Identification of Lactococcus garvieae by PCR

Abstract

Lactococcus garvieae junior synonym Enterococcus seriolicida) is an emerging zoonotic agent isolated from economically important fish (rainbow trout and yellowtail), from cattle, and from humans. Clindamycin susceptibility is the only phenotypic test which can differentiate L. garvieae from Lactococcus lactis, another emerging agent in humans. A PCR assay for the identification of L. garvieae was developed and resulted in an amplified fragment of 1,100 bp in size. The PCR assay was shown to be specific to L. garvieae. The PCR assay was positive for all the L. garvieae strains tested, which originated from three different continents (Asia, Australia, and Europe). The PCR assay was negative for the phenotypically similar L. lactis and for all the other fish pathogens tested, including Streptococcus iniae and Aeromonas salmonicida. The PCR assay was applied to plasma obtained from diseased animals and was found sensitive enough to detect bacteria from 1 microl of plasma. The PCR assay that was developed is the only practical test besides the clindamycin test which can specifically identify the zoonotic agent L. garvieae and which can differentiate it from L. lactis.

FIG. 1

Specificity of the L. garvieae PCR assay. Agarose gel (2%)-resolved ethidium bromide-stained PCR products from template DNA of various needle-touched bacterial colonies are shown. Lanes: 1, L. garvieae ATCC 43921^T; 2, E. seriolicida ATCC 49156^T; 3, L. garvieae ITP 2001 (Italy); 4, L. garvieae S1449 (Japan); 5, L. garvieae 88/1400 (Australia); 6, L. garvieae Sp 1 (Spain); 7, L. lactis NCFB 604^T; 8, L. piscium NCFB 2778^T; 9, V. salmoninarum NCFB 2777^T; 10, S. iniae ATCC 29178^T; 11, S. difficile CIP 103769^T; 12, A. salmonicida 3173/86; N, negative control (no DNA); M, DNA molecular weight marker VI. The arrow indicates the band of the expected length of 1,100 bp.

FIG. 2

Field application and sensitivity of the L. garvieae PCR assay. (a) PCR product from 1 μl of fish plasma. Lanes: 1 to 3, diseased fish; 4, antibiotic-treated fish. (b) PCR assay sensitivity evaluated with a serially diluted bacterial suspension. Lanes: 1, 3,600 CFU; 2, 360 CFU; 3, 36 CFU; 4, 4 CFU; 5, 0.4 CFU. The arrows indicate the band of the expected length of 1,100 bp. Lane M contains DNA molecular weight marker VI.