D. W. Bruno

A colour atlas of salmonid diseases

Normal teleost histology autopsy procedures disease diagnosis viral diseases bacterial diseases fungal diseases parasitic diseases metazoa diseases due to nutritional imbalance tumour malformations environmental factors affecting fish health problems associated with poor husbandry disease of uncertain aetiology staining techniques for light microscopy formulae for fixatives, declarification and other solutions for light microscopy.

Diseases and disorders of finfish in cage culture

Overview of cage cutlure marine and brackish water - coldwater fishes freshwater - coldwater fishes marine and brackish water - warmwater fishes fresh water - warmwater fishes noninfectious disorders emerging infectious diseases.

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.

Comparative susceptibility of Arctic char (Salvelinus alpinus), rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) to the Scottish isolate of infectious salmon anaemia virus

Experimental infection studies were conducted to determine the susceptibility of pathogen-free Arctic char (Salvelinus alpinus), rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta) to the Scottish isolate of infectious salmon anaemia virus (ISAV). No mortalities were recorded in any of these species following infection by intra-peritoneal injection, although transient drops in haematocrit were recorded. Sub-samples of all species were screened for the presence of ISAV using RT-PCR and virus culture. While virus was unculturable from all samples at 40 days post-infection, virus was detectable using RT-PCR in all species. Clearance of virus following experimental infection did however progress at a greater rate in Arctic char than in rainbow trout and brown trout. The potential for this species to act as a long-term carrier of ISAV, and thus exert a role in the epizootiology of ISAV in Scotland may be less than in other salmonid species. The increasing commercial production of Arctic char render these findings significant to the future management of ISAV in Scotland.

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.

A method to measure an indicator of viraemia in Atlantic salmon using a reporter cell line

RTG-P1 is a transgenic fish cell line producing luciferase under the control of the IFN-induced Mx rainbow trout gene promoter. This cell line was used to measure viraemia of Salmonid alphavirus (SAV), the cause of Salmon Pancreas Disease (SPD), a serious disease in farmed Atlantic salmon. Two SAV genotype 1 (SAV1) isolates were used in this study, F93-125 (tissue-culture adapted, from Ireland) and 4640 (from a field case in Scotland). The kinetics and magnitude of luciferase activity were monitored versus the time of infection. During a direct infection experiment, the induction of luciferase significantly increased 16- and 4-fold after incubation for 6 days with F93-125 at 15 and 20°C, respectively. Filtration and heat treatment experiments demonstrated that the luciferase induction in RTG-P1 was dependent on viral replication. Unlike many cell lines used in fish viral diagnostic, RTG-P1 is not sensitive to salmonid serum, therefore, viraemia could be successfully monitored on serum collected from fish during a cohabitation challenge with 4640 isolate. A peak of viraemia could be detected 16 days post IP inoculation of the shedders. This novel cost-effective method to measure viraemia does not rely on development of cytopathic effect (CPE) in culture, is compatible with non-lethal blood collections in fish and can be used to assign emerging diseases to a viral aetiology.

Hyperpigmentation in North Sea dab Limanda limanda. I. Spatial and temporal patterns and host effects

Hyperpigmentation is a term describing a specific pigment anomaly affecting common dab Limanda limanda in the North Sea and, less frequently, in adjacent areas, e.g. the English Channel, Irish and Celtic Seas, western Baltic Sea and Icelandic waters. Other North Sea flatfish species are also affected, but at a markedly lower prevalence. The condition is characterised by the occurrence of varying degrees of green to black patchy pigment spots in the skin of the upper (ocular) body side and pearly-white pigment spots in the skin of the lower (abocular) body side. In the course of fish disease monitoring programmes carried out by Germany and the UK (England and Scotland), a pronounced spatial pattern of hyperpigmentation has been detected in the North Sea. An increase in prevalence has been recorded in almost all North Sea areas studied in the past 2 decades. The prevalence recorded in hot spot areas of the condition increased from 5 to >40% between 1988 and 2009. Analysis of the German data indicates that the prevalence and intensity (degree of discolouration) of hyperpigmentation increase with size and age, indicating a temporal progression of the condition with size and age. Intense hyperpigmentation is associated with increased growth (length) and decreased condition factor. Potential causes of the condition (UV-B radiation nutrition, water temperature increase, demographic changes) and, in particular, of the spatial/temporal patterns recorded as well as the relationship to host-specific factors (sex, age, length, growth, condition factor) are discussed.

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.

Common dab, Limanda limanda (L.), as a natural carrier of salmonid alphavirus (SAV) from waters off north‐west Ireland

Pancreas disease (PD) and sleeping disease (SD) are important, largely chronic conditions of farmed Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss), respectively, and are caused by salmonid alphavirus (SAV) (Togaviridae) (McLoughlin & Graham 2007). Six SAV subtypes have been distinguished to date utilizing the partial E2 and nsP3-gene sequence data with subtypes I, IV, V and VI present in Scotland and Ireland, subtype II dominant in the UK and continental Europe and subtype III restricted to Norway (Fringuelli et al. 2008). The first evidence of a wild reservoir for SAV was reported by Snow et al. (2010) where SAV V was detected in several species of flatfish (Pleuronectidae), and later on, Bruno et al. (2014) and McCleary et al. (2014) confirmed the presence of SAV I, II and V in common dab and plaice. Also for first time the virus was successfully cultured on a salmonidderived cell line (Bruno et al. 2014). The present article aims to survey SAV in common dab from waters off the north-west coast of Ireland and report on apparent prevalence in wild fish. In total, 120 common dab were collected in Irish waters approximately 70 nautical miles from the north-west coast in February 2012 (Fig. 1). Gross changes consistent with epidermal hyperplasia/papilloma, lymphocystis and acute/healing skin ulcers and hyperpigmentation on the ocular side from individual dab were noted. Total RNA was extracted from 5 mg of heart tissue (QIAsymphony RNA Kit; Qiagen), genomic DNA was removed (DNA-free DNA Removal Kit; Life Technologies), and cDNA was synthesized (Snow et al. 2010). A nsP1 RT-qPCR assay (Hodneland & Endresen 2006) and 12s RNA endogenous control assay (McCleary et al. 2014) were run on the LightCycler 480 detection system (Roche) using Quanta Custom Toughmix (Quanta Biosciences). A sample was recorded as SAV positive when crossing point (Cp) values were generated in all three replicates. Partial E2 gene was amplified using a nested PCR and sequenced according to Bruno et al. (2014). Virus culturing was attempted using heart and kidney tissue shown positive by nsP1 RT-qPCR. The prevalence and 95% confidence intervals (CI) were estimated from a generalized linear model assuming a binomial error distribution and comprising the intercept only as the explanatory variable (Nelder & Wedderburn 1972). Four dab tested SAV positive by nsP1 RT-qPCR assay in all triplicates (Table 1). This corresponds to an apparent prevalence of SAV type I of 3.3% (95% CI 1.3%–8.5%). No cytopathic effect was observed in the tissue culture, and supernatant tested negative for SAV by RT-qPCR. Sequencing was attempted from all positive samples, and a partial E2 sequence (KP091737) was obtained for Correspondence: J Simons, Marine Scotland Science, 375 Victoria Road, Aberdeen AB11 9DB, UK (e-mail: judy.simons@scotland.gsi.gov.uk)

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 hosts 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.