Multi-locus sequence typing of Ehrlichia ruminantium strains from geographically diverse origins and collected in Amblyomma variegatum from Uganda

Abstract

Background: The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater in ruminants. A better understanding of the population genetics of its different strains is, however, needed for the development of novel diagnostic tools, therapeutics and prevention strategies. Specifically, the development of effective vaccination policies relies on the proper genotyping and characterisation of field isolates. Although multi-locus sequence typing (MLST) has been recently developed, only strains from geographically restricted collections have been analysed so far. The expansion of the MLST database to include global strains with different geographic origins is therefore essential. In this study, we used a panel of reference strains from geographically diverse origins and field samples of E. ruminantium detected from its vector, Amblyomma variegatum, in heartwater-endemic areas in Uganda.

Results: A total of 31 novel alleles (six, four, six, three, two, five, three, and two for gltA, groEL, lepA, lipA, lipB, secY, sodB, and sucA loci, respectively) and 19 novel sequence types (STs) were identified. Both neighbour-joining and minimum spanning tree analyses indicated a high degree of genetic heterogeneity among these strains. No association was observed between genotypes and geographic origins, except for four STs from West African countries. When we performed six different tests for recombination (GeneConv, Bootscan, MaxChi, Chimaera, SiScan, and 3Seq) on concatenated sequences, four possible recombination events were identified in six different STs. All the recombination breakpoints were located near gene borders, indicating the occurrence of intergenic recombination. All four STs that localized to a distinct group in clustering analysis showed evidence of identical recombination events, suggesting that recombination may play a significant role in the diversification of E. ruminantium.

Conclusions: The compilation of MLST data set across the African continent will be particularly valuable for the understanding of the existing genetic diversity of field isolates in African countries. Comprehensive information on the degree of cross-protection between strains and further understanding of possible relationships between genotypes and phenotypes such as vaccine efficacy are expected to lead to the development of region-specific vaccination strategies.

Figure 1

Minimum-spanning tree based on MLST profiles. Each circle in the tree represents a different ST. The white, red, and blue circles represent reference strains, A. variegatum samples from Uganda, and A. variegatum samples from Burkina Faso, respectively. Circle size is proportional to the numbers of strains or tick samples belonging to an ST. Numbers between circles represent the number of allelic differences. Two or more STs differing at less than two loci are regarded as a group and are distinguished by a different colour. The strength of the link (bold, plain, or discontinuous) indicates the degree of genetic similarity (number of common alleles) between STs.

Figure 2

Neighbour-net network constructed from concatenated sequences obtained from all eight loci. The eight housekeeping genes were concatenated in the order gltA-sucA-lepA-sodB-lipA-secY-lipB-groEL. Significant evidence of recombination was obtained by the PHI test (P = 0.0).

Figure 3

Detection of recombination events. (A) Schematic representation of four recombination events identified by RDP3. Each gray bar represents a concatenated sequence of a recombinant. Colour bars indicate the insertion of foreign sequences. The event numbers and the putative origins of foreign sequences are shown in the box. The gene order of the concatenated sequence is shown on the top and the borders are shown as vertical lines. (B) Mosaic structure in recombination event 2. The alignment was conducted on the concatenated sequences of one recombinant (Strain 1062) and two parental STs (Kerr Seringe and Uganda S001). Only the polymorphic sites are shown, and the position on the sequence is shown above each site. A foreign sequence is indicated as red on yellow. The gene order of the concatenated sequence is shown on the top and the borders are shown as vertical lines.