Tick-borne haemoparasite occurrence and Anaplasma species diversity in selected South African wild rodents and domestic dogs

Abstract

Since its first discovery, Anaplasma phagocytophilum has gained interest in both veterinary (and human) medicine as the causative agent of tick-borne fever in ruminants and granulocytic anaplasmosis in humans and in a wide variety of domestic animals. The enzootic cycle includes rodents, wild ungulates and possibly birds as reservoir hosts. Reports of human granulocytic anaplasmosis occurring in Africa have been few. In South Africa there have been no official diagnoses of A. phagocytophilum in humans. The first report of the molecular detection of a bacterium closely related to A. phagocytophilum in South Africa was in whole blood specimens from dogs in 2005, which was designated Anaplasma sp. South African dog strain. More recently, the same Anaplasma sp. South African dog strain as well as A. phagocytophilum and an Orientia tsutsugamushi-like partial 16S rRNA sequence were detected in several dog samples collected in the Mnisi community, Bushbuckridge, Mpumalanga, South Africa. Little is known about the significance of this finding. The aim of this study was, therefore, to determine the occurrence of A. phagocytophilum in selected small rodent species collected from four South African provinces and in dogs from the Mnisi community, and to molecularly characterize the Anaplasma species found. Molecular analysis was carried out on 37 wild rodent blood samples (Rhabdomys pumilio, Rhabdomys dilectus, Micaelamys namaquensis and Myotomys unisulcatus) collected from four provinces within South Africa and 56 domestic dog blood samples collected from the Mnisi community, Bushbuckridge, Mpumalanga, South Africa. All samples were screened for the presence of Anaplasma, Ehrlichia, Theileria and Babesia species using the Reverse Line Blot (RLB) hybridization assay. The same samples were also subjected to real-time quantitative PCR (qPCR) analysis for the specific detection of A. phagocytophilum. The RLB results have shown single infections of A. bovis (3.3%), Babesia microti (3.3%) and Ehrlichia ruminantium (3.3%) in R. pumilio (four-striped grass mouse) while Anaplasma sp. Omatjenne (33.1%) and E. ruminantium (33.1%) were detected in M. namaquensis (Namaqua rock mouse). There were 83.3% (n = 25) mixed infections in R. pumilio; of which B. microti was the highest (56.7%). There were no mixed infections in the other three rodent species. For both R. dilectus (mesic four-striped grass rat) and M. unisalcatus (Karoo bush rat), PCR products only hybridized with the genus-specific probes, and with none of the species-specific probes; indicating the presence of novel species or a variant of a species. In dogs, single infections of B. microti (3.6%), B. rossi (3.6%) and E. canis (21.4%) were detected. Mixed infections were present in 50% of the dog samples. The RLB assay only detected 4.3% A. phagocytophilum positive samples in both rodents and dogs whereas qPCR detected 57% A. phagocytophilum DNA positive samples in both rodents and dogs; most propably due to the A. phagocytophilum real-time PCR probe cross-reacting with Anaplasma sp. Zambian and South African dog strain DNA. The pathogen’s 16S rRNA gene was subsequently amplified, cloned and sequenced from eight samples (7 dogs; 1 rodent) that tested positive by the qPCR assay. A total of 36 recombinant sequences were obtained from the eight samples. BLASTn homology searches showed that the obtained sequences had 98-100% sequence identity to published sequences of A. phagocytophilum, A. platys, Anaplasma sp. from Zambian and South African dogs; and A. bovis. The observed sequence similarities were confirmed by Neighbor-joining and Maximum Likelihood phylogenetic analyses. The study highlighted the importance of wild rodents and dogs as reservoir species for haemoparasites that are of medical and veterinary importance. We have shown that: (i) wild rodents and domestic dogs in South Africa habour many tick-borne pathogens, some of which are animal and human pathogens, further studies should be carried out to determine the risk of these infections to human health; (ii) the qPCR assay was more sensitive than the RLB assay in detecting A. phagocytophilum infections, however, the specificity of the assay should be confirmed; (iii) sequence analyses confirmed the presence of A. phagocytophilum DNA in a dog; and (iv) Anaplasma sp. Zambian and South African dog strain seems to be a common species in South African dogs, more studies are needed to determine the taxonomic status and epidemiology of these species in South Africa.