Molecular detection, genetic diversity and phylogenetic analysis of anaplasma marginale infecting cattle in South Africa
Mutshembele, Awelani Mirinda
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Bovine anaplasmosis caused by Anaplasma marginale is endemic in South Africa. This endemicity is due to presence of tick vectors that transmit A. marginale the causal agent of the disease and the high seroprevalence in Limpopo, Free State and North West provinces. To date, the genetic diversity of A. marginale isolates infecting cattle in all South African provinces, except Free State, are generally unknown. Recently, vaccines based on the A. marginale major surface protein 1 a (MSP1 a) has been proposed as a strategy for controlling bovine anaplasmosis. However, characterization of genetic diversities of the A. marginale isolates in these regions is still needed before this protein can be used for vaccine development. Therefore, the aim of this study was to determine the prevalence, genetic diversity and phylogenetic relationship of A. marginale infecting cattle in all South African provinces except the Free State. A total of 280 whole blood samples were collected from cattle in all provinces with exception of the Free State. Twenty six districts and municipalities were included in this sampling. Anaplasma marginale genomic DNA was then extracted from the blood sample using ZR Genomic DNA 1M Tissue Miniprep (Zymo Research, CA, USA). A polymerase chain reaction (PCR) was done with primers targeting msp1a and msp4 genes and the PCR products were sequenced using genetic analyser (ABI, Life technologies, CA, USA). The generated sequences were analysed by bioinformatics and their phylogeny as well as genetic diversity index (GDI) was determined based on the sequences of msp1a and msp4 genes. Overall, the prevalence of A. marginale infection in cattle was 76% in all provinces except for Northern Cape Province where the prevalence was zero. The prevalence per province was as follows: Eastern Cape 19.1 %, Gauteng 9.6%, KwaZulu-Natal 23.0%, Limpopo 15.3%, Mpumalanga 10.1%, North West 12.4% and Western Cape 10.5%. The msp1 a revealed genetic variability with regions of different types of tandem repeats. Some repeats were conserved amongst the A. marginale strains and revealed low variable peptides in the MSP1 a tandem repeats. A polynomial correlation (R2=0. 76) was observed between the GDI and anaplasmosis prevalence per province. Interestingly, provinces with the highest prevalence were not the ones with highest or lowest GDI. The analysis of msp4 gene sequences, which provided evolutionary information about geographically distinct A. marginale strains, was used in the present study for phylogenetic analysis of samples from Limpopo (LP), Mpumalanga (MP), North West (NW), Gauteng (GP), KwaZulu-Natal (KZN), Eastern Cape (EC) and Western Cape (WC) provinces of South Africa. Two clades were observed which consisted of first clade (LP, NW, GP, KZN and WC) and second clade (MP and EC) isolates. In addition when DNA sequence variation of msp4 gene was analysed in combination with isolates from other countries outside South Africa, important phylogeographic information was observed. The South African strains had 100% identity with isola tes from Kenya, Zimbabwe and Australia. Good representation of the Southern and Northern Hemispheres was observed and demonstrated that the msp4 gene was a good phylogeographic marker. These results indicated that A. marginale is widespread in South Africa, and suggested that the analysis of msp1a and msp4 gene sequences provided an understanding of the phylogeny and epidemiology of A. marginale in South Africa.