Patricia A. Noguera

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.

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.

Use of Salmon Cardiac Primary Cultures (SCPCs) of different genotypes for comparative kinetics of mx expression, viral load and ultrastructure pathology, after infection with Salmon Pancreas Disease Virus (SPDV)

ABSTRACT In vitro fish based models have been extensively applied in human biomedical research but, paradoxically, less frequently in the research of fish health issues. Farmed Atlantic salmon can suffer from several viral conditions affecting the heart. Therefore, species‐specific, cardiac in vitro models may represent a useful tool to help further understanding and management of these diseases. The mechanisms underlying genotype based resistance are complex and usually rely on a combined effect of elements from both the innate and adaptive immune response, which are further complicated by external environmental factors. Here we propose that Salmon Cardiac Primary Cultures (SCPCs) are a useful tool to investigate these mechanisms as the basis for genotypic differences between Atlantic salmon families in susceptibility to cardiotropic viral disease. Using SCPCs produced from two different commercially available Atlantic salmon embryonated ova (Atlantic Ova IPN sensitive” (S) and “Atlantic QTL‐innOva® IPN/PD” (R)), the influence of host genotype on the viral load and mx expression following Salmon Pancreas Disease Virus infection was assessed over a 15 day period. Both R and S SCPCs groups were successfully infected. A measurable difference between groups of viral nsP1 and host antiviral mx gene expression was observed (i.e. a later, but larger onset of mx expression in the R group). Mx expression peaks were followed by a decrease in viral nsP1 in both groups. Additionally, ultrastructural examination of infected SCPCs allowed the description of degenerative changes at the individual cell level. The SCPC model presents some advantages, over current fish cell culture monolayers and in vivo material, such as the presence of different cell components normally present in the target organ, as well as the removal of a layer of functional complexity (acquired immunity), making it possible to focus on tissue specific, early innate immune mechanisms. These preliminary results highlight the importance of considering genetic origin when selecting the fish source for the production of SCPCs, as well as their usefulness as screening tools for assessment of genotypic differences in disease resistance. HighlightsSCPCs represent as a useful tool to investigate fish cardiotropic viral conditions.Using a disease resistance trait, genotypic screening with SCPCs seems possible.Host mx and viral nsP1 expressions showed differences after SPDV infection.There was a later but larger onset of mx expression in the resistant genotype SCPCs.Ultrastructural examination shows single cell degenerative changes from 24hpi.

Fungal and Related Oomycete Infections

The oomycetes (fungal-like) occur as primary and secondary agents of infection among wild and farmed salmonids in fresh and sea water. These infections can prove fatal, at least for farmed fish, if preventative measures are not undertaken. Molecular sequencing has improved our understanding of the phylogenetic relationships within this taxonomically diverse group with current evidence indicating these organisms evolved from simple holocarpic marine parasites. This chapter covers the commonly encountered oomycetes infections in salmonids.

Production Diseases and Other Disorders

Production related diseases and disorders cover a large area that the authors believe deserves increased attention, although for some areas we cannot cover the subject in great depth. These refer to a wide range of conditions that may or not be attributed to a biological agent with examples discussed in this chapter covering environmental related conditions, vaccination, developmental and congenital abnormalities and disorders, dietary imbalance, disorders affecting the heart and the eye, general skeletal abnormalities, and deformities among eggs and fry and predators.

Post-mortem Examination and the Recognition of Tissue Abnormalities

Necropsy is an essential part of an investigation of fish health of wild and farmed fish. Information on management practices and diet, detailed history of mortality, changes in fish behaviour, stock weight and length, management practices (recent or former transport, grading or treatments) as well as feeding response, are all important factors normally available with farmed fish. For wild fish, as much information as possible should also be collected and in both scenarios, water temperature, chemical and physical characteristics should be recorded with notes of any concurrent or recent event affecting other species in the area. The chapter describes the procedures of necropsy with particular reference to obtaining samples of the most common tissues collected for histological examination.

Pathology and Disease Diagnosis

A methodical approach is a prerequisite for an accurate diagnosis and requires a description of the tissue changes that occur following infectious and non-infectious conditions. Cells have a limited repertoire of morphological response to injury which is linked to biochemical mechanisms that determine the outcome of cell damage, thus accounting for the appearance of cells within lesions inducing general pathological changes, rather than those that are pathognomonic. This chapter covers the different types of cell and tissue responses to acute or chronic injury.