Detection of salmonid alphavirus RNA in wild marine fish: implications for the origins of salmon pancreas disease in aquaculture

Abstract

Salmonid alphaviruses (SAVs), which include the aetiological agents of salmon pancreas disease (SPD) in farmed Atlantic salmon Salmo salar L. and sleeping disease (SD) in rainbow trout Oncorhynchus mykiss (Walbaum), are significant viral pathogens of European salmonid aquaculture. SAV is horizontally transmitted and the virus can survive for extended periods in seawater. A lack of convincing evidence for vertical transmission coupled to the fact that the SPD virus (SPDV) occurs in historically infected sites irrespective of fallow period duration suggests that a substantial reservoir of infection exists in the marine environment. We used a highly sensitive real-time PCR (qPCR) assay targeting a region of the SAV nsP1 gene to screen wild marine fish species for the presence of SAV in an attempt to identify such a potential reservoir. Screened fish species were caught in the vicinity of aquaculture activity in an area with a previous history of SAV infection (Shetland Isles, Scotland). SAV RNA was detected in internal organs (kidney and heart) from the flatfish species common dab Limanda limanda, long rough dab Hippoglossoides platessoides, and plaice Pleuronectes platessa. Based on these findings, sampling was extended to an area remote from aquaculture activity (Stonehaven Bay, NE coast of Scotland) from where heart tissues obtained from common dab also tested positive. While no virus could be cultivated from these samples, qPCR detections were shown to be SAV-specific by sequencing of an alternative gene region (E2) to that targeted by the qPCR assay. Analysis of these nucleotide sequences revealed minor differences to those previously obtained from farmed salmon, and subsequent phylogenetic analysis of an E2 dataset demonstrated a subtype V-like sequence.

Identification of a wild reservoir of salmonid alphavirus in common dab Limanda limanda, with emphasis on virus culture and sequencing

“…The generally high Ct values re ported in the current study are indicative of a low level of viral RNA in heart tissue. This confirms and extends previous work that identified the presence of SAV RNA in heart and kidney tissue collected from flatfish in Scottish coastal waters, but which failed to isolate the virus (Snow et al 2010). Based on the presence of SAV in dab in a locality remote from salmonid aquaculture, we concluded that a reservoir of SAV in non-salmonid wild fish probably existed prior to the largescale development of salmonid aquaculture in Scotland (Nash 2011).…”

“…A previous study using RT-qPCR detected a potential wild reservoir of SAV in several flatfish species, but failed to provide follow-up evidence in support of this result, e.g. successful isolation of the virus (Snow et al 2010). The objectives of the current study were (1) to determine the prevalence of SAV subtypes in common dab from Scottish and international waters, (2) to culture any identified SAVs in vitro and (3) to examine common dab tissues from selected sampling areas for the presence of histopathological changes consistent with pancreas disease.…”

“…As common dab are able to tolerate brackish waters and may enter freshwater river systems in coastal areas, they could encounter subtypes other than those frequently attributed to marine sal mon. Snow et al (2010) noted the occurrence of SAV RNA by RT-qPCR in pooled tissue from the long rough dab Hippoglossoides plates so ides, common dab and the plaice Pleuro nectes platessa; therefore, further re search is re quired to understand the relevance of common dab and other flatfish as a wild re servoir of SAV and to assess the potential of horizontal transmission with other wild or farmed fish. Such understanding would be advantageous in order to improve management practices on farms, as well as to interpret virus evolution and adaptation to new hosts.…”

“…Total RNA was eluted in a final volume of 100 µl. cDNA synthesis was performed using the Taqman Reverse Transcription Reagent Kit and random hexamers (Applied Biosystems) according to Snow et al (2010). For detection of the SAV target in heart tissue an nsP1 RT-qPCR assay was used, with a cut off Ct value of 39.3 (see for details on assay validation).…”

“…For detection of the SAV target in heart tissue an nsP1 RT-qPCR assay was used, with a cut off Ct value of 39.3 (see for details on assay validation). RT-qPCR reactions were run on an ABI Prism 7000 detection system, using cycling conditions and the reaction set up according to Snow et al (2010). Negative controls were routinely included at the stages of extraction, reverse transcription and RT-qPCR.…”