Development of a Discriminating Method for Pathogenic Bacteria that Cause Lactococcal Infections in Marine Fish Farms in Japan

Abstract

Pathogenic bacteria that cause lactococcal infections in marine aquaculture in Japan are currently classified into three types based on serotype and genetic characterization: Lactococcus garvieae type I, L. formosensis (formerly, L. garvieae type II), and L. garvieae type III. ​Although a slide agglutination test with rabbit antiserum is used to discriminate these three types, its accuracy is insufficient for clinical diagnosis owing to the existence of isolates that show agglutination with more than one diagnostic antiserum. ​Furthermore, a previously developed PCR assay was not able to distinguish L. garvieae type I from L. garvieae type III, a newly emerging pathogen. ​In this study, primers specific to each type were designed and applied in a newly developed multiplex PCR assay to discriminate these three types of lactococcal pathogens isolated from diseased fish. ​Amplification products with the expected sizes of 262, 1,007, and 500 bp could be obtained for L. garvieae type I, L. formosensis, and L. garvieae type III, respectively. ​Amplification products could not be obtained from isolates of Lactococcus spp. from non-marine fish species. ​These results show that the multiplex PCR assay can discriminate these three pathogens that cause lactococcal infections in fish farms.