Susceptibility of goldsinny wrasse, Ctenolabrus rupestris L. (Labridae), to viral haemorrhagic septicaemia virus (VHSV) genotype III: Experimental challenge and pathology.

Cleaner fish, such as wrasse, are being increasingly used to combat the sea lice infestation of Atlantic salmon (Salmo salar L.) in many European countries. To determine susceptibility of the goldsinny wrasse (Ctenolabrus rupestris L.) and pathogenesis of the viral haemorrhagic septicaemia virus (VHSV) genotype III isolate 12-654, previously associated with VHSV infection in the Shetland Islands in 2012, fish were experimentally challenged by intraperitoneal injection (IP), bath immersion and cohabitation routes. Cumulative proportion of moribund wrasse reached 17% following the virus immersion challenge while by the IP-route moribunds exceeded 50% within 14days post-challenge. Typical signs of VHS as reported in rainbow trout (Oncorhynchus mykiss), were not observed in moribund goldsinny wrasse. The most pronounced histopathological changes, consistent regardless of the route of infection, were observed within the heart and included atrium myofibril degeneration, focal infiltration and multifocal necrosis, with prominent swelling of the endocardium and occasional detachment. Pathological changes in the atrium were associated with presence of the viral antigen as confirmed by a positive immunohistochemical staining. Virus clearance and heart tissue recovery were noted although further experiments are required to confirm these observations. The results of a cohabitation experiment confirmed that goldsinny wrasse shed viable virus and therefore represent a risk of virus transmission to other VHSV susceptible species. Similarities between the pathology in goldsinny wrasse induced through the controlled experimental challenges and that of wrasse spp. from an infection occurrence in Shetland are discussed.

Differential characterization of emerging skin diseases of rainbow trout--a standardized approach to capturing disease characteristics and development of case definitions.

Farmed and wild salmonids are affected by a variety of skin conditions, some of which have significant economic and welfare implications. In many cases, the causes are not well understood, and one example is cold water strawberry disease of rainbow trout, also called red mark syndrome, which has been recorded in the UK since 2003. To date, there are no internationally agreed methods for describing these conditions, which has caused confusion for farmers and health professionals, who are often unclear as to whether they are dealing with a new or a previously described condition. This has resulted, inevitably, in delays to both accurate diagnosis and effective treatment regimes. Here, we provide a standardized methodology for the description of skin conditions of rainbow trout of uncertain aetiology. We demonstrate how the approach can be used to develop case definitions, using coldwater strawberry disease as an example.

Hyperpigmentation in North Sea dab Limanda limanda. II. Macroscopic and microscopic characteristics and pathogen screening.

An increasing trend in the prevalence of hyperpigmentation in the common dab Limanda limanda from the North Sea prompted us to investigate the potential role of infectious agents as causes or contributing factors to the condition. Dab representing 3 severity grades of hyperpigmentation were sampled for virology, bacteriology, histopathology and ultrastructure assessments. No cytopathic effect was recorded during virology testing, and bacteriological results showed no differences between normal and hyperpigmented dab. Histopathological assessment showed that the most significant changes occurred in the dermis as a result of chromatophore hyperplasia, namely melanophores and iridophores, alongside loose melanin granules. Dermal lymphocytic infiltration occasionally expanding into the epidermis and the underlying musculature was more frequent in highly pigmented dab than in normal fish, suggesting an active immune response. Ultrastructure studies showed additional disruption of the epithelial layer, with loose melanin granules between cells and a number of single or aggregated melanocytes. Dab representing different grades of hyperpigmentation kept in the laboratory alongside normal fish for a monitoring period of 18 mo showed no changes in their pigment distribution pattern, nor occurrence of new pigment in the normal fish. The current investigation found no association of hyperpigmentation in the common dab with infectious agents; therefore, understanding the cause of the condition remains a challenge which can now more reliably focus on a non-infectious origin hypothesis.

Detection of salmonid alphavirus RNA in wild marine fish: implications for the origins of salmon pancreas disease in aquaculture.

Salmonid alphaviruses (SAVs), which include the aetiological agents of salmon pancreas disease (SPD) in farmed Atlantic salmon Salmo salar L. and sleeping disease (SD) in rainbow trout Oncorhynchus mykiss (Walbaum), are significant viral pathogens of European salmonid aquaculture. SAV is horizontally transmitted and the virus can survive for extended periods in seawater. A lack of convincing evidence for vertical transmission coupled to the fact that the SPD virus (SPDV) occurs in historically infected sites irrespective of fallow period duration suggests that a substantial reservoir of infection exists in the marine environment. We used a highly sensitive real-time PCR (qPCR) assay targeting a region of the SAV nsP1 gene to screen wild marine fish species for the presence of SAV in an attempt to identify such a potential reservoir. Screened fish species were caught in the vicinity of aquaculture activity in an area with a previous history of SAV infection (Shetland Isles, Scotland). SAV RNA was detected in internal organs (kidney and heart) from the flatfish species common dab Limanda limanda, long rough dab Hippoglossoides platessoides, and plaice Pleuronectes platessa. Based on these findings, sampling was extended to an area remote from aquaculture activity (Stonehaven Bay, NE coast of Scotland) from where heart tissues obtained from common dab also tested positive. While no virus could be cultivated from these samples, qPCR detections were shown to be SAV-specific by sequencing of an alternative gene region (E2) to that targeted by the qPCR assay. Analysis of these nucleotide sequences revealed minor differences to those previously obtained from farmed salmon, and subsequent phylogenetic analysis of an E2 dataset demonstrated a subtype V-like sequence.

Liver involvement in post-smolt Atlantic salmon, Salmo salar L., infected with infectious pancreatic necrosis virus (IPNV): a retrospective histopathological study.

Histological changes associated with infectious pancreatic necrosis virus (IPNV) infection have historically been described for the pancreas and gut, but any involvement of the liver was poorly acknowledged or described. The aims of this study were to find robust evidence that the reported increase in liver pathology in Atlantic salmon post-smolts in natural outbreaks was effectively related to IPNV infection and retrospectively to report when such a shift in the involvement of the liver had taken place, supported by a histopathological description for a differential diagnosis. The study reports new findings concerning the dynamics of liver pathology development, with apoptosis, demonstrated by histological and immunological techniques, described as the most relevant and particular feature. Immunohistochemical examination of affected liver suggests apoptosis is not only the result of the virus infection itself but triggered through the action of the host's innate immune response. Liver involvement contributes to the nature of infection and becomes an important factor in the disease process. Additionally, it was established that the increase in infectious pancreatic necrosis prevalence is correlated with a new distinct pattern of outbreak distribution throughout the year. The role of smolt category (i.e. S1, S1/2 or S0), hence timing of seawater transfer as a strong correlating factor, is discussed.

Comparative experimental transmission of cardiomyopathy syndrome (CMS) in Atlantic salmon Salmo salar.

Cardiomyopathy syndrome (CMS) has been recorded in wild and farmed Atlantic salmon Salmo salar. Characteristic heart lesions primarily involving the myocardium are reported in natural outbreaks with associated mortality. To date, no experimental trials have reproduced these lesions in the laboratory. The present study reports on the first successful experimental transmission of CMS in Atlantic salmon in Scotland, with full development of the histological lesions that are described for the syndrome. Tissue homogenates of CMS-infected fish indicative of mild and severe lesions from Scottish and Norwegian natural outbreaks, respectively, were injected into naïve fish, and both induced heart lesions consistent with CMS. Lesion development was earlier and progression faster in the fish group receiving the Norwegian homogenate, but equivalent in both groups by the end time point of the experiment. The study demonstrated that the reported condition for both countries is identical, as evaluated through light microscopy, and that tissue homogenates from either mild or severely affected fish contain the transmissible agent.

Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections.

The identification of protozoan and metazoan parasites is traditionally carried out using a series of classical keys based upon the morphology of the whole organism. However, in stained tissue sections prepared for light microscopy, taxonomic features will be missing, thus making parasite identification difficult. This work highlights the characteristic features of representative parasites in tissue sections to aid identification. The parasite examples discussed are derived from species affecting finfish, and predominantly include parasites associated with disease or those commonly observed as incidental findings in disease diagnostic cases. Emphasis is on protozoan and small metazoan parasites (such as Myxosporidia) because these are the organisms most likely to be missed or mis-diagnosed during gross examination. Figures are presented in colour to assist biologists and veterinarians who are required to assess host/parasite interactions by light microscopy.

Infectious pancreatic necrosis virus in Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland: virus identification, histopathology, immunohistochemistry and genetic comparison with Scottish mainland isolates.

During mid-June 1999 peak mortalities of 11% of the total stock per week were seen at a sea cage site of Atlantic salmon, Salmo salar L., post-smolts in the Shetland Isles, Scotland. Virus was isolated on chinook salmon embryo (CHSE) cells in a standard diagnostic test and infectious pancreatic necrosis virus (IPNV) identified by enzyme-linked immunosorbent assay. IPNV was confirmed as serogroup A by a cell immunofluorescent antibody test using the cross-reactive monoclonal antibody AS-1. Four weeks after the main outbreak, virus titres in surviving moribund fish were assayed at >10(10) TCID50 g(-1) kidney. Histopathology of moribund fish was characterized by pancreatic acinar cell necrosis and a marked catarrhal enteritis of the intestinal mucosa. In the liver, necrosis, leucocytic infiltration and a generalized cell vacuolation were noted. IPNV-specific immunostaining was demonstrated in pancreas, liver, heart, gill and kidney tissue. The nucleotide sequence of the coding region of segment A was determined from the Shetland isolate. A 1180 bp fragment of the VP2 gene of this isolate was compared with a 1979 reference isolate from mainland Scottish Atlantic salmon, La/79 and another more recent mainland isolate, 432/00. Both A2 isolates were derived from carrier fish without signs of IPN and serotyped by a plaque neutralization test. The Shetland isolate shows a different nucleotide and amino acid sequence compared with the two isolates from carrier fish. These latter isolates showed identical amino acid sequences in the fragment examined, despite the 21 years separating the isolations. Sequence comparisons with other A2 (Sp) isolates on the database confirm all three Scottish isolates are A2 (Sp).

An evaluation of current diagnostic tests for the detection of infectious salmon anaemia virus (ISAV) following experimental water-borne infection of Atlantic salmon, Salmo salar L.

Four commonly used diagnostic tests [reverse transcription polymerase chain reaction (RT-PCR), indirect fluorescent antibody test (IFAT), virus culture and light microscopy] were evaluated for their ability to detect infectious salmon anaemia virus (ISAV) or tissue pathology following experimental infection of Atlantic salmon. Fish were infected with ISAV by water-borne exposure which mimics the route of natural infection. Forty-five per cent of pre-clinical fish tested yielded positive results by RT-PCR for at least one of the organs tested (kidney, heart, gill, liver, blood). No significant difference was detected between organs in the number or time of first occurrence of positive result. Virus culture identified a total of 14% of pre-clinical fish as ISAV-infected. The presence of ISAV in heart tissue was particularly notable (13% of fish sampled) as was the inability to culture virus from spleen tissue. In the case of IFAT, 15% of fish sampled were positive, although tissue other than kidney proved unsuitable for use in this method. Only limited ISAV-specific pathology was detectable by histological examination of fish prior to the onset of clinical disease. These findings reveal important information regarding the optimal choice of both tissue sample and diagnostic test for the routine diagnosis of ISAV.

Investigation into the susceptibility of saithe Pollachius virens to infectious salmon anaemia virus (ISAV) and their potential role as a vector for viral transmission.

Wild-caught saithe Pollachius virens were experimentally exposed to an isolate of infectious salmon anaemia virus (ISAV) of Norwegian origin. Mortality attributable to ISAV did not occur following exposure by intra-peritoneal (i.p.) injection of virus or by cohabitation with ISAV-infected Atlantic salmon Salmo salar. Despite the individual testing of 120 ISAV-exposed saithe, ISAV was not detectable using RT-PCR, the most sensitive ISAV diagnostic tool demonstrated to date. Furthermore, saithe exposed to ISAV-infected salmon were not capable of transmitting virus when transferred to tanks containing naïve salmon. Thus saithe appear to be resistant to this Norwegian isolate of ISAV and incapable of supporting its replication. Saithe which co-exist with salmon in and around aqua-culture facilities are considered unlikely to have a significant impact on the epizootiology of ISAV.

Gyrodactyloides bychowskii (Monogenea: Gyrodactylidae) from sea-caged Atlantic salmon Salmo salar in Scotland: occurrence and ribosomal RNA sequence analysis.

Gyrodactyloides bychowskii has been recorded in Scottish waters for the first time. The parasite was found on the gills of Atlantic salmon reared in seawater. An integrated morphological and molecular examination of the parasite was carried out. Prevalence of the parasite was greatest in February and declined to 0 by June in 1999. During 2000, parasites were located in March and November. An overdispersion of parasites was recorded, with intensity of infection reaching over 200 parasites per gill arch in some fish. Parasitised gill tissue showed hyperplasia and hypertrophy but not always at the site of parasite attachment. The internal transcribed spacer of the ribosomal RNA gene array was amplified by PCR and sequenced. This sequence shared greatest similarity with the internal transcribed spacer of Gyrdicotylus gallieni, followed by Gyrodactylus species. This is the first molecular analysis of this parasite and provides sequence data that may be used in comparison of G. bychowskii from other locations or in phylogenetic analysis of this group of Monogenea.

Experimental infection models and susceptibility of Atlantic salmon Salmo salar to a Scottish isolate of infectious salmon anaemia virus.

Infection models for both fresh and seawater salmon were established using a Scottish isolate of infectious salmon anaemia virus (ISAV). Modes of infection were intra-peritoneal injection, cohabitation and immersion exposure, and a range of doses was tested. Development of these models using a Scottish isolate of ISAV provided an approximation of the minimum infective dose leading to mortality under different infection regimens. The models also allow prediction of the time to first mortality and an estimation of expected total mortality following the various routes of infection. Such knowledge is important to the development of anti-ISAV vaccines and to future studies aimed at understanding the biology of ISAV in general.

The histopathology and ultrastructure of steatitis affecting common dab Limanda limanda.

This study presents a new description, based on histopathological and ultrastructural studies, of a disease affecting the common dab Limanda limanda (L.). The condition can be recognised by the presence of multiple orange or yellow lesions in the pterygophorial region of the fish. The principal histopathological features are necrosis of fat cells, extensive macrophage infiltration leading to the formation of granulomatous structures, and the accumulation of lipopigment by lipid peroxidation. Based on this description, the condition has been diagnosed as steatitis. Although pathology associated with lipid peroxidation is the dominant characteristic of the lesions examined, it is proposed that this process is secondary to necrosis of the adipose tissue. The aetiology is discussed in the light of these observations. In addition, the first record of this condition affecting long rough dab Hippoglossoides platessoides (Fabricius) is made.

Susceptibility of juvenile and sub-adult Atlantic halibut (Hippoglossus hippoglossus L.) to infection by Vibrio anguillarum and efficacy of protection induced by vaccination.

Experimental bath challenge of juvenile and sub-adult Atlantic halibut with Vibrio anguillarum induced severe mortalities of 47 and 80%, respectively. However, animals vaccinated with a commercial V. anguillarum vaccine demonstrated excellent protection against the disease (100% RPS). This study also describes the gross pathology and histological changes associated with this infection. A loss of coordination, haemorrhage at the fin base and splenomegaly were frequent findings. Serum agglutinating activity demonstrated a rise following vaccination, the mean log2 titre rising from 3.8 to 8.4. This was associated with a significant rise in antibody-mediated complement killing ability of immune serum when compared to non-immune serum.

Pathology attributed to Mycobacterium chelonae infection among farmed and laboratory-infected Atlantic salmon Salmo salar.

This study was promoted following concern over increasing mortality on 2 farms rearing Atlantic salmon Salmo salar in the Shetland Isles, Scotland. A Mycobacterium sp. was isolated from moribund, market-sized Atlantic salmon. Biochemical tests, lipid analysis and PCR (polymerase chain reaction) techniques confirmed the bacterium to be Mycobacterium chelonae. Multiple greyish-white miliary granuloma-like nodules were observed in several tissues. Dense hard-packed nodules contained abundant acid-fast bacteria. Atlantic salmon injected with M. chelonae remained sub-clinically infected, demonstrating the chronic nature of this disease. The source of the pathogen was not identified.

Vibrio viscosus in farmed Atlantic salmon Salmo salar in Scotland: field and experimental observations.

Winter mortality occurred in market-sized (2 to 3 kg) Atlantic salmon Salmo salar reared in sea cages in Scottish waters. Many of the fish had skin ulcers. Internally prominent dark-brown petechiae or ecchymotic haemorrhage was observed. Splenomegaly was associated with congestion and widespread necrosis. A Vibrio sp. was isolated from internal organs. Biochemically isolates of the bacterium were similar to a previously described bacterium, Vibrio viscosus, recorded in a phenotypic study from farmed salmon in Norway. This work examines the occurrence of V. viscosus in marine-reared Atlantic salmon for the first time in Scottish waters. An experimental study reproduced the field observations and Koch's postulates were fulfilled. The histopathology associated with natural infection was compared with that in laboratory-infected fish.