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. 2001 Jan;39(1):119-28.
doi: 10.1128/JCM.39.1.119-128.2001.

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

S M Alexander  1 , T H Grayson, E M Chambers, L F Cooper, G A Barker, M L Gilpin
Affiliations

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

S M Alexander et al. J Clin Microbiol. 2001 Jan.

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 IS994. 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 IS994 profiles.

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Figures

FIG. 1
FIG. 1
The RAPD patterns of seven isolates of R. salmoninarum. The patterns were generated using primers P1 (A), P2 (B), P3 (C), P4 (D), P5 (E), P6 (F), OPB1 (G), and OPA9 (H). Lanes 1 to 7 correspond to isolates 980297#97, F95, F85, F82, F60, F47, and F3, respectively. Lanes M, 100-bp (A to F) or 1-kb (G and H) DNA ladder (Gibco BRL).
FIG. 2
FIG. 2
The tDNA PCR profiles of 60 isolates of R. salmoninarum. The patterns were generated using consensus primer sets T3B-T5A (A), T5A-T5B (B), T3A-T3B (C), and T3A-T5A (D to F). (A and F) Lanes 1 to 20, isolates 960046, F-120-87(P-2), F-130-87(P-4), F-138-87(O-78), F-273-87(P-19), F-283-87(P-10), F-358-87(P-13), S-182-90(P-7), Rs 9, Rs 19, Rs 61, Rs 116, Rs 122, Rs 125, Rs 126, 3015-86, 4451-86, RS-TSA, FT10, and BY1996, respectively. (C and E) Lanes 1 to 20, isolates MT452, MT1363, MT410, Siletz, Marion Forks, Little Goose, CCM6205, 84-019-OC, SS-ChS-94-1, Cow ChS94 P22, Idaho 91-126, RFL-643.94#1, CCM6206, Round Butte, NCIMB2235, AcF6-1, DR143, DR384, 980002, and 960023, respectively. (B and D) Lanes 1 to 20, isolates 970083-88, 970083-102, 980106#1.1.5, 980036-150, 980036-87, 970419-1.2.3, 970153-19, A6, A80, MT409, MT417, MT239, MT426, NCIMB1111, NCIMB1112, NCIMB1113, NCIMB1114, NCIMB1115, NCIMB1116, and MT420, respectively. Lanes M, 100-bp DNA ladder (Gibco BRL).
FIG. 3
FIG. 3
The tDNA PCR profiles of 22 isolates of R. salmoninarum generated when using specific primers A35K+754 and T25D−120 (A), T17C+80 and T25E−128 (B), A35K+754 and T17C−135 (C), T3C+42 and T25E−128 (D), and T25E−128 (E). Lanes 1 to 22 correspond to the isolate numbers in Table 2. Lanes 23, negative control; lanes M, 100-bp DNA ladder (Gibco BRL).
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