Species-specific polymerase chain reaction primer sets for the diagnosis of Tenacibaculum maritimum infection

Abstract

In this study the specificity and sensitivity of 2 primer pairs, MAR1-MAR2 and Mar1-Mar2, for the detection of Tenacibaculum maritimum were evaluated in parallel using 79 T. maritimum strains isolated from different fish species, as well as 53 representatives of related and unrelated bacterial species. Both primer pairs were species-specific for T. maritimum, since no amplification products were obtained from chromosomal DNA of the non-T. maritimum bacteria tested. However, whereas MAR1-MAR2 identified all the T. maritimum strains studied, producing a unique and clear PCR band of the expected 1088 bp length, the Mar1-Mar2 primer pair failed to amplify the 400 bp specific band in 3 sole isolates. To verify if these strains belonged to T. maritimum species, 2 endonucleases (PvuI and SacII) were selected as the most adequate enzymes to confirm the specificity of the MAR1-MAR2 amplified fragment. The digestion patterns obtained with both endonucleases supported the assignation of all the strains to T. maritimum. The sensitivity of both PCR detection methods was also different, showing a reduction of sensitivity in at least one order of magnitude of the Mar1-Mar2 primer pair in comparison with MAR1-MAR2. When the MAR-MAR2 PCR protocol was applied to different seeded turbot tissues, the detection limit was 10 2 to 10 4 T. maritimum cells per reaction. In addition, a nested PCR protocol for detection of this pathogens based on MAR1-MAR2 was developed, which increased the sensitivity by approximately 2 orders of magnitude, ranging from 1 to 250 T. maritimum cells per reaction depending on the tissue employed. The tissues that allowed the most easy detection of T. maritimum were the skin and mucus. Based on the findings reported here, we propose the nested PCR protocol as the most adequate for an accurate detection of T. maritimum in diagnostic pathology as well as in epidemiological studies of gliding bacterial disease of marine fish.

Tenacibaculosis infection in marine fish caused by Tenacibaculum maritimum: a review

“…Recent research comparing the specificity and sensitivity of these 2 primer pairs demonstrated that the Toyama PCR procedure is the most adequate for an accurate detection of Tenacibaculum maritimum in diagnostic pathology, as well as in epidemiological studies of marine tenacibaculosis (Avendaño-Herrera et al 2004c). Although this method proved useful in detecting acute T. maritimum infections in fish, the level of sensitivity was not sufficient to detect the pathogen when present in very low number in asymtomatic or carrier fish.…”

“…Then, Bader & Shotts (1998) also selected a pair of T. maritimum species-specific PCR primers Mar1 (5´-TGTAGCTTGCTACAGATGA-3´) and Mar2 (5´-AAATACCTACTCGTAGGTACG-3´), positions 77 to 98 and 1060 to 1081, respectively, from unique sequence stretches within this gene, delimiting a 400 bp DNA fragment. Unfortunately, the authors only tested these primers with pure cultures, and not with infected fish tissues.Recent research comparing the specificity and sensitivity of these 2 primer pairs demonstrated that the Toyama PCR procedure is the most adequate for an accurate detection of Tenacibaculum maritimum in diagnostic pathology, as well as in epidemiological studies of marine tenacibaculosis (Avendaño-Herrera et al 2004c). Although this method proved useful in detecting acute T. maritimum infections in fish, the level of sensitivity was not sufficient to detect the pathogen when present in very low number in asymtomatic or carrier fish.…”

Simultaneous evaluation of four PCR primer sets for the diagnosis of Streptococcus phocae infection

“…The detection limits of each primer set (cpn60F-cpn60R, sodAF-sodAR, cae1-caeVQ2 and PX1-PXVQ2) were evaluated in sensitivity assays using pure or mixed cultures as described by Avendaño-Herrera et al (2004). Separate bacterial suspensions of the 5 Streptococcus phocae strains were prepared to contain 10 9 cells ml -1 (McFarland Scale 4) and were 10-fold diluted in 0.85% sterile saline solution from 10 8 to 10 cells ml -1…”

Multilocus Sequence Analysis of the Marine Bacterial Genus Tenacibaculum Suggests Parallel Evolution of Fish Pathogenicity and Endemic Colonization of Aquaculture Systems

“…Furthermore, the data can easily be stored in databases, compared across experiments, and progressively enriched by the addition of new isolates. The generality of MLSA also makes it complementary to more rapid and less expensive detection methods, such as PCR or immunohistochemical assays already proposed for T. maritimum (33)(34)(35)(36) and T. soleae (37). To illustrate the applicability of our approach at the genus level, we include in this report the results of MLSA on a selection of isolates responsible for recent tenacibaculosis outbreaks in Norwegian salmon farms (38) and in Italian sea bass and sea bream farms.…”