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 fromLactococcus 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 similarL. 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 μl 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.

Genetic investigation within Lactococcus garvieae revealed two genomic lineages

“…From these sources, we selected 24 new ecotypes that were studied in comparison with previously isolated dairy and fish ecotypes ( Table 1). All new isolates were properly identified by specific PCR, giving the expected amplification product of 1100 bp belonging to the 16S rRNA gene (Zlotkin et al, 1998).…”

“…All samples were aseptically collected and transported in isothermal boxes to the laboratory. For L. garvieae isolation, food samples (25 g) were enriched in 1 : 9 (w/w) M17 broth (Difco, Detroit, MI) supplemented with 1 g L À1 glucose (M17-G) at 37°C for 24 h. After enrichment, total DNA was extracted as reported below and the presence of L. garvieae was established through a species-specific PCR assay, as reported by Zlotkin et al (1998). For each sample positive to the species-specific amplification, L. garvieae selection was attempted on M17-G agar.…”

A real-time PCR assay for detection and quantification ofLactococcus garvieae

“…Several molecular diagnostic studies have been developed for L. garvieae-specific detection in aquacultures. The primers, pLG-1 and pLG-2 (c. 1100 bp fragment) from the 16S rRNA gene and SA1B10-1-F and SA1B10-1-R (709 bp fragment) from the dihydropteroate synthase gene, could differentiate it from other closely related genera using a conventional PCR-based method (Zlotkin et al 1998;Aoki et al 2000). However, these techniques lack assay specificity for the detection of the L. garvieae genomic DNA and can yield false-positive results, such as Tetragenococcus solitarius (data not shown).…”

Differences between Lactococcus garvieae isolated from the genus Seriola in Japan and those isolated from other animals (trout, terrestrial animals from Europe) with regard to pathogenicity, phage susceptibility and genetic characterization

“…All the isolates were reconfirmed as L. garvieae at our laboratory based on Gram-staining; growth on Bile Esculin Azide Agar (BEA; Becton, Dickinson and Company, Sparks, MD, USA); a-haemolysis on a Todd Hewit Broth (THB; Becton, Dickinson and Company) plate, supplemented with 5% defibrinated sheep blood and 1AE5% agar; and the polymerase chain reaction (PCR) with specific primer as described previously (Zlotkin et al 1998). These isolates were cultured in THB for 20 h at 25°C, suspended in 10% skimmed milk (Becton, Dickinson and Company) solution, and then stored at )80°C until they were used for the tests.…”