Evaluation of five housekeeping genes for genotyping southern African Ehrlichia ruminantium strains

Heartwater is a tick-borne disease caused by the bacterium Ehrlichia ruminantium. It affects domestic animals such as cattle, sheep and goats as well as a range of wild animals. The disease is characterised by various clinical signs including high fever, exaggerated blinking, a stiff gait, convulsions, loss of appetite, heavy breathing, hanging head, depression, anorexia, hyperaesthesia, lacrimation and recumbency. The pathogen is transmitted to hosts by tick vectors in the genus Amblyomma and disease distribution corresponds to vector distribution. The disease is endemic to the majority of sub-Saharan Africa, excluding only the very dry south-west, as well as the islands of Grande Comore, Madagascar, Mauritius, Réunion, and São Tomé. Heartwater is also present in the Caribbean islands of Guadeloupe, Antigua and Marie-Galante. Measures to control heartwater include tick control, immunisation, farming with resistant stock and the use of antibiotics. Multiple serological and molecular tests have been developed for diagnostic and phylogenetic purposes but genotyping of the E. ruminantium genome is limited to ftsZ, gltA, groEL, groESL, lepA, lipA, lipB, nuoB, pCS20 gene region, secY, sodB and sucA, with little success in observing a rich genetic constitution. These genes demonstrate genetic variation, but not enough to adequately differentiate between E. ruminantium field strains in southern Africa. This gap in knowledge establishes the need to identify new genes that can provide us with a more in-depth and comprehensive understanding of said genetic diversity. Therefore, the aim of this study is to investigate the genetic constitution of five housekeeping genes of southern African E. ruminantium isolates by evaluating their capability to differentiate between strains. Gene alignments were made, primers were designed in a nested approach, PCR was conducted, and sequencing results of PCR amplicons were phylogenetically analyzed individually and via MLST for the genes carA, glnA, guaB, hemC and pyrD. The results indicate that carA, glnA and guaB are most efficient at differentiating at strain level; hemC does not adequately differentiate at strain level; and pyrD failed at sequencing and could therefore not be analyzed phylogenetically. The concatenated sequence tree of genes carA, glnA, guaB and hemC gave an overall better understanding of the differentiation of southern African strains as it delivers 18 different genotypes from the 18 field samples.