Study on tick-borne pathogens and tick-borne diseases using molecular tools with emphasis on Anaplasma spp. and Ehrlichia spp. in ticks and domestic ruminants in Ethiopia

Although tick-borne pathogens are among the most important constraints in the domestic ruminant industry causing huge economic losses, little information is available on their occurrence and distribution in Ethiopia. Within this doctoral study, three cross-sectional epidemiological studies using molecular techniques were conducted, one on ticks collected from cattle and sheep, one on domestic ruminants and one on unfed ticks collected from the field. The survey conducted on ticks collected from cattle and sheep using a molecular analysis of 18S rDNA identified the occurrence of Theileria buffeli/orientalis, Theileria velifera, and Theileria ovis in Rhipicephalus evertsi evertsi and Rhipicephalus decoloratus. According to the 16s rDNA PCR and sequencing, six species of Bartonella, three species of Rickettsia (Rickettsia africae, Rickettsia felis and Rickettsia sp.), Anaplasma ovis, Ehrlichia ruminantium, Ehrlichia spp., Anaplasma spp. and Borrelia burgdorferi s.l. were identified in Rhipicephalus spp. This is the first report of the occurrence of A. ovis, Bartonella elizabethae, Bartonella bovis, Bartonella koehlerae, Bartonella quintana and Bartonella vinsonii berkhoffii in Ethiopia. Our finding highlights the risk of infection of animals and humans with zoonotic tick-borne bacteria in Ethiopia. At the end of the first survey, we focused on the molecular identification of Ehrlichia spp. and Anaplasma spp., in which we developed a semi-nested PCR amplifying 925bp of 16S rDNA. A pair of primers designated EBR2 and EBR3 was designed from the Anaplasma 16S rDNA sequences for simultaneous detection of Ehrlichia spp. and Anaplasma spp. Individual species of Ehrlichia and Anaplasma were identified by restriction with MboII, HhaI and MspI enzymes, including mixed infections. Analysis of Amblyomma spp. from various parts of Ethiopia for bacterial pathogens using this molecular method revealed the occurrence of Anaplasma marginale, Anaplasma phagocytophilum, Anaplasma centrale, Anaplasma (formerly Ehrlichia) sp. Omatjenne and E. ruminantium. By helping animal health professionals in their decision-making regarding the diagnosis and control of anaplasmosis and heartwater, this improved molecular method will help the government in establishing a disease-monitoring program in the dairy and beef industries. The second part involves surveys of A. phagocytophilum and A. sp. Omatjenne infection in cattle in Africa and that of Anaplasma spp. and Ehrlichia spp. in cattle, sheep and goats in Ethiopia. In the international survey, respectively 19 (2.7%) and 45 (6.5%) samples yielded positive signals for A. phagocytophilum and A. sp. Omatjenne. Anaplasma sp. Omatjenne was detected in all countries except Tanzania while A. phagocytophilum was detected only in samples originating from Ethiopia. Out of the 922 blood samples from cattle, sheep and goats from five different localities in Ethiopia analyzed by 16S rDNA PCR, 523 (56.7%) tested positive. Overall, 67.4% of cattle, 65% of sheep and 18% of goats tested positive for one or more Anaplasma spp. No positive result was observed for Ehrlichia spp. using the 16S rDNA analysis. RFLP analysis identified A. marginale, A. ovis, A. phagocytophilum, A. centrale and A. sp. Omatjenne. We conclude that infection of domestic ruminants with Anaplasma spp. is widespread in Ethiopia. Livestock improvement plans through introduction of improved breeds should be aware of this and take the necessary precautions to minimize losses associated with anaplasmosis. First reports of infection of domestic ruminants with A. phagocytophilum, A. ovis and A. sp. Omatjenne are provided for Ethiopia. In addition, the occurrence of A. phagocytophilum together with A. sp. Omatjenne outside Europe and South Africa is made for the first time. Random samples of 493 animals were tested with a pCS20 PCR for the identification of E. ruminantium. Three samples from 75 (4%) cattle gave a positive result. Ehrlichia ruminantium was identified in five of 13 clinical cases of heartwater in dairy cows (38.46%) and 30 of 72 (41.67%) clinically affected Boer goats. Identification of ticks that carry pathogens of veterinary and public-health importance, belonging to the genus Anaplasma, is an initial step to identify tick vectors that play a role in the epidemiology of the diseases they cause. A molecular investigation was carried out to identify Anaplasma spp. carried by unfed field ticks with an emphasis on A. phagocytophilum and A. ovis. A total of 240 unfed ticks (adults and nymphs) were collected and analyzed using PCR-RFLP. Anaplasma ovis was identified in R. evertsi, Amblyomma spp. and Hyalomma spp. Anaplasma phagocytophilum was detected only in Rhipicephalus pulchellus. Our finding identified potential vectors of A. ovis to be further confirmed by experimental study. In conclusion, this doctoral study identified tick-borne pathogens of veterinary and public health importance in Ethiopia. An improved molecular method was developed for simultaneous detection of Anaplasma spp. and Ehrlichia spp. and it was used in epidemiological studies in domestic ruminants and ticks. A high prevalence of anaplasmosis and a low prevalence of heartwater were observed. The occurrence of infection in cattle with A. sp. Omatjenne was identified in certain African countries. Furthermore, candidate vectors of A. ovis were identified. Further studies on the medical significance of the identified zoonotic pathogens, search for vectors of A. phagocytophilum and experimental studies for the confirmation of the vectors of A. ovis are warranted. Lastly, an improvement of molecular methods is needed for the detection of carriers of E. ruminantium.