examined by TaqMan qPCR assays. A significant up-regulation of the gene expression of C3, lysozyme, IL-1ß and T-bet and down-regulation of TGFß and TLR3 was observed in PSD fish compared to control fish. MHCI gene expression was up-regulated only in severe PSD lesions. Histological examinations of the epidermis showed a significant increase in the number of eosinophil cells and dendritic melanocytes in PSD fish. In severe lesions, mild diffuse lymphocyte infiltration was observed. IgT and CD8 positive cells were detected locally in the skin of PSD fish by in situ hybridisation (ISH), however, the gene expression of those genes was not different from control fish. Total IgM in serum of diseased animals was not different from control fish, measured by a sandwich ELISA, nor was significant up regulation of IgM gene expression in PSD lesions observed. Taken together, these results show activation of the complement pathway, up-regulation of a Th17 type response and eosinophilia during PSD. This is typical of a response to extracellular pathogens (i.e. bacteria and parasites) and allergens, commonly associated with acute dermatitis.

A Genome-Wide Association Study for Host Resistance to Ostreid Herpesvirus in Pacific Oysters (Crassostrea gigas).

Ostreid herpesvirus (OsHV) can cause mass mortality events in Pacific oyster aquaculture. While various factors impact on the severity of outbreaks, it is clear that genetic resistance of the host is an important determinant of mortality levels. This raises the possibility of selective breeding strategies to improve the genetic resistance of farmed oyster stocks, thereby contributing to disease control. Traditional selective breeding can be augmented by use of genetic markers, either via marker-assisted or genomic selection. The aim of the current study was to investigate the genetic architecture of resistance to OsHV in Pacific oyster, to identify genomic regions containing putative resistance genes, and to inform the use of genomics to enhance efforts to breed for resistance. To achieve this, a population of ∼1,000 juvenile oysters were experimentally challenged with a virulent form of OsHV, with samples taken from mortalities and survivors for genotyping and qPCR measurement of viral load. The samples were genotyped using a recently-developed SNP array, and the genotype data were used to reconstruct the pedigree. Using these pedigree and genotype data, the first high density linkage map was constructed for Pacific oyster, containing 20,353 SNPs mapped to the ten pairs of chromosomes. Genetic parameters for resistance to OsHV were estimated, indicating a significant but low heritability for the binary trait of survival and also for viral load measures (h2 0.12 - 0.25). A genome-wide association study highlighted a region of linkage group 6 containing a significant QTL affecting host resistance. These results are an important step toward identification of genes underlying resistance to OsHV in oyster, and a step toward applying genomic data to enhance selective breeding for disease resistance in oyster aquaculture.

Molecular Characterization of an Endozoicomonas-Like Organism Causing Infection in the King Scallop (Pecten maximus L.).

One of the fastest growing fisheries in the UK is the king scallop (Pecten maximus L.), also currently rated as the second most valuable fishery. Mass mortality events in scallops have been reported worldwide, often with the causative agent(s) remaining uncharacterized. In May 2013 and 2014, two mass mortality events affecting king scallops were recorded in the Lyme Bay marine protected area (MPA) in Southwest England. Histopathological examination showed gill epithelial tissues infected with intracellular microcolonies (IMCs) of bacteria resembling Rickettsia-like organisms (RLOs), often with bacteria released in vascular spaces. Large colonies were associated with cellular and tissue disruption of the gills. Ultrastructural examination confirmed the intracellular location of these organisms in affected epithelial cells. The 16S rRNA gene sequences of the putative IMCs obtained from infected king scallop gill samples, collected from both mortality events, were identical and had a 99.4% identity to 16S rRNA gene sequences obtained from "Candidatus Endonucleobacter bathymodioli" and 95% with Endozoicomonas species. In situ hybridization assays using 16S rRNA gene probes confirmed the presence of the sequenced IMC gene in the gill tissues. Additional DNA sequences of the bacterium were obtained using high-throughput (Illumina) sequencing, and bioinformatic analysis identified over 1,000 genes with high similarity to protein sequences from Endozoicomonas spp. (ranging from 77 to 87% identity). Specific PCR assays were developed and applied to screen for the presence of IMC 16S rRNA gene sequences in king scallop gill tissues collected at the Lyme Bay MPA during 2015 and 2016. There was 100% prevalence of the IMCs in these gill tissues, and the 16S rRNA gene sequences identified were identical to the sequence found during the previous mortality event.IMPORTANCE Molluscan mass mortalities associated with IMCs have been reported worldwide for many years; however, apart from histological and ultrastructural characterization, characterization of the etiological agents is limited. In the present work, we provide detailed molecular characterization of an Endozoicomonas-like organism (ELO) associated with an important commercial scallop species.

Rapid and Sensitive Detection of Lymphocystis Disease Virus Genotype VII by Loop-Mediated Isothermal Amplification.

Lymphocystis disease virus (LCDV) infections have been described in gilthead seabream (Sparus aurata L.) and Senegalese sole (Solea senegalensis, Kaup), two of the most important marine fish species in the Mediterranean aquaculture. In this study, a rapid, specific, and sensitive detection method for LCDV genotype VII based on loop-mediated isothermal amplification (LAMP) was developed. The LAMP assay, performed using an apparatus with real-time amplification monitoring, was able to specifically detect LCDV genotype VII from clinically positive samples in less than 12 min. In addition, the assay allowed the detection of LCDV in all asymptomatic carrier fish analysed, identified by qPCR, showing an analytical sensitivity of ten copies of viral DNA per reaction. The LCDV LAMP assay has proven to be a promising diagnostic method that can be used easily in fish farms to detect the presence and spread of this iridovirus.

Puffy Skin Disease Is an Emerging Transmissible Condition in Rainbow Trout Oncorhynchus mykiss Walbaum.

The transmission of puffy skin disease (PSD) to rainbow trout Oncorhynchus mykiss Walbaum was tested in the laboratory by conducting co-habitation challenges with puffy skin (PS)-affected fish (Trojans) collected from the field. Two separate challenges were conducted using Trojans sourced from two different sites and diploid (first trial) or triploid (second trial) naïve fish. PSD-specific clinical signs were observed in both groups of naïve fish, with 66% of the fish sampled during the challenges showing signs of varying severity. The first clinical features of PSD were presented as white oval skin patches on one or both flanks 15-21 days post-challenge (dpc). The extent of the lesions ranged from 10 to 90% of the body surface, depending on the severity of the lesion. Both the severity and number of affected fish increased during the challenge. Macroscopically, oedema of the skin and multifocal petechial haemorrhaging were observed towards the end of the trials. Abnormal fish behaviour consisting of "flashing" and excessive mucous production was noted from 15 dpc onwards. Fish with severe PSD lesions also displayed inappetence and associated emaciation. Rodlet cells were observed in 41% of the fresh skin scrapes analysed from the second trial. Histologically epidermal oedema was observed in 31% of the naive fish showing gross pathology, with additional 12% displaying epidermal hyperplasia, mostly observed at the end of the challenge. Other concomitant features of the PSD lesions in challenged fish were epithelial erosion and sloughing, and occasionally mild or focal inflammation. No consistent pathology of internal organs was observed. The parasites Ichthyophthirius multifiliis and Ichthyobodo necator were observed in skin samples of a proportion of naïve challenged fish and in Trojans but not in control fish. The presence of these and other known fish pathogens in the skin of PSD-fish was confirmed by high-throughput sequencing analysis. In summary, we have demonstrated that PSD is a transmissible condition. However, even though a number of known fish pathogens were identified in the skin tissues of PSD-fish, the actual causative infectious agent(s) remain(s) unknown.

Gene expression comparison of resistant and susceptible Atlantic salmon fry challenged with Infectious Pancreatic Necrosis virus reveals a marked contrast in immune response.

BACKGROUND: Infectious Pancreatic Necrosis (IPN) is a highly contagious birnavirus disease of farmed salmonid fish, which often causes high levels of morbidity and mortality. A large host genetic component to resistance has been previously described for Atlantic salmon (Salmo salar L.), which mediates high mortality rates in some families and zero mortality in others. However, the molecular and immunological basis for this resistance is not yet fully known. This manuscript describes a global comparison of the gene expression profiles of resistant and susceptible Atlantic salmon fry following challenge with the IPN virus. RESULTS: Salmon fry from two IPNV-resistant and two IPNV-susceptible full sibling families were challenged with the virus and sampled at 1 day, 7 days and 20 days post-challenge. Significant viral titre was observed in both resistant and susceptible fish at all timepoints, although generally at higher levels in susceptible fish. Gene expression profiles combined with gene ontology and pathway analyses demonstrated that while a clear immune response was observed in both resistant and susceptible fish, there were striking differences between the two phenotypes. The susceptible fish showed marked up-regulation of genes related to cytokine activity and inflammatory response that evidently failed to protect against the virus. In contrast, the resistant fish demonstrated a less pronounced immune response including up-regulation of genes relating to the M2 macrophage system. CONCLUSIONS: While only the susceptible phenotype shows appreciable mortality levels, both resistant and susceptible fish can become infected with IPNV. Susceptible fish are characterized by a much larger, yet ineffective, immune response, largely related to cytokine and inflammatory systems. Resistant fish demonstrate a more moderate, putative macrophage-mediated inflammatory response, which may contribute to their survival.

Isolation and culture of Sphaerothecum destruens from Sunbleak (Leucaspius delineatus) in the UK and pathogenicity experiments in Atlantic salmon (Salmo salar).

The sunbleak (Leucaspius delineatus), a cyprinid fish native to continental Europe and now established in the UK, is experiencing population decline which appears to be linked to the spread of the invasive Asian cyprinid (Pseudorasbora parva). A population of sunbleak in the UK has previously been identified as infected with S. destruens at low prevalence. Because Sphaerothaecum destruens has, on occasion, caused severe disease in cultured and wild salmonids the aim of this work was to establish laboratory cultures of S. destruens from sunbleak in the UK and use these cultures in challenge experiments to determine if the UK isolate of S. destruens from cyprinid species is a potential threat to Atlantic salmon (Salmo salar). The first isolation and culture of S. destruens in the UK and from a cyprinid species is described. Cultured S. destruens spores from sunbleak are infective to EPC, CHSE and FHM cells, replicating most rapidly in FHM and EPC cells. Spores can be induced to zoosporulate in water forming motile, uni-flagellated zoospores. Challenge experiments indicated the spores are able to replicate and disperse in Atlantic salmon and are associated with increased mortality (up to 90%) when injected intraperitonealy.