Multiple locus VNTR analysis highlights that geographical clustering and distribution of Dichelobacter nodosus, the causal agent of footrot in sheep, correlates with inter-country movements

Abstract

Dichelobacter nodosus is a Gram-negative, anaerobic bacterium and the causal agent of footrot in sheep. Multiple locus variable number tandem repeat (VNTR) analysis (MLVA) is a portable technique that involves the identification and enumeration of polymorphic tandem repeats across the genome. The aims of this study were to develop an MLVA scheme for D. nodosus suitable for use as a molecular typing tool, and to apply it to a global collection of isolates. Seventy-seven isolates selected from regions with a long history of footrot (GB, Australia) and regions where footrot has recently been reported (India, Scandinavia), were characterised. From an initial 61 potential VNTR regions, four loci were identified as usable and in combination had the attributes required of a typing method for use in bacterial epidemiology: high discriminatory power (D>0.95), typeability and reproducibility. Results from the analysis indicate that D. nodosus appears to have evolved via recombinational exchanges and clonal diversification. This has resulted in some clonal complexes that contain isolates from multiple countries and continents; and others that contain isolates from a single geographic location (country or region). The distribution of alleles between countries matches historical accounts of sheep movements, suggesting that the MLVA technique is sufficiently specific and sensitive for an epidemiological investigation of the global distribution of D. nodosus.

Keywords: Dichelobacter nodosus; Global distribution; MLVA; Ovine footrot.

Fig. 1

Diversity of global D. nodosus populations. (A) Minimum-spanning tree of D. nodosus MLVA data. Numbers indicate MLVA types and circle size is proportional to the numbers of isolates of each type (range 1–5). Single locus variants are connected by thick black lines and double locus variants by narrow grey lines. Circle colours indicate the country each MLVA type was isolated from: Australia (green), GB (dark blue), India (grey), Norway (yellow), Sweden (light blue); putative ancestral MLVA types have a thick black border. MLVA type membership of Structure clusters is highlighted in maroon (Cluster I), light purple (Cluster II), orange (Cluster III) and brown (Cluster IV). (B) Distruct plots of Structure output from K = 2 (upper image) to K = 4 (lower image) following analysis of 77 isolates of D. nodosus. Each isolate is represented by a single vertical line indicating its membership in each of K independent clusters. Geographic populations indicated along the bottom are separated by vertical black lines.