Monitoring the immune response in fish over the progression of a disease is traditionally carried out by experimental infection whereby animals are killed at regular intervals and samples taken. We describe here a novel approach to infectiology for salmonid fish where blood samples are collected repeatedly in a small group of PIT-tagged animals. This approach contributes to the reduction of animals used in research and to improved data quality. Two groups of 12 PIT-tagged Atlantic salmon (Salmo salar) were i.p infected with Infectious Salmon Anaemia Virus (ISAV) or culture medium and placed in 1 m3 tanks. Blood samples were collected at 0, 4, 8, 12, 16, 21 and 25 days post infection. The viral load, immune and stress response were determined in individual fish by real-time quantitative PCR (QPCR) on the blood cells, as well as the haematocrit used as an indicator of haemolysis, a clinical consequence of ISAV infection. “In-tank” anaesthesia was used in order to reduce the stress related to chase and netting prior to sampling. The data were analysed using a statistical approach which is novel with respect to its use in fish immunology. The repeated blood collection procedure did not induce stress response as measured by HSP70 and HSP90 gene expression in the un-infected animals. A strong increase in viraemia as well as a significant induction of Mx and γIP gene expression were observed in the infected group. Interleukin 10 was found induced at the later stage of the infection whereas no induction of CD8 or γ IFN could be detected. These results and the advantages of this approach are discussed.

Amoebic gill disease (AGD) in farmed Atlantic salmon is caused by the amoeba Paramoeba perurans. The recent establishment of in vitro culture techniques for P. perurans has provided a valuable tool for studying the parasite in detail. In this study, flow cytometry was used to generate clonal cultures from single-sorted amoeba, and these were used to successfully establish AGD in experimental Atlantic salmon. The clonal cultures displayed differences in virulence, based on gill scores. The P. perurans load on gills, determined by qPCR analysis, showed a positive relationship with gill score, and with clonal virulence, indicating that the ability of amoebae to proliferate and/or remain attached on gills may play a role in virulence. Gill scores based on gross signs and histopathological analysis were in agreement. No association between level of gill score and specific gill arch was observed. It was found that for fish with lower gill scores based on histopathological examination, gross examination and qPCR analysis of gills from the same fish were less successful in detecting lesions and amoebae, respectively.

Summary Genetic sequence data from pathogens presents a novel means to investigate the spread of infectious disease between infected hosts, or infected premises, complementing traditional contact-tracing approaches, and much recent work has gone into the development of methods for this purpose. The objective is to recover the epidemic transmission tree, which identifies who infected whom. This paper reviews the various approaches that have been taken. The first step is to define a measure of difference between sequences, and factors such as recombination and convergent evolution must be taken into account. Three broad categories of method, of increasing complexity, exist: those that assume no within-host genetic diversity or mutation, those that assume no within-host diversity but allow mutation, and those which allow both. Until recently, the assumption was usually made that every host in the epidemic could be identified, but this is now being relaxed, and some methods are intended for sparsely sampled data, concentrating on the identification of pairs of sequences that are likely to be the result of direct transmission rather than inferring the complete transmission tree. Many of the procedures described here are available to researchers as free software.

Viral haemorrhagic septicaemia virus (VHSV) was isolated from five species of wrasse (Labridae) used as biological controls for parasitic sea lice predominantly, Lepeophtheirus salmonis (Krøyer, 1837), on marine Atlantic salmon, Salmo salar L., farms in Shetland. As part of the epidemiological investigation, 1400 wild marine fish were caught and screened in pools of 10 for VHSV using virus isolation. Eleven pools (8%) were confirmed VHSV positive from: grey gurnard, Eutrigla gurnardus L.; Atlantic herring, Clupea harengus L.; Norway pout, Trisopterus esmarkii (Nilsson); plaice, Pleuronectes platessa L.; sprat, Sprattus sprattus L. and whiting, Merlangius merlangus L. The isolation of VHSV from grey gurnard is the first documented report in this species. Nucleic acid sequencing of the partial nucleocapsid (N) and glycoprotein (G) genes was carried out for viral characterization. Sequence analysis confirmed that all wild isolates were genotype III the same as the wrasse and there was a close genetic similarity between the isolates from wild fish and wrasse on the farms. Infection from these local wild marine fish is the most likely source of VHSV isolated from wrasse on the fish farms.

Observations from the field and experimental evidence suggest that different strains of infectious salmon anaemia virus (ISAV) can induce disease of varying severity in Atlantic salmon. Variation in host mortality and dissemination of ISAV isolates with high and low virulence was investigated using immersion challenge; from which mortality, pathological, immunohistochemical and preliminary molecular results have been previously published. Here, real-time RT-PCR analysis and statistical modelling have been used to further investigate variation in virus load and the response of four select immune genes. Expression of type I and II interferon (IFN), Mx and γIFN induced protein (γIP) to high and low pathogenic virus infection were examined in gill, heart and anterior kidney. In addition, a novel RNA species-specific assay targeting individual RNA types was used to investigate the separate viral processes of transcription and replication. Unexpectedly, the low virulent ISAV (LVI) replicated and transcribed more rapidly in the gills compared to the highly virulent virus (HVI). Subsequently LVI was able to disseminate to the internal organs more quickly and induced a more rapid systemic immune response in the host that may have offered some protection. Contrary to this, HVI initially progressed more slowly in the gills resulting in a slower generalised infection. However HVI ultimately reached a higher viral load and induced a greater mortality.