Studies on resistance to infectious pancreatic necrosis virus in Atlantic salmon (Salmo salar)

Abstract

Infectious pancreatic necrosis virus (IPNV) is responsible for serious mortalities in postsmolt Atlantic salmon (Salmo salar) annually in both Scotland and Norway. The huge financial losses involved and the lack of control measures have prompted attempts to produce IPN vaccines and disease resistant strains of fish. A characteristic of IPNV is its ability to persist in a latent carrier state and to ensure stocks of fish are truly virus free there is a need for highly sensitive diagnostic methods. In this study microsatellites were employed to assign fish susceptible to IPN in the field to families. Laboratory challenges were performed on 24 families and various humoral immune parameters were measured. A variety of diagnostic techniques were also developed, their sensitivity and specificity evaluated and compared to tissue culture. Parental Atlantic salmon and offspring susceptible to IPN in the field were genotyped using a published multiplex microsatellite PCR adapted for use with fluorescent genotyping systems. The reliability and utility of the genotyping system was evaluated with positive results. The majority of susceptible fish could not be assigned to parents since the fish were found to be from a mixed stock. Various experimental challenges with IPNV infected tissue homogenate were performed on post-smolts, however they did not produce mortality or induce clinical IPN. As a result of this virus titre was used as an indirect measurement of disease resistance. Atlantic salmon post-smolts were found to effectively clear cell cultured IPNV, isolate Br from Shetland. A second Shetland isolate (Os) was found to actively replicate in the fish while isolate Br was rapidly cleared. To increase virulence isolate Os was passaged once further through fish prior to use in challenging 24 families of PIT 0 8 tagged Atlantic salmon. At week 3 mean viral titres for each family ranged from 10 to 1011 TCID5o/g kidney. Using these data six families were targeted to follow through to week 7, two with low titres, two with medium titres and two with high titres of IPNV. 0 8 15 At week 7 virus titres had decreased to between 10 ' and 1 0 '' TCIDso/g kidney, many of the fish in the families with low IPNV titres at week 3 had cleared the virus. Lysozyme and neutralising antibody responses were measured, however little or no correlation was found between either parameter and IPNV titre at weeks 3 or 7. The control fish were found to be positive for IPNV despite earlier negative testing. Reasons for this were unclear, the fish may have been exposed to the virus through the seawater inflow or alternatively the fish may have been carriers of the virus. Diagnostic techniques employed to detect IPNV were RT-PCR, ELISA and immunohistochemistry; cell culture was used as a comparison to the RT-PCR and ELISA. The RT-PCR was developed using two published IPNV primer sets both of which amplified regions on VP2, they were found to be specific to IPNV and together amplified 9 out of 10 serotypes of IPNV. The most sensitive primer set detected between 107 and 108 TCID50 IPNV/ml in spiked tissue, in comparison to the ELISA which detected 1012'5 TCID50 IPNV/ml in tissue homogenate. The AS-1 monoclonal antibody was used successfully in immunohistochemistry to identify IPNV in fixed tissue. The antibody was found to identify IPNV in pancreatic lesions from infected fish, but did not detect the virus in the gut. All of these methods were found to be less sensitive than cell culture, however they are very rapid and ELISA and RT-PCR may be used to confirm IPNV infection once CPE has been visualised in cell culture.