Genetic diversity in Babesia bovis from southern Africa and estimation of B. bovis infection levels in cattle using an optimised quantitative PCR assay

• Published in: Ticks and tick-borne diseases 2023-03, Vol.14 (2), p.102084-102084, Article 102084
• Main Authors: Byaruhanga, Charles, Makgabo, S. Marcus, Choopa, Chimvwele N., Mulandane, Fernando C., Vorster, Ilse, Troskie, Milana, Chaisi, Mamohale E., Collins, Nicola E.
• Format: Article
• Language: English
• Subjects: 18S rRNA; Africa, Southern - epidemiology; Animals; Babesia bovis - genetics; Babesiosis - diagnosis; Babesiosis - epidemiology; Babesiosis - parasitology; Cattle; Cattle Diseases - diagnosis; Cattle Diseases - epidemiology; Cattle Diseases - parasitology; Genetic Variation; msa-2b; Phylogeny; Quantification; Real-time PCR; Real-Time Polymerase Chain Reaction - veterinary; Reverse line blot; RNA, Ribosomal, 18S - genetics; South Africa
• ISSN: 1877-959X; 1877-9603
• DOI: 10.1016/j.ttbdis.2022.102084

Babesia bovis is a causal agent of bovine babesiosis, a disease which leads to mortality and morbidity and impacts the cattle industry worldwide. We amplified, cloned and sequenced the B. bovis merozoite surface antigen-2b (msa-2b) gene (∼940 bp) and the near full-length 18S rRNA gene (∼1600 bp) from cattle samples from South Africa and Mozambique to determine sequence variation between B. bovis parasites in the region. A TaqMan quantitative real-time PCR (qPCR) assay (18S rRNA gene) was optimised for the detection of B. bovis and estimation of parasitaemia in field samples from cattle from southern Africa. Phylogenetic analysis grouped the Msa-2b sequences in six clades and these were 59.7 to 99.6% identical to reference sequences. Sequence variation amongst B. bovis 18S rRNA sequences was found at 2 to 36 positions, and the sequences were 97 to 99% identical to published sequences. Mismatches between the B. bovis 18S rRNA sequences and a previously published qPCR forward primer (BoF) were observed; therefore, we developed a new forward primer (BoF2), and optimised the qPCR assay. Six 10-fold dilution series of B. bovis infected erythrocytes (2 × 108 to 2 × 103 infected red blood cells [iRBC]/ml) were analysed in triplicate in each of six separate qPCR runs, to determine the efficiency of the assay. The qPCR assay amplified the B. bovis 18S rRNA gene with 92.0 to 94.9% efficiency. The detection limit of the qPCR assay was approximately 6 iRBCs/μl. The performance of the optimised assay to diagnose B. bovis in field samples was assessed by testing DNA from 222 field samples of cattle from South Africa and Mozambique using three methods: the optimised qPCR assay, the reverse line blot (RLB) hybridisation assay, and the previously published qPCR assay. The detection rate of B. bovis using the optimised qPCR assay (31.1%, 69/222) was significantly higher (p