Salmon gill poxvirus disease in Atlantic salmon fry as recognized by improved immunohistochemistry also demonstrates infected cells in non-respiratory epithelial cells.

Gill diseases cause serious losses in farming of Atlantic salmon and the number of agents involved increases. Salmon gill poxvirus (SGPV) and the gill disease in causes where SGPV apparently was the only disease-causing agent were initially characterized. Recently, it was further shown that SGPV can be a common denominator in widely different multifactorial gill diseases. Here, we present the challenge of diagnosing gill disease with SGPV in salmon fry of 0,3-5 grams. Apoptosis of gill lamellar epithelial cells and hemophagocytosis was also observed in fry similar to findings in smolts and grow-out fish. Using our newly developed immunohistochemistry method, we further demonstrate that some of the apoptotic epithelial cells covering the oral cavity were positive for SGPV. Thus, SGPV is not restricted to respiratory epithelium alone and may infect the fish at very early life stages. Furthermore, as the cases examined here are from Norway, Faroe Island and Scotland, we show that SGPV is more widespread than previously reported.

Ectopic epithelial cell clusters in salmonid intestine are associated with inflammation.

An epizootic incidence of intestinal adenocarcinomas was reported in brood fish of Atlantic salmon (Salmo salar L.) in 2009. The condition was associated with a specific diet inducing enteritis and morphological changes. Here, two field trials of fish up to slaughter size were initiated. In Trial 1, two different feed recipes were used. Feed I was predominantly based on marine ingredients, whereas plant ingredients were limited to soy protein concentrate and wheat. Feed II was lower in fishmeal and without soya protein, which was substituted with plant proteins from other sources. In Trial 2, a commercial feed (Feed III) was included. No macroscopic tumours were observed in 300 fish (Trial 1). At the end of both trials, samples from five different segments of the gastrointestinal tract of a total of 39 fish were investigated with morphological methods. Here, we show the presence of ectopic proliferating epithelial cells only occurring in inflamed intestine and predominantly in the second segment of the mid-intestine. Presence of ectopic epithelial cells in submucosal inflammatory foci may indicate early stages in tumorigenesis, but other possibilities such as proliferative enteric disorders cannot be excluded. Together with inflammation, carcinogenesis should be a focus of investigation in future feed trials.

Salmon gill poxvirus, a recently characterized infectious agent of multifactorial gill disease in freshwater- and seawater-reared Atlantic salmon.

Gill diseases cause considerable losses in Norwegian salmon farming. In 2015, we characterized salmon gill poxvirus (SGPV) and associated gill disease. Using newly developed diagnostic tools, we show here that SGPV infection is more widely distributed than previously assumed. We present seven cases of complex gill disease in Atlantic salmon farmed in seawater and freshwater from different parts of Norway. Apoptosis, the hallmark of acute SGPV infection, was not easily observed in these cases, and qPCR analysis was critical for identification of the presence of SGPV. Several other agents including Costia-like parasites, gill amoebas, Saprolegnia spp. and bacteria were observed. The studied populations experienced significant mortalities, which increased to extreme levels when severe SGPV infections coincided with smoltification. SGPV infection appears to affect the smoltification process directly by affecting the gills and chloride cells in particular. SGPV may be considered a primary pathogen as it was often found prior to identification of complex gill disease. It is hypothesized that SGPV-induced gill damage may impair innate immunity and allow invasion of secondary invaders. The distinct possibility that SGPV has been widely overlooked as a primary pathogen calls for extended use of SGPV qPCR in Atlantic salmon gill health management.

Atlantic salmon, Salmo salar L. are broadly susceptible to isolates representing the North American genogroups of infectious hematopoietic necrosis virus.

Beginning in 1992, three epidemic waves of infectious hematopoietic necrosis, often with high mortality, occurred in farmed Atlantic salmon Salmo salar L. on the west coast of North America. We compared the virulence of eleven strains of infectious hematopoietic necrosis virus (IHNV), representing the U, M and L genogroups, in experimental challenges of juvenile Atlantic salmon in freshwater. All strains caused mortality and there was wide variation within genogroups: cumulative mortality for five U-group strains ranged from 20 to 100%, four M-group strains ranged 30-63% and two L-group strains varied from 41 to 81%. Thus, unlike Pacific salmonids, there was no apparent correlation of virulence in a particular host species with virus genogroup. The mortality patterns indicated two different phenotypes in terms of kinetics of disease progression and final per cent mortality, with nine strains having moderate virulence and two strains (from the U and L genogroups) having high virulence. These phenotypes were investigated by histopathology and immunohistochemistry to describe the variation in the course of IHNV disease in Atlantic salmon. The results from this study demonstrate that IHNV may become a major threat to farmed Atlantic salmon in other regions of the world where the virus has been, or may be, introduced.

Host tropism of infectious salmon anaemia virus in marine and freshwater fish species.

The aquatic orthomyxovirus infectious salmon anaemia virus (ISAV) causes a severe disease in farmed Atlantic salmon, Salmo salar L. Although some ISA outbreaks are caused by horizontal transmission of virus between farms, the source and reservoir of the virus is largely unknown and a wild host has been hypothesized. Atlantic salmon are farmed in open net-pens, allowing transmission of pathogens from wild fish and the surrounding environment to the farmed fish. In this study, a large number of fish species were investigated for ISAV host potential. For orthomyxoviruses, a specific receptor binding is the first requirement for infection; thus, the fish species were investigated for the presence of the ISAV receptor. The receptor was found to be widely distributed across the fish species. All salmonids expressed the receptor. However, only some of the cod-like and perch-like fish did, and all flat fish were negative. In the majority of the positive species, the receptor was found on endothelial cells and/or on red blood cells. The study forms a basis for further investigations and opens up the possibility for screening species to determine whether a wild host of ISAV exists.

Immersion challenge with low and highly virulent infectious salmon anaemia virus reveals different pathogenesis in Atlantic salmon, Salmo salar L.

The salmonid orthomyxovirus infectious salmon anaemia virus (ISAV) causes disease of varying severity in farmed Atlantic salmon, Salmo salar L. Field observations suggest that host factors, the environment and differences between ISAV strains attribute to the large variation in disease progression. Variation in host mortality and dissemination of ISAV isolates with high and low virulence (based on a previously published injection challenge) were investigated using immersion challenge. Virus dissemination was determined using real-time PCR and immunohistochemistry in several organs, including blood. Surprisingly, the low virulent virus (LVI) replicated and produced nucleoprotein at earlier time points post-infection compared to the virus of high virulence (HVI). This was particularly noticeable in the gills as indicated by different viral load profiles. However, the HVI reached a higher maximum viral load in all tested organs and full blood. This was associated with a higher mortality of 100% as compared to 20% in the LVI group by day 23 post-infection. Immersion challenge represented a more natural infection method and suggested that specific entry routes into the fish may be of key importance between ISAV strains. The results suggest that a difference in virulence is important for variations in virus dissemination and pathogenesis (disease development).

Infectious salmon anaemia - pathogenesis and tropism.

Infectious salmon anaemia (ISA) is a serious disease of farmed Atlantic salmon caused by the aquatic orthomyxovirus infectious salmon anaemia virus (ISAV). ISA was first detected in Norway in 1984 and was characterized by severe anaemia and circulatory disturbances. This review elucidates factors related to the pathogenesis of ISA in Atlantic salmon, the dissemination of the virus in the host and the general distribution of the 4-O-acetylated sialic acids ISAV receptor. The knowledge contributes to the understanding of this disease, and why, almost 30 years after the first detection, it is still causing problems for the aquaculture industry.

Outbreak of viral haemorrhagic septicaemia (VHS) in seawater-farmed rainbow trout in Norway caused by VHS virus Genotype III.

We describe the finding of a novel viral haemorrhagic septicaemia virus (VHSV) Genotype III strain that caused disease of both a neurological and septicaemic nature in seawater-farmed rainbow trout Oncorhynchus mykiss in Storfjorden, Norway. In November 2007, an outbreak of VHS associated with slightly elevated mortality was confirmed at a seawater site rearing rainbow trout (90 to 440 g). Within 3 to 4 mo, the disease was recognised in 3 neighbouring sea sites with ongrowing rainbow trout. The clinical, gross pathological and histopathological findings were in accordance with VHS, and the diagnosis was confirmed by the detection of VHSV in brain and internal tissues by immunohistochemistry, cell culture and reverse transcriptase PCR (RT-PCR). Sequence analysis of the G-gene revealed that the isolated virus clustered with VHSV Genotype III and that the Norwegian isolate represents a unique strain of VHSV. The pathogenicity of the virus strain to rainbow trout and Atlantic salmon Salmo salar was examined using infection experiments. In immersion trials, the Norwegian isolate produced a cumulative mortality of 70% in rainbow trout, while nearly 100% mortality was obtained after intraperitoneal injection of the virus. For Atlantic salmon, no mortality was observed in immersion trials, whereas 52% mortality was observed after intraperitoneal injection. The Norwegian isolate thus represents the first VHSV of Genotype III pathogenic to rainbow trout.

First cases of amoebic gill disease (AGD) in Norwegian seawater farmed Atlantic salmon, Salmo salar L., and phylogeny of the causative amoeba using 18S cDNA sequences.

Amoebic gill disease (AGD) was observed in seawater farmed Atlantic salmon at four geographically distant locations on the western coast of Norway. To the best of our knowledge, these are the first detected AGD outbreaks in Norway. The outbreaks lasted for 7-12 weeks in late autumn 2006 and were for the most part concurrent. The crude, cumulative mortality was in the range of 12-20% at three farms and 82% at a fourth. The histopathology showed uniform parasomal amoebae in lesions characteristic for AGD. Another gill disease, proliferative gill inflammation (PGI), was also present to a variable degree and the distinction between the two gill problems is discussed. Seawater temperatures were 3.5 degrees C higher than average before disease outbreaks, which subsided in early winter. The geographical and time pattern of these outbreaks strongly indicates simultaneous infection from the marine environment. Two contiguous 18S cDNA sequences, obtained by reverse transcriptase PCR from gill tissue with AGD-related lesions, showed highest similarity (99.2%) to a newly recognized species designated Neoparamoeba perurans and maximum likelihood analysis demonstrates that they represent Norwegian strains of this Neoparamoeba lineage.

Myocardial glycogen storage disease in farmed rainbow trout, Oncorhynchus mykiss (Walbaum).

This paper is the first description of a spontaneous glycogen-storage disease in a lower vertebrate, as previous descriptions deal with humans and other mammals, or fish where the condition has been experimentally induced. Affected farmed rainbow trout experienced increased mortality from 60 days post-startfeeding and displayed clinical signs of heart failure with abnormal behaviour, exophthalmia, distended abdomen and ventral skin petechiation. Necropsy revealed alterations in cardiac shape with distended atria and rounded ventricles. Microscopically, the compact wall of the ventricle was absent, uneven or thinner than normal. The cardiac myocytes contained extensive amounts of glycogen in cytoplasmic vacuoles as demonstrated by periodic acid-Schiff staining that was abolished by saliva-diastase pretreatment on serial sections. Associated lesions included conspicuous subepicardial and myocardial vascularization, epicardial thickening and necrosis of the ventricular compactum/spongiosum interphase. The lesions in cardiac myocytes had a striking resemblance to glycogenosis type II (Pompe disease), a rare autosomal recessive lysosomal storage disease in humans. This condition was more severe and mortality was higher in a replicate/parallel fish group treated perorally with 17alpha-methyltestosterone to produce all-female progeny, indicating that the hormone treatment aggravated the condition resulting in earlier and more severe manifestation of the disease in this group.

Immunohistochemical identification of Renibacterium salmoninarum by monoclonal antibodies in paraffin-embedded tissues of Atlantic salmon (Salmo salar L.), using paired immunoenzyme and paired immunofluorescence techniques.

Renibacterium salmoninarum was identified in situ by immunoenzymatic and immunofluorescence techniques in paraffin-embedded tissue specimens collected during a natural outbreak of bacterial kidney disease (BKD) and from an experimental infection in Atlantic salmon (Salmo salar L.). Monoclonal antibodies (MAbs) 4D3 and 2G5 were used in this study, both specific for the 57-58-kD outer membrane protein (p57) of the bacterium. Both MAbs revealed positive staining in ethanol-fixed tissue specimens, but only the epitope identified by MAb 4D3 was formalin resistant. Pretreatment with trypsin did not reestablish the antigenicity for the epitope identified by Mab 2G5. Paired immunoenzymatic staining for identification of the bacterium in sequential incubation steps on ethanol-fixed tissue specimens using an avidin-biotin-peroxidase system was obtained after serial dilution of the Mab (2G5) or the chromagen, amino ethyl carbazole, in the first sequence. Paired immunofluorescence staining with well-balanced color mixing was easily obtained on ethanol-fixed tissue specimens using sequential incubations. Single exposures gave blue (aminomethyl coumarin acetic acid) and green (fluorescein isothiocyanate) fluorescence for MAbs 2G5 and biotinylated 4D3, respectively. Color mixing was revealed as a turquoise staining. Studies on method sensitivity was performed by incorporating a known amount of a protein preparation of p57 into an inert matrix, creating an artificial test substrate.(ABSTRACT TRUNCATED AT 250 WORDS)