Identification and molecular characterization of zoonotic rickettsiae and other vector-borne pathogens infecting dogs in Mnisi, Bushbuckridge, Mpumalanga Province, South Africa

Abstract

Dogs play an important role in the lives of humans, and because of this close contact they share with humans, they can serve as sentinels for infections to humans. Rickettsiae are small, obligate intracellular bacteria that are vector-borne and can cause mild to fatal diseases in humans worldwide. There is little information on the zoonotic rickettsial pathogens that may be harboured by dogs from rural localities in South Africa. Thus the primary aim of this study was to identify and characterise rickettsial pathogens infecting dogs using molecular techniques by screening blood samples and ectoparasites collected from domestic dogs in the Bushbuckridge area, Mpumalanga province of South Africa. A total of 141 blood samples, 103 ticks and 43 fleas were collected from owned, free-roaming dogs in the Mnisi community area in Bushbuckridge Local Municipality, Mpumalanga Province, South Africa under the project “Health and Demographic Surveillance System (HDSS) in dogs” (protocol number: V033/11). Samples were collected from October 2011 through May 2012. Ectoparasites identified are as follows: the tick species include Haemaphysalis elliptica, Amblyomma hebraeum, Rhipicephalus sanguineus, Rhipicephalus simus, and an unspeciated Ixodes spp. Fleas includes Ctenocephalides felis strongylus and Echidnophaga gallinacea. DNA was extracted from blood and ectoparasites and screened for the presence of Ehrlichia, Anaplasma, Theileria and Babesia species infections using the reverse line blot (RLB) hybridization assay. Almost half of the blood samples reacted with the genus-specific probes for Ehrlichia/Anaplasma (49%), and 16% of the samples investigated were positive for Ehrlichia canis DNA. Babesia rossi (9%) and Babesia vogeli (4%) were detected among the 14% of samples that were positive for the genus-specific probe 1 of Babesia. Furthermore, 14% of the samples were positive for the genus-specific probes of Theileria/Babesia, 2% were positive for the genus-specific probe of Theileria, and 1% for the genus-specific probe 2 of Babesia. Haemoparasite DNA could not be detected in 36% of the samples investigated. Results of the RLB assay for ectoparasites showed that 30% of tick pools were positive for the genus-specific probes of Ehrlichia/Anaplasma species, Babesia 1 and 2 (30%) and Theileria (10%) while haemoparasite DNA could not be detected in 30% of the tick pools. Haemoparasite species DNA detected from tick pools includes Neoehrlichia spp., Ehrlichia ruminantium, and Babesia microti. Flea pools were positive for the genus-specific probes of Babesia 1 and 2 (50%). Specific haemoparasite species were not detected from the flea pools. Rickettsia genus and species-specific qPCR assays were performed to detect zoonotic rickettsiae from DNA obtained from blood and ectoparasite samples. Rickettsiae were not detected in blood samples but in 70% of tick pools analyzed using a genus-specific qPCR assay, based on the 17-kD antigen gene. Rickettsial infections were detected in tick pools, namely Haemaphysalis elliptica, Amblyomma hebraeum, Rhipicephalus sanguineus, Rhipicephalus simus, and Ixodes, and in flea pools Ctenocephalides felis strongylus and Echidnophaga gallinacea. Using the species-specific assays, three pools of ticks (two Amblyomma hebraeum pools and a Haemaphysalis elliptica pool) tested positive on a R. africae genotype-specific qPCR assay, while flea pools (Ctenocephalides felis strongylus and Echidnophaga gallinacea) were positive on the R. felis qPCR assay. Blast analysis of nucleotide sequences of the V1 hypervariable region of the 16S rRNA gene of five selected Ehrlichia/Anaplasma positive samples on the RLB hybridization assay revealed an Anaplasma sp. South Africa dog strain 98% related to Anaplasma phagocytophilum in four of the five samples and an Anaplasma phagocytophilum strain in one sample. Furthermore, Rickettsia massiliae and Orientia tsutsugamushi sequences were also identified in two additional samples. Analysis of nucleotide sequences obtained from tick and flea pool DNA samples positive on R. africae and R. felis qPCR assays revealed Rickettsia africae and Candidatus Rickettsia asemboensis a new Rickettsia felis-like organism respectively. Analysis of nucleotide sequences of the 18S rRNA gene of three selected B. rossi positive samples on the RLB hybridization assay confirmed detection of B. rossi in all selected samples. The detection of zoonotic rickettsiae in canine blood and their ectoparasites suggests that dogs may play an important role in the lifecycle of these organisms; these data also provide preliminary information about the diversity of rickettsiae and other vector-borne pathogens circulating in domestic dogs and their ectoparasites in Bushbuckridge, South Africa. In conclusion the detection of zoonotic rickettsiales from blood samples of domestic dogs and their ectoparasites in a rural community in South Africa highlights the potential risk of human infection that may occur from these pathogens.