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Molecular epidemiology of bovine anaplasmosis in South Africa

Bovine anaplasmosis, caused by Anaplasma marginale, is one of the most economically important tick-borne diseases of ruminants. Anaplasma centrale causes a milder form of the disease, and is used as a live vaccine against bovine anaplasmosis. A duplex quantitative real-time polymerase chain reaction (qPCR) assay was recently shown to be more sensitive that the nested PCR and reverse line blot hybridization assays in detecting A. marginale in cattle samples. In this study, the level of variation in the qPCR target regions of the msp1b (A. marginale) and groEL (A. centrale) genes from cattle field samples was assessed by amplicon sequencing. The A. centrale groEL was conserved and different from A. marginale. A total of 186 msp1b sequences were obtained from 40 samples, yielding 11 variants within the qPCR target region. Evaluation of the most diverse variants indicated that the polymorphisms had no significant effect on the efficiency of the assay. The duplex qPCR assay was then used to screen 517 cattle samples from all nine provinces of South Africa for the presence of these organisms. A. marginale and A. centrale were detected in 56.8% and 17.2% of the samples, respectively; 81 (15.3%) samples had mixed infections. A. marginale is widespread in cattle in eight of the nine provinces of South Africa, confirming that the organism is endemic in the distribution area of the vector ticks. The presence of A. centrale in many unvaccinated cattle suggests that there is a natural transmission cycle of this organism in South Africa.


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

Abstract: 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 (226 bp 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%), T. 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. Theileria 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.

Pub.: 29 Jun '16, Pinned: 06 Jun '17

Characterization of Anaplasma marginale subspecies centrale strains using msp1aS genotyping reveals a wildlife reservoir.

Abstract: Bovine anaplasmosis caused by the intraerythrocytic rickettsial pathogen Anaplasma marginale is endemic in South Africa. Anaplasma marginale subspecies centrale (A. centrale) also infects cattle, however, it causes a milder form of anaplasmosis and is used as a live vaccine against A. marginale. There has been less interest in the epidemiology of A. centrale, and, as a result, there are few reports detecting natural infections of this organism. When detected in cattle, it is often assumed that it is due to vaccination, and in most cases it is reported as co-infection with A. marginale without characterization of the strain. In this study a total of 380 blood samples from wild ruminant species and cattle collected from Biobanks, National Parks, and other regions of South Africa were used in duplex real-time PCR assays to simultaneously detect A. marginale and A. centrale. PCR results indicated high occurrence of A. centrale infections ranging from 25-100% in National Parks. Samples positive for A. centrale were further characterized using the msp1aS gene, a homolog of msp1α of A. marginale which contains repeats at the 5' end that are useful for genotyping strains. A total of 47 Msp1aS repeats were identified which corresponded to 32 A. centrale genotypes detected in cattle, buffalo and wildebeest. RepeatAnalyzer was used to examine strain diversity. Our results demonstrate a diversity of A. centrale strains from cattle and wildlife hosts from South Africa and indicate the utility of msp1aS as a genotypic marker for A. centrale strain diversity.

Pub.: 22 Jul '16, Pinned: 06 Jun '17