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

Samuel Duodu,Andreas Sjödin,Pär Larsson,Mats Forsman,Duncan J Colquhoun,Xihe Wan,Nora Martinussen Tandstad,Kerstin Myrtennäs

doi:10.1186/1746-6148-9-252

Abstract

BackgroundFrancisellosis, 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.ResultsThe 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.ConclusionsThe 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.

Highlights

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
Francisellosis in farmed Atlantic cod Gadhus morhua L. was first reported in 2004/2005, when an outbreak caused by F. noatunensis subsp. noatunensis was discovered in Norway [2,3]
multiple-locus variable-number of tandem repeat analysis (MLVA) The MLVA assay developed in this study is based on capillary electrophoresis of the five most polymorphic loci identified within available fish-pathogenic Francisella genomes

Summary

Introduction

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. Francisellosis, caused by the bacterium Francisella noatunensis subsp. 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. Francisella noatunensis is an emergent fish pathogen of major concern. It causes francisellosis, a systemic bacterial disease characterized by the presence of multi-organ granuloma with high morbidity and varying associated mortality levels [1]. Francisellosis in farmed Atlantic cod Gadhus morhua L. was first reported in 2004/2005, when an outbreak caused by F. noatunensis subsp. An increasingly applied molecular typing tool for discrimination of bacterial species with stable clonal population structures is multiple locus variable-number tandem repeat analysis (MLVA). The different variants are most commonly resolved by standard agarose gel electrophoresis or capillary electrophoresis on a DNA sequencer [10]

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