A PCR protocol (McIntosh et al., 1996. Appl. Environ. Microbiol. 62, 3929-2932), designed to detect the P57 gene of Renibacterium salmoninarum, causative agent of Bacterial Kidney Disease, was modified slightly by redesign of the forward primer and recommended conditions to prevent possible false positives observed when tested against pure cultures of Yersinia ruckeri. The modified PCR, in combination with an improved DNAzol (TM)-based DNA extraction technique, was very specific and sensitive (detecting between 5 and 72 R. salmoninarum cfu per mg head kidney tissue). It could detect infected fish using only 50 mg head kidney material, making the technique Suitable for sampling small fish. It was also shown that samples Could be pooled from up to five rainbow trout prior to PCR without noticeably affecting the sensitivity of the assay, providing at least 50 mg head kidney tissue was included from each of the fish that were pooled. Crown Copyright (c) 2008 Published by Elsevier B.V. All rights reserved.

ABSTRACT A means for distinguishing between clinical isolates of Renibacterium salmoninarum that is based on the PCR amplification of length polymorphisms in the 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 consensus tRNA gene primers. Twenty-one PCR products were sequenced from five isolates of R. salmoninarum from the United States, England, and Scotland, and four complete tRNA genes and spacer regions were identified. Sixteen specific PCR primers were designed and tested singly and in all possible pairwise combinations for their potential to discriminate between isolates from recent clinical outbreaks of bacterial kidney disease (BKD) in the United Kingdom. Fourteen of the isolates were cultured from kidney samples taken from fish displaying clinical signs of BKD on five farms, and some of the isolates came from the same farm and at the same time. The tDNA-ILP profiles separated 22 clinical isolates into nine groups and highlighted that some farms 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 fragment length profiles developed using insertion sequence IS 994 . Our method enabled us to make divisions between closely related clinical isolates of R. salmoninarum that have identical exact tandem repeat (ETR-A) loci, rRNA intergenic spacer sequences, and IS 994 profiles.