DAO 74:209-223 (2007)  -  doi:10.3354/dao074209

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

Kunio Suzuki1,*, D. K. Sakai2

1Eastern Hokkaido Inland Water Fisheries Section, The Hokkaido Fish Hatchery, Notorominato 1-1, Abashiri, Hokkaido 093-0131, Japan
2Faculty of Bio-industry, Tokyo University of Agriculture, Yasaka 196, Abashiri, Hokkaido 099-2493, Japan

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 real-time 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 × 101 to 5.0 × 109 copies µl–1) with high correlation (R2 = 0.999). The mRNA concentration correlated with CFU in kidney tissue from fish infected by i.p. injection (R2 = 0.924), by bath infection (R2 = 0.502) and in culture (R2 = 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 × 102 to 5.6 × 105 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.

KEY WORDS: Real-time PCR · msa gene mRNA · Renibacterium salmoninarum · Bacterial kidney disease · Viability · Chum salmon

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