Presence of selected pathogens on the gills of five wrasse species in western Norway.

The objective of this study was to identify gill pathogens in Labridae (wrasse) species used as cleaner fish to control salmon louse in western Norwegian aquaculture. Wrasse are often moved over long distances, raising issues of fish health, welfare and pathogen transmission. Histological examination and real-time RT-PCR analysis of the gills from Centrolabrus exoletus, Ctenolabrus rupestris, Labrus bergylta, L. mixtus and Symphodus melops revealed several pathogens: a new species of Ichthyobodo, Paramoeba perurans, microsporidia, trichodinids, Hatschekia spp., Candidatus Similichlamydia labri and 2 putative new species of Chlamydiae. Cand. S. labri or closely related bacteria were present on most wrasse specimens. Epitheliocysts on the gills of L. mixtus contained large inclusions (120 µm) with actiniae radiating from the inclusion membrane. A possible member of the Candidatus family Parilichlamydiaceae was present at a high prevalence on the gills of L. mixtus, L. bergylta and C. rupestris. Sequencing the 16S rRNA gene showed 93.9% similarity to Cand. S. labri and 96.8% similarity to Cand. Parilichlamydia carangidicola from the gills of Seriola lalandi. This bacterium probably represents a new species within the order Chlamydiales, family Cand. Parilichlamydiaceae. The other Chlamydiae detected on gills of S. melops could represent a new species in Cand. genus Syngnamydia. Ichthyobodo sp. and Paranucleospora theridion were detected on the gills of nearly all individuals, while Paramoeba spp. were detected on the gills of L. bergylta and L. mixtus. Trichodinids, microsporidia and parasitic copepods had low prevalence. Viral haemorrhagic septicaemia virus was not detected.

Diseases of farmed Atlantic salmon Salmo salar associated with infections by the microsporidian Paranucleospora theridion.

The microsporidian Paranucleospora theridion was discovered in Atlantic salmon Salmo salar suffering from proliferative gill disease in a marine farm in western Norway in 2008. The parasite develops in cells of the reticuloendothelial system, cells important for normal immune function. The aim of this study was to see if P. theridion could play a part in some of the diseases with unclear causes in salmon production in Norway, i.e. proliferative gill disease (PGI), pancreas disease (PD), heart and skeletal muscle inflammation (HSMI) and cardiomyopathy syndrome (CMS). P. theridion was present in all areas with salmon farming in Norway, but high prevalence and densities of the parasite in salmon and salmon lice were only seen in southern Norway. This region is also the main area for PGI and PD in Norway. Quantification of pathogens associated with PGI, PD, HSMI and CMS diagnoses showed that P. theridion levels are high in southern Norway, and may therefore play a role in susceptibility and disease development. However, among the different diagnoses, fish with PGI are particularly heavily infected with P. theridion. Therefore, P. theridion appears as a possible primary agent in cases with high mortality in connection with PGI in western Norway.

Prevalence of infectious salmon anaemia virus (ISAV) in wild salmonids in western Norway.

Studies of infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Ireland, Canada, the USA and Chile, suggest that natural reservoirs for this virus can be found on both sides of the North Atlantic. Based on existing information about ISAV it is believed to be maintained in wild populations of trout and salmon in Europe. It has further been suggested that ISAV is transmitted between wild hosts, mainly during their freshwater spawning phase in rivers, and that wild salmonids, mainly trout, are possible carriers of benign wild-type variants of ISAV. Change in virulence is probably a result of deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates after transmission to farmed salmon. Hence, it has been suggested that the frequency of new outbreaks of ISA in farmed salmon could partly reflect natural variation in the prevalence of ISAV in wild populations of salmonids. The aims of the present study were to screen for ISAV in wild salmonids during spawning in rivers and to determine the pathogenicity of resultant isolates from wild fish. Tissues from wild salmonids were screened by RT-PCR and real-time PCR. The prevalence of ISAV in wild trout Salmo trutta varied from 62 to 100% between tested rivers in 2001. The prevalence dropped in 2002, ranging from 13 to 36% in the same rivers and to only 6% in 2003. All ISAV were nonpathogenic when injected into disease-free Atlantic salmon, but were capable of propagation, as indicated by subsequent viral recovery. However, non-pathogenic ISAV has also been found in farmed salmon, where a prevalence as high as 60% has been registered, but with no mortalities occurring. Based on the results of the present and other studies, it must be concluded that vital information about the importance of wild and man-made reservoirs for the emergence of ISA in salmon farming is still lacking. This information can only be gained by further screening of possible reservoirs, combined with the development of a molecular tool for typing virulence and the geographical origin of the virus isolates.

Emergence and maintenance of infectious salmon anaemia virus (ISAV) in Europe: a new hypothesis.

The present study describes the use of molecular methods in studying infectious salmon anaemia virus (ISAV), an important pathogen of farmed salmon in Norway, Scotland, the Faeroe Islands, Canada, USA and Chile. The nucleotide sequences of the haemagglutinin gene (HA) from 70 ISAV isolates have been analysed for phylogenetic relationship and the average mutation rate of nucleotide substitutions calculated. The isolates constitute 2 major groups, 1 European and 1 North American group. The isolate from Chile is closely related to the North American isolates. The European isolates can be further divided into 3 separate groups reflecting geographical distribution, time of collection, and transmission connected with farming activity. Based on existing information about infectious salmon anaemia (ISA) and new information emerging from the present study, it is hypothesised that: (1) ISAV is maintained in wild populations of trout and salmon in Europe; (2) it is transmitted between wild hosts mainly during their freshwater spawning phase in rivers; (3) wild salmonids, mainly trout, possibly carry benign wild-type ISAV isolates; (4) a change (mutation) in virulence probably results from deletions of amino acid segments from the highly polymorphic region (HPR) of benign wild-type isolates; (5) ISA emerges in farmed Atlantic salmon when mutated isolates are transmitted from wild salmonids or, following mutation of benign isolates, in farmed salmon after transmission from wild salmonids; (6) farming activity is an important factor in transmission of ISAV between farming sites in addition to transmission of ISAV from wild salmonids to farmed salmon; (7) transmission of ISAV from farmed to wild salmonids probably occurs less frequently than transmission from wild to farmed fish due to lower frequency of susceptible wild individuals; (8) the frequency of new outbreaks of ISA in farmed salmon probably reflects natural variation in the prevalence of ISAV in wild populations of salmonids.

Haemorrhagic smolt syndrome (HSS) in Norway: pathology and associated virus-like particles.

Atlantic salmon Salmo salar pre-smolt, smolt and post-smolt, with clinical signs of haemorrhagic smolt syndrome (HSS) have been found in several locations along the Norwegian coast (Rogaland to Troms). Affected fish had pale gills and bleeding at the fin bases, but seemed to be in good physical condition with no obvious weight loss. The internal organs and body cavity showed distinct bleedings. Petechiae were found on the gastrointestinal tract, swim bladder and peritoneum, visceral adipose tissue, heart and somatic musculature. The liver was bright yellow and sometimes mottled with petechiae and ecchymoses. Acitic fluid was found in the visceral cavity and fluid was also present in the pericardial cavity. Histological examination revealed haemorrhage in most organs. The glomeruli were degenerated and the renal tubules were filled with erythrocytes. The aims of this study were to describe the pathology and discover, if possible, the aetiology of the HSS. Tissues were collected for light and transmission electron microscopy (TEM), immunofluorescence (IFAT), reverse transcription (RT)-PCR diagnostics (screening for infectious salmon anaemia virus [ISAV], viral haemorrhagic septicaemia virus [VHSV], salmon pancreas disease virus [SPDV], sleeping disease virus [SDV] and infectious haematopoetic necrosis virus [IHNV]), and tissue homogenates (heart, liver, kidney and spleen) were sterile-filtered and inoculated into cell cultures. Homogenates made from several tissues were also injected intraperitoneally into salmon and rainbow trout Oncorhynchus mykiss. The diagnostic tests revealed no consistent findings of any pathogens, with the exception of TEM which showed 2 types of virus-like particles: Type I was 50 to 60 nm in diameter and Type II about 50 nm in diameter. These virus-like particles were found in salmon from all farms affected by HSS and screened by TEM. Several different cells, blood vessel endothelial cells, endocardial cells, heart myofibres, and leukocytes were associated with the 2 virus-like particles. The Type I particle seems to be an infectious pancreatic necrosis (IPN)-like virus, while (based on the number of target cells, particle morphology, budding and uptake into target cells) Type II particle could be a togavirus.

Transmission of infectious salmon anaemia virus (ISAV) in farmed populations of Atlantic salmon (Salmo salar).

In the present study, 24 smolt production sites were screened for the presence of infectious salmon anaemia virus (ISAV) with the help of a specific real-time RT PCR assay, and 22 of these sites had smolts that were positive. If these smolt production sites are representative for the prevalence of ISAV in Norwegian smolts, then most marine production sites must be considered to be positive for ISAV. In addition, 92 European ISAV isolates have been genotyped based on the hemagglutinin-esterase gene (HE), and their distribution pattern was analysed. This pattern has been coupled to information about the origin of smolt, eggs, and broodfish in those cases where it has been possible to obtain such information, and with information about ISAV in neighbouring farms. The pattern suggests that an important transmission route for the ISAV could be that the salmon farming industry in Norway is circulating some of the isolates in the production cycle, i.e. some sort of vertical or transgenerational transmission may occur. It has also been shown that avirluent ISAV isolates are fairly common in Norwegian farmed salmon. Based on this, it is hypothesized that the change from avirulent to virulent ISAV isolates is a stochastic event that is dependent on the replication frequency of the virus and the time available for changes in a highly polymorphic region (HPR) of the HE gene to occur. This, and the possibility that only avirluent ISAV isolates are vertically transmitted, may explain why ISA most often occurs at marine sites and why no more than about 15 farms get ISA every year in Norway.