Molecular detection of haemoparasites and characterization of Theileria parva from cattle in Mashonaland East Province, Zimbabwe

Abstract

Tick-borne haemoparasites (TBH) greatly threaten cattle production and cause economic losses to the cattle industry in Zimbabwe. In Mashonaland East Province, severe cattle mortalities have been occurring due to infection with TBH, especially Theileria parva (T. parva). Despite the huge challenge posed by tick-borne diseases (TBD) to cattle production, very little work has been conducted to detect TBH in cattle in Zimbabwe utilising molecular techniques, particularly in hotspots like Mashonaland East Province. Furthermore, no attempts have been made to characterise T. parva in cattle despite its importance in disease outbreaks in the province. The objectives of the current study were, to determine whether cattle at Goromonzi and Seke Districts in Mashonaland East Province, Zimbabwe are infected with Theileria, Babesia, Anaplasma and Ehrlichia parasites using the Reverse Line Blot - Polymerase Chain Reaction (RLB-PCR) hybridization assay. Furthermore, to characterise the p67 antigen gene from T. parva positive samples. A total of 264 communal cattle were sampled in Goromonzi and Seke districts of Zimbabwe, and their blood was spotted onto Flinders Technology Associates (FTA) cards. Deoxyribonucleic acid (DNA) extracted from FTA cards was screened for the presence of TBH using the RLB. Samples that hybridised to the T. Parva species-specific probe were subjected to hybridization probe-based real-time PCR to confirm T. Parva infections. The T. parva strains were characterised by amplification and sequencing of the p67 gene. About 95.5% (252/264) of cattle sampled were positive for TBH as detected by the RLB hybridisation assay. Using the T. parva-specific qPCR, 40 (15.2%) of the 264 sampled cattle were positive, which was similar (p> 0.05) to the detection rate using the RLB hybridisation assay (41 or 15.5%) by hybridisation to the T. parva species-specific probe, although the latter is known to be less sensitive. Overall, infection with multiple TBH were detected in 24.6% (65/264) of the cattle samples analysed. Theileria parva (29%), which is pathogenic parasite was detected at a higher rate in Goromonzi than in Seke district, whereas Anaplasma sp. Omatjenne (24%) which is non-pathogenic parasite was detected at a higher rate in Seke than in Goromonzi district (p < 0.05). The size of the p67 PCR product obtained from amplification was approximately 900 base pairs (bp) which was associated with the cattle-derived allele 1 that has a 129 bp insert deletion. The p67 sequences were aligned against cattle-derived and buffalo-derived allele 1 references from the Genbank, and they showed 99% similarity to the sequence of a cattle-derived isolate T. parva Muguga isolate (accession number: M67476) and 97% similarity to sequences from buffalo-derived isolates KNP_MN_C81_3 (Mukolwe et al. 2020), KNP_MN_C89_2 (accession number MT199346) and KNP_MN_C108_6 (Mukolwe et al. 2020). In conclusion, Theileria, Babesia, Anaplasma and Ehrlichia parasites infect cattle in Seke and Goromonzi districts. Cattle-derived T. parva with the p67 allele genotype 1 is responsible for theileriosis outbreaks and massive cattle mortality in the two districts sampled. In Zimbabwe ticks are controlled by dipping. It is recommended that farmers and state veterinary authorities improve on their control measures to control ticks through intensified dipping and by vaccination of cattle to reduce the impact of TBH on the cattle. Further studies are required to determine the extent of spread of TBH and characterisation of the T. parva countrywide.