Genetic diversity and geographic relationships amongst southern African Amblyomma ticks, Rickettsia africae and Ehrlichia ruminantium

Amblyomma, the third largest genus in the Ixodidae, are known for their vibrant decorative appearance and aggressive hunting behaviour. The genus comprises 24 species found across the African continent, with 17 identified as inhabiting various ecological niches within the southern regions of Africa. Of these species, Amblyomma hebraeum and Amblyomma variegatum have been well studied due to their widespread geographical range and their status as competent vectors of pathogens including Ehrlichia ruminantium, the causative agent of heartwater, and Rickettsia africae, the causative agent of African tick bite fever (ATBF) in humans. Heartwater is a lethal disease in ruminants, estimated to cause an economic loss of 44.4 million US$ annually in South Africa and 5.6 million US$ annually in Zimbabwe. ATBF is a non-lethal disease, mainly afflicting individuals travelling to southern Africa. In this study, Amblyomma ticks were collected from livestock in Angola, South Africa, Zambia and Zimbabwe and from both wildlife and livestock in Mozambique. The collected ticks were morphologically identified with identification keys and morphological characteristics were documented by photography. Primary screening for E. ruminantium was conducted, targeting the pCS20 gene fragment and strain characterization of 100 positives was conducted with Ampliseq technology. A subsample of ticks was screened with the use of the ompA gene for R. africae, while characterization was conducted on 50 positives with ompA, ompB, gltA and omp genes. Tick genetic diversity was evaluated using 12S, 16S, coi and ITS2 genes. In total, 7,773 Amblyomma ticks were collected and identified as belonging to four species: Amblyomma eburneum, A. hebraeum, Amblyomma pomposum and A. variegatum. The phylogenetical analysis of the four Amblyomma species illustrated clear separations between A. eburneum and A. hebraeum, while A. pomposum and A. variegatum clustered together. Little intraspecies variation was also observed, where ticks of the same species from different countries cluster together indiscriminately. Using phylogenetic, pairwise distance and automatic barcode gap discovery analyses, we are unable to molecularly distinguish between A. pomposum and A. variegatum. Ehrlichia ruminantium infection rates in ticks per country ranged from 7.7% to 36.5%. Genotyping analysis indicated the clustering of the sequences with several strains, including the Gardel, Grootvallei and Springbokfontein1. The Ampliseq analysis had amplification failures as documented with the genes used, emphasizing the requirement for the development of new markers for phylogenetic characterization. Infection rates of R. africae ranged from 11.4% to 35.6% per country. The phylogenetical analysis depicts little variation and illustrates that the genes used were not sufficient enough to distinguish between strains. This study is one of the largest performed in this region of Africa, depicting the genetic variation of E. ruminantium, R. africae and the four collected Amblyomma species. The infection rates obtained during this study provides new and current insight of the pathogen distributions ranges in southern Africa. Greater attention should be given to the neglected Amblyomma species and their role in the distribution and maintenance of pathogens.