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.

Expression of Mx mRNA following infection with IPNV is greater in IPN-susceptible Atlantic salmon post-smolts than in IPN-resistant Atlantic salmon parr.

The Mx response was compared in parr and post-smolt Atlantic salmon following intra-peritoneal injection of the same dose of Infectious Pancreatic Necrosis Virus (IPNV) per g of fish. Mx gene expression, measured by quantitative RT-PCR in liver, showed a maximum level 3days after injection in parr with undetectable levels on day 7. In post-smolts, similar levels as in parr were attained on day 3, but levels then continued to rise on day 5 and 7 to about 10 times higher than the peak level in parr. Poly I:C injected parr showed Mx levels similar to IPNV injected post-smolts. Mortality from IPN in post-smolts occurred on days 6 and 7. Levels of IPN VP2 transcripts in parr were very low and did not increase with time, suggesting viral replication was low. Individual variation in levels of Mx and IPN VP2 gene transcripts was very high in post-smolts and although data is limited there was an inverse relationship between the levels of Mx and VP2, suggesting that individuals with high Mx levels on day 5 may be able to prevent viral replication. This contrasts with the response in parr, where IPN-resistance was not associated with a high Mx response.

Expression kinetics of interferon and interferon-induced genes in Atlantic salmon (Salmo salar) following infection with infectious pancreatic necrosis virus and infectious salmon anaemia virus.

Infectious pancreatic necrosis virus (IPNV) and infectious salmon anaemia virus (ISAV) are economically important pathogens of the salmonid aquaculture industry. Atlantic salmon were challenged by intraperitoneal injection (i.p.) with either virus followed by time-course sampling. Cohabiting fish in the IPNV challenge were also sampled. Kidney tissue was analysed using a TaqMan real-time PCR assay to measure the expression of a range of host immune genes in relation to the endogenous control, elongation factor 1 alpha (ELF). Host genes measured included Mx, type I and type II interferon (IFN), gammaIFN induced protein (gammaIP), interleukin-1 beta (IL-1beta) and tumour necrosis factor alpha (TNF-alpha). Viral levels were also measured. In i.p. injected fish, both viruses greatly induced expression of Mx, gammaIP, type I and type II IFN by day 6 post-infection, however only ISAV caused substantial mortality. Some differences between the expression kinetics produced by both viruses were noted. Infection with ISAV increased IL-1beta expression following day 6, but no effect was seen in fish infected with IPNV. Neither virus induced TNF-alpha expression. This study confirms the presence of both type I and type II IFN responses and their induced genes in Atlantic salmon upon infection with an orthomyxovirus and a birnavirus.

Development, application and validation of a Taqman real-time RT-PCR assay for the detection of infectious salmon anaemia virus (ISAV) in Atlantic salmon (Salmo salar).

Infectious salmon anaemia (ISA) is a disease of cultured Atlantic salmon (Salmo salar) which was successfully eradicated from Scotland following its emergence in 1998. The rapid deployment of sensitive diagnostic methods for the detection of ISA virus (ISAV) was fundamental to the swift eradication of ISA disease in Scotland and continues to be of crucial importance to surveillance of the aquaculture industry. This study reports the development, validation, application and interpretation of two independent, highly sensitive and specific semi-quantitative Taqman real-time RT-PCR (qRT-PCR) methods for the detection of ISAV. Such technology offers considerable advantages over conventional RT-PCR methods in current routine use for ISAV surveillance. These include an increased sensitivity, enhanced specificity, semi-quantification using endogenous controls, a lack of subjectivity in results interpretation, speed of processing and improved contamination control.

Molecular markers for gyrodactylids (Gyrodactylidae: Monogenea) from five fish families (Teleostei).

Thirty-one gyrodactylid species from five families of freshwater fish were examined and variable region V4 of the 18S small subunit ribosomal RNA gene and ribosomal RNA internal transcribed spacers ITS1 and ITS2 were sequenced. Both the V4 region and spacers ITS1 and ITS2 proved useful for gyrodactylid diagnosis. Sequences of these fragments exhibited interspecific variations and allowed clear determination at the species level. In some cases, the length of the ITS1 PCR fragment provided useful genetic markers. Species that yielded a short ITS1 fragment also showed distinct groupings in ITS2 and V4 sequences that were markedly different to sequences from species that contain a long ITS1. Repetitive sequences located in the ITS1 of Gyrodactylus gobii and Gyrodactylus vimbi accounted for some of the variations in length of PCR products. There was no evidence for intraspecific variation within these regions and short tandem repeats were not found in the other species studied. The number of polymorphic and intraspecific variations in nucleic acid sequences was low, therefore these variations did not affect species determination of gyrodactylids. Minor differences in the sequences between Western and Eastern European populations were detected for Gyrodactylus salaris/Gyrodactylus thymalli, Gyrodactylus teuchis and Gyrodactylus truttae, but these do not affect species diagnosis based on ribosomal DNA sequence. These results confirm the utility of both variable region V4 and the ITS as molecular markers for Gyrodactylus species.