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

Abstract

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.

Figure 1

Electropherograms showing PCR fragments of all five VNTR loci co-amplified in a single PCR reaction. The fragments were resolved by size and dye colour using capillary electrophoresis.

Figure 2

Minimum spanning tree (MST) of MLVA showing the genetic relatedness among the isolates included in this study. A categorical coefficient and the priority rule using the highest number of single-locus changes was used for generation of the minimum spanning tree. Each circle in the tree represents a different MLVA type (MVT), with the different MVTs indicated by the number in the circle. MVTs differing by a single VNTR locus are represented by thick short lines, while double-locus variants are shown by thin longer lines and dotted lines for those differing by more than two loci. The number of loci that differ between two MVTs is indicated on the lines linking them together. Clusters were defined as MVTs having a maximum of three differing loci and a minimum cluster size of two. The Norwegian isolates grouped into three main clusters (I, II and III). Cluster I is shaded pink, Cluster II in green and cluster III in violet.

Figure 3

Map of Norway showing the geographical distribution of F. noatunensis subsp. noatunensis MLVA clonal groups generated by minimum spanning tree (MST) analysis. Cluster I is shaded pink, cluster II in green and cluster III in violet.

Figure 4

Dendrogram showing the genetic relatedness among selected isolates based on PFGE analysis. The dendrogram was constructed using the Dice coefficient correlation and WARD algorithm. The Norwegian isolates grouped into two clusters (I and II).