An improved multiple-locus variable-number of tandem repeat analysis (MLVA) for the fish pathogen Francisella noatunensis using capillary electrophoresis

Background: Francisellosis, caused by the bacterium Francisella noatunensis subsp. noatunensis, remains a serious threat to Atlantic cod (Gadhus morhua) farming in Norway and potentially in other countries. As outbreak strains appear clonal in population structure, access to highly discriminatory typing tools is critical for understanding the epidemiology of francisellosis infections in aquaculture. In this study, a simplified multiple-locus variable-number of tandem repeat analysis (MLVA) targeting five highly polymorphic variable number of tandem repeat (VNTR) loci in a single multiplex PCR was developed to rapidly discriminate between outbreak strains. Results: The assay resulted in identification of at least 13 different allelic profiles or subpopulations among 91 F. noatunensis isolates from farmed cod in Norway. The VNTR loci appear relatively stable, with isolates originating from individual outbreaks showing identical MLVA profiles following repeated passage. MLVA displayed greater discriminatory power than pulse-field gel electrophoresis (PFGE). Both MLVA and PFGE show good epidemiological concordance by their abilities to separate outbreak strains from epidemiologically unrelated isolates. Conclusions: The MLVA method presented here is robust, easy to perform and provides a good alternative to other typing systems for F. noatunensis subsp. noatunensis and epidemiological study of francisellosis in cod.