Real-time PCR for quantification of viable Renibacterium salmoninarum in chum salmon Oncorhynchus keta

Abstract

Quantification of msa gene mRNA of Renibacterium salmoninarum, the causative agent of bacterial kidney disease (BKD), was investigated using reverse transcription followed by real-time PCR assay on R. salmoninarum in culture, and in experimentally challenged chum salmon Oncorhynchus keta fry kidney tissues (total of 70 samples) after intraperitoneal (i.p.) injection and bath infection. Correlations of msa gene mRNA concentrations with culturable cell concentrations (as colony forming units [CFU]), determined by drop-plate culture method on selective kidney disease medium (SKDM) agar through a 12 wk incubation time, and msa gene DNA concentrations by realtime PCR assay were examined. Furthermore, ovarian fluid samples from wild chum salmon adults with no clinical signs of disease were collected from 8 rivers and from clinically infected kokanee O. nerka and masu salmon O. masou that were reared in 1 and 2 hatcheries, respectively (total of 414 samples). All samples were examined by nested PCR assay. Then, positive samples were examined by real-time PCR assays for mRNA and DNA; mRNA was detectable at 8 log units (5.0 × 10 1 to 5.0 × 10 9 copies µl -1 ) with high correlation (R 2 = 0.999). The mRNA concentration correlated with CFU in kidney tissue from fish infected by i.p. injection (R 2 = 0.924), by bath infection (R 2 = 0.502) and in culture (R 2 = 0.888). R. salmoninarum was detected and quantified by real-time PCR assay for mRNA in ovarian fluid samples in both subclinically infected chum salmon adults and clinically infected kokanee and masu salmon adults; detection rates ranged from 0 to 44.4% and concentrations ranged from 9.7 × 10 2 to 5.6 × 10 5 copies µl -1. These results indicate that real-time PCR assay for the mRNA is a rapid, sensitive and reliable method to detect and quantify the viability of R. salmoninarum in kidney and ovarian fluid samples of salmonid fishes with both clinical and subclinical infection of the pathogen.

Interpreting dual ELISA and qPCR data for bacterial kidney disease of salmonids

“…Detection of bacterial DNA or protein is not necessarily indicative of viable cells, which can be confirmed by growth in culture. Analysis for bacterial RNA, which is extremely labile and produced only by living cells, has been proposed as a marker for viable Renibacterium salmoninarum (Cook & Lynch 1999, Suzuki & Sakai 2007 Suzuki & Sakai (2007) observed elevated DNA concentrations but minimal or no mRNA or culture growth during the early course of a disease challenge (see also our Fig. 5B), indicating that the DNA signal can dominate during expanding infections.…”

“…Teska et al 1995, Griffiths et al 1996, Miriam et al 1997, Suzuki & Sakai 2007. In the context of previous studies using multiple assays, a conceptual model for interpreting our ELISA and qPCR results may be developed.…”

Pathological and immunological responses associated with differential survival of Chinook salmon following Renibacterium salmoninarum challenge

“…Since these assays measure RNA rather than DNA, RT-qPCR may be better able to discriminate live from dead bacteria. One caveat is that differential regulation of R. salmoninarum genes may occur during the infection cycle; however, a previous study in coho salmon (Oncorhynchus kisutch) found good correlation between msa mRNA and genomic DNA copy number in infected samples (Suzuki & Sakai 2007). Our RT-qPCR methodology has been previously described in detail (Purcell et al 2004).…”

Development of a real-time PCR assay for identification and quantification of the fish pathogen Francisella noatunensis subsp. orientalis

“…and viruses (Bode et al 2004, Kocagoz et al 2005, Takahashi et al 2007, Tomaso et al 2007, Abril et al 2008, Kidd et al 2008. In recent years, fish disease diagnosticians have used this technique to identify and quantify bacterial, viral, and parasitic fish pathogens such as Aeromonas salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, Henneguya ictaluri, largemouth bass virus, and recently Francisella piscicida in Norwegian cod (Balcázar et al 2007, Getchell et al 2007, Panangala et al 2007, Suzuki & Sakai 2007, Griffin et al 2008, Ottem et al 2008. The high sensitivity, high specificity, and short turnaround time for results make this technique an attractive replacement method for conventional diagnostic techniques (Espy et al 2006).…”