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. 2011 Jan 24:7:5.
doi: 10.1186/1746-6148-7-5.

Multiple-locus, variable number of tandem repeat analysis (MLVA) of the fish-pathogen Francisella noatunensis

Øyvind J Brevik  1 , Karl F Ottem, Are Nylund
Affiliations

Multiple-locus, variable number of tandem repeat analysis (MLVA) of the fish-pathogen Francisella noatunensis

Øyvind J Brevik et al. BMC Vet Res. .

Abstract

Background: Since Francisella noatunensis was first isolated from cultured Atlantic cod in 2004, it has emerged as a global fish pathogen causing disease in both warm and cold water species. Outbreaks of francisellosis occur in several important cultured fish species making a correct management of this disease a matter of major importance. Currently there are no vaccines or treatments available. A strain typing system for use in studies of F. noatunensis epizootics would be an important tool for disease management. However, the high genetic similarity within the Francisella spp. makes strain typing difficult, but such typing of the related human pathogen Francisella tullarensis has been performed successfully by targeting loci with higher genetic variation than the traditional signature sequences. These loci are known as Variable Numbers of Tandem Repeat (VNTR). The aim of this study is to identify possible useful VNTRs in the genome of F. noatunensis.

Results: Seven polymorphic VNTR loci were identified in the preliminary genome sequence of F. noatunensis ssp. noatunensis GM2212 isolate. These VNTR-loci were sequenced in F. noatunensis isolates collected from Atlantic cod (Gadus morhua) from Norway (n = 21), Three-line grunt (Parapristipoma trilineatum) from Japan (n = 1), Tilapia (Oreochromis spp.) from Indonesia (n = 3) and Atlantic salmon (Salmo salar) from Chile (n = 1). The Norwegian isolates presented in this study show both nine allelic profiles and clades, and that the majority of the farmed isolates belong in two clades only, while the allelic profiles from wild cod are unique.

Conclusions: VNTRs can be used to separate isolates belonging to both subspecies of F. noatunensis. Low allelic diversity in F. noatunensis isolates from outbreaks in cod culture compared to isolates wild cod, indicate that transmission of these isolates may be a result of human activity. The sequence based MLVA system presented in this study should provide a good starting point for further development of a genotyping system that can be used in studies of epizootics and disease management of francisellosis.

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Figures

Figure 1
Figure 1
The geographical location of sampling sites of the Norwegian F. noatunensis ssp. noatunensis isolates (n = 21). Accurate location of FnnMR-011/07 and FnnMR-013/07 is unknown. Isolates from production sites of cultured cod is predominantly of clade I and III. I = clade I, II = clade II, III = clade III, W = wild caught, broodfishF, type strainT
Figure 2
Figure 2
The relationship of the isolates included in this study. Relationship among the 33 Francisella isolates based on allelic differences at seven VNTR loci. This unrooted ultrametric NJ tree show known topography a) between the species of F. philomiragia (Fp) and F. noatunensis, b) between the subspecies F. noatunensis ssp. orientalis (Fno) and F. noatunensis ssp. noatunensis (Fnn) and c) among the F. noatunensis ssp. noatunensis isolates from Norway and Chile (FnnC-UA2660). Two previously unrecognized clades are evident within the Norwegian Fnn isolates. Bootstrap values were not calculated as there were too few characters and taxa in the dataset for correct estimation of sampling error W = wild caught, broodfishF, type strainT
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