Virus isolation vs RT‐PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions?

Abstract

This study compared the results of reverse transcription-polymerase chain reaction (RT-PCR) and traditional virus isolation on cell culture in detection of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis virus (IHNV). RT-PCR was used for 172 tissue sample pools (total of 859 fish) originating from a field survey on the occurrence of VHSV and IHNV in farmed and wild salmonids in Switzerland. These samples represented all sites with fish that were either identified as virus-positive by means of virus isolation (three sites, four positive tissue sample pools) and/or demonstrated positive anti-VHSV-antibody titres (83 sites, 121 positive blood samples) in a serum plaque neutralization test (SPNT). The RT-PCR technique confirmed the four VHSV-positive tissue sample pools detected by virus isolation and additionally identified one VHSV-positive sample that showed positive anti-VHSV-AB titres, but was negative in virus isolation. With IHNV, RT-PCR detected two positive samples not identified by virus isolation while in these fish the SPNT result had been questionable. One of the IHNV-positive samples represents the first detection of IHNV-RNA in wild brown trout in Switzerland. Compared to SPNT, the RT-PCR method detected, as with virus isolation, a much lower number of positive cases; reasons for this discrepancy are discussed. Our results indicate that RT-PCR can not only be successfully applied in field surveys, but may also be slightly more sensitive than virus isolation. However, in a titration experiment under laboratory conditions, the sensitivity of RT-PCR was not significantly higher when compared with virus isolation.

Screening for Viral Hemorrhagic Septicemia Virus in Marine Fish along the Norwegian Coastal Line

“…As in the present study an increased number of positive pooled samples (n = 4) were found using RT-PCR compared to cell culture isolation. Experimental testing of the sensitivity of cell culturing versus PCR-methodology has demonstrated RT-PCR the most sensitive [51], [52]. The development of PCR assays with higher sensitivity and a broader detection range to several genotypes of VHSV has made this the preferred method for VHSV detection in most laboratories [36].…”

Survey of viral haemorrhagic septicaemia virus in wild fishes in the southeastern Black Sea

“…However, due to the unstable nature of RNA and the risk of contamination, Knüsel et al (2007) suggested that RT-mPCR is more suitable for laboratory samples instead of testing on field samples. Consequently, in this study, we developed an RT-mPCR method with commonly used RT-PCR kits for the detection of VHSV and IPNV.…”

To pool or not to pool? Guidelines for pooling samples for use in surveillance testing of infectious diseases in aquatic animals

“…However, these assumptions depend on the prevalence of the infectious agent in the source population and the concentration of target analyte in each specimen included in the pool (Johnson et al, ; Zainathan, Carson, Crane, & Nowak, ). In low prevalence scenarios, pools of tissues or swabs from non‐infected animals can dilute an already low concentration of an infectious agent that may be close to the detection limit or cut‐off value used to classify a test result as positive or negative (Corsin et al, ; East, ; Jansen et al, ; Johnson et al, ; Knüsel et al, ; Oidtmann et al, ). Another important factor that is often driven by prevalence and affected by analyte concentration is the number of positives per pool.…”