Detection of salmon pancreas disease virus in the faeces and mucus of Atlantic salmon, Salmo salar L., by real‐time RT‐PCR and cell culture following experimental challenge

Abstract

Salmonid alphavirus (SAV) (salmon pancreas disease virus [SPDV]), Genus Alphavirus, Family Togaviridae) is the aetiological agent of the economically important conditions referred to as pancreas disease in Atlantic salmon, Salmo salar L., and sleeping disease in rainbow trout, Oncorhynchus mykiss (Walbaum) (Nelson et al. 1995; Castric et al. 1997; Rowley et al. 1998; Taksdal et al. 2007; Aunsmo et al. 2010). Experimental studies have shown that SAV can be transmitted directly through water from infected to susceptible fish (McLoughlin et al. 1996; Graham et al. 2011), and epidemiological and modelling studies have confirmed the importance of transmission of infection through water in field outbreaks of pancreas disease (Kristoffersen et al. 2009; Viljugrein et al. 2009; Aldrin et al. 2010). In a previous study (Graham et al. 2011), it was shown that SAV RNA could be detected by RT-PCR in the faeces and mucus of Atlantic salmon infected by a natural route in a cohabitant challenge. Shedding was detectable for periods of 2–4 weeks and this occurs 2–6 weeks after cohabitation with shedders. This suggested that these matrices could be important in the horizontal transmission of infection through water between cages and sites and that their dispersal should be considered when modelling such transmission events. The current study was conducted to determine whether live virus could be cultured from faeces and mucus samples, confirming that these are potential routes of shedding and transmission from infected fish. Samples were collected from Atlantic salmon cohabiting with shedder fish infected intraperitoneally 3 weeks earlier with a SAV subtype 3 strain as previously described (Graham et al. 2011). This earlier study had indicated that this was the time point at which the prevalence of SAV RT-PCR signals in faeces and mucus was highest, and also corresponded to the peak of viraemia in cohabitant fish. Mucus samples (n = 19) were collected by scraping the blunt side of a scalpel blade along the lateral line from the gills to the tail, while faeces (n = 5) were extruded by gently squeezing the ventral abdomen in a caudal direction. Each sample was divided into two, with one aliquot untreated for subsequent virus isolation and one aliquot being added to RNAlater for subsequent RT-PCR testing. Untreated samples were stored below 60 °C, while samples in RNAlater were stored at 2–8 °C prior to shipping to the testing laboratory. RNA extraction from faeces and mucus samples in RNAlater and real-time RT-PCR testing were conducted as previously described (Graham et al. 2011). For virus isolation, faeces and mucus Correspondence D A Graham, Fish Diseases Unit, Agrifood and Biosciences Institute, Stoney Rd, Stormont, Belfast BT4 3SD, UK (e-mail: david@animalhealthireland.ie)