Variation in the spacer regions separating tRNA genes in Renibacterium salmoninarum distinguishes recent clinical isolates from the same location

A means for distinguishing between clinical isolates of Renibacterium salmo ninarum that is based on the PCR amplification of length polymorphisms in t he tRNA intergenic spacer regions (tDNA-ILPs) was investigated. The method used primers specific to nucleotide sequences of R. salmoninarum tRNA genes and tRNA intergenic spacer regions that had been generated by using consen sus tRNA gene primers. Twenty-one PCR products were sequenced from five iso lates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen speci fic PCR primers were designed and tested singly and in all possible pairwis e combinations for their potential to discriminate between isolates from re cent clinical outbreaks of bacterial kidney disease (BKD) in the United Kin gdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isola tes came from the same farm and at the same time. The tDNA-ILP profiles sep arated 22 clinical isolates into nine groups and highlighted that some farm s may have had more than one source of infection. The grouping of isolates improved on the discriminatory power of previously reported typing methods based on randomly amplified polymorphic DNA analysis and restriction fragme nt length profiles developed using insertion sequence IS994. Our method ena bled us to make divisions between closely related clinical isolates of R. s almoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA inte rgenic spacer sequences, and IS994 profiles.