Molecular investigation of tick-borne haemoparasite infections among transhumant zebu cattle in Karamoja Region, Uganda

Tick-borne diseases (TBDs) are a major constraint to cattle production in pastoral areas in Africa. Although information on tick-borne infections is important to prioritise control approaches, it is limited for transhumant zebu cattle in Karamoja, Uganda. We conducted a study to determine the occurrence and level of tick-borne infections among cattle in Karamoja Region. A total of 240 cattle were selected for blood collection using systematic sampling in 20 randomly-selected herds in two districts. The hypervariable V4 region of the 18S rRNA gene for Theileria/Babesia and the V1 region of the 16S rRNA gene for Ehrlichia/Anaplasma were amplified and hybridised to genus- and species-specific oligonucleotide probes on a reverse line blot (RLB) membrane. A duplex quantitative real-time polymerase chain reaction (qPCR) assay based on msp1β and groEL genes was used for the detection of Anaplasma marginale and A. centrale, while monoplex qPCR assays were used for the detection of Ehrlichia ruminantium (226bp fragment of the pCS20 region) and Theileria parva (18S rRNA gene). The RLB hybridisation assay demonstrated the presence of tick-borne haemoparasites in all but one sample (99.6%), mostly as mixed infections (97.5%). The most frequently detected species were Theileria mutans (88.3%, 95% confidence interval: 84.6-91.7%), A. marginale (73.8%: 68.3-78.8%), Theileria velifera (71.3%: 65.8-76.7%) and Anaplasma sp. Omatjenne (63.3%: 57.5-68.8%). Other virulent pathogens, namely Babesia bigemina (5.0%) and T. parva (2.9%), were also detected with RLB, but not E. ruminantium. The proportions of qPCR positive samples were 82.9% (A. marginale), 12.1% (A. centrale), 3.3% (T. parva), and 1.7% (E. ruminantium). The full-length 18S rRNA genes from 6 out of 47 samples that were positive on RLB for the Babesia genus-specific probe and not for any of the Babesia species-specific probes were amplified, cloned and sequenced. The sequences were used to construct phylogenetic trees. Variations (5 to 9 nucleotides) in the 18S rRNA gene sequences of B. bigemina were identified, when compared with B. bigemina sequences from other parts of the world. Three nucleotide differences in the B. bigemina probe region may explain the failure of the RLB hybridisation assay to detect B. bigemina in some samples. T. mutans and B. bigemina sequences grouped in separate clades from previously published sequences. In conclusion, this study demonstrated high and widespread occurrence, and sequence variation of tick-borne haemoparasites among cattle in the pastoral area of Karamoja, which is useful for diagnosis and control of TBDs.