Multilocus variable-number tandem-repeat genotyping of Renibacterium salmoninarum, a bacterium causing bacterial kidney disease in salmonid fish.

BACKGROUND: Bacterial kidney disease (BKD), caused by Renibacterium salmoninarum, is a bacterial disease of fish, which is both geographically widespread and difficult to control. Previously, application of various molecular typing methods has failed to reliably discriminate between R. salmoninarum isolates originating from different host species and geographic areas. The current study aimed to utilize multilocus variable number tandem repeats (VNTR) to investigate inter-strain variation of R. salmoninarum to establish whether host-specific populations exist in Atlantic salmon and rainbow trout respectively. Such information would be valuable in risk assessment of transmission of R. salmoninarum in a multispecies aquaculture environment. RESULTS: The present analysis utilizing sixteen VNTRs distinguished 17 different haplotypes amongst 41 R. salmoninarum isolates originating from Atlantic salmon and rainbow trout in Scotland, Norway and the US. The VNTR typing system revealed two well supported groups of R. salmoninarum haplotypes. The first group included R. salmoninarum isolates originating from both Atlantic salmon and rainbow trout circulating in Scottish and Norwegian aquaculture, in addition to the type strain ATCC33209T originating from Chinook salmon in North America. The second group comprised isolates found exclusively in Atlantic salmon, of mainly wild origin, including isolates NCIB1114 and NCIB1116 associated with the original Dee disease in Scotland. CONCLUSIONS: The present study confirmed that VNTR analysis can be successfully applied to discriminate R. salmoninarum strains. There was no clear distinction between isolates originating from Atlantic salmon and rainbow trout as several haplotypes in group 1 clustered together R. salmoninarum isolates from both species. These findings indicate a potential exchange of pathogens between Atlantic salmon and rainbow trout in Scottish and Norwegian aquaculture during the last 20 years. In a scenario of expansion of rainbow trout farming into the marine environment, appropriate biosecurity measures to minimize disease occurrence are advised. The present results also suggest that R. salmoninarum isolates circulating in European aquaculture over the last 20 years are genetically distant to the wild strains originally causing BKD in the rivers Dee and Spey.

A strand specific real-time RT-PCR method for the targeted detection of the three species (vRNA, cRNA and mRNA) of infectious salmon anaemia virus (ISAV) replicative RNA.

Three species of viral-derived RNA (vRNA, cRNA and mRNA) are produced during an infectious salmon anaemia virus (ISAV) infection. Conventional real-time RT-PCR (RT-qPCR) targeting ISAV segment 8 provides a very sensitive method for the detection of ISAV RNA, however it does not differentiate between these three individual RNA species. In this study, strand-specific tagged primers have been utilised in the RT reaction to specifically produce cDNA corresponding to each of the 3 viral RNA types produced from ISAV segment 8 for the subsequent detection by real-time PCR. The RNA species-specific assay was successfully used to specifically distinguish synthetic T7-produced RNA transcripts representing the 3 species of ISAV RNA at levels up to approximately 10(5)-fold higher than the other types. In addition, the method was applied to investigate the production of segment 8 RNA in time-course tissue culture experiments performed at optimal (15°C), sub-optimal (20°C) and inadequate (25°C) temperatures for replication or in the presence of a chemical inhibitor to vary the RNA populations and investigate its effectiveness. Variation in RNA production was observed between the optimal and sub-optimal temperatures and in the presence of the chemical inhibitor. Production of all RNA species was completely inhibited at 25°C indicating the potential usefulness of the assay as a tool in the understanding of ISAV replication and transcription dynamics.

Surveillance for infectious salmon anaemia virus HPR0 in marine Atlantic salmon farms across Scotland.

Infectious salmon anaemia virus (ISAV) is a serious and commercially important pathogen of Atlantic salmon. Multiple viruses have been defined based on a highly polymorphic region (HPR) of the haemagglutinin-esterase (HE) protein encoded by genomic segment 6. The viruses causing disease outbreaks in farms to date all have deletions in this region with respect to a putative ancestral variant with a longer HPR (HPR0). The presence of HPR0 nucleic acid has been detected in many countries including Scotland, where it has mostly been associated with healthy wild and farmed fish. Pathogenic ISAVs appear to have been derived from HPR0 ancestors on multiple independent occasions, which suggests that the presence of HPR0 could represent a risk factor in the re-emergence of infectious salmon anaemia (ISA) disease. In order to better understand this potential risk factor, anonymous samples of gill and heart tissues from marine Atlantic salmon farms throughout Scotland were collected and screened for the presence of ISAV RNA. Since it has not been possible to isolate HPR0 in conventional ISA-permissive cell cultures, a sensitive real-time RT-PCR method was employed for the detection of viral RNA. DNA sequencing was carried out on the positive samples to determine their HPR sequence. ISAV RNA was detected in 6 samples originating from 4 different locations and sequence analysis indicated the viruses were of the HPR0 type. Full length segment 6 sequence analysis of 1 positive sample indicated that it was most similar to a European genotype sequence previously obtained from North America.