Molecular detection, genetic diversity and phylogenetic analysis of anaplasma marginale infecting cattle in South Africa

[摘要] Bovine anaplasmosis caused by Anaplasma marginale is endemic in South Africa. Thisendemicity is due to presence of tick vectors that transmit A. marginale the causal agentof the disease and the high seroprevalence in Limpopo, Free State and North Westprovinces. To date, the genetic diversity of A. marginale isolates infecting cattle in allSouth African provinces, except Free State, are generally unknown. Recently, vaccinesbased on the A. marginale major surface protein 1 a (MSP1 a) has been proposed as astrategy for controlling bovine anaplasmosis. However, characterization of geneticdiversities of the A. marginale isolates in these regions is still needed before this proteincan be used for vaccine development. Therefore, the aim of this study was to determinethe prevalence, genetic diversity and phylogenetic relationship of A. marginale infectingcattle in all South African provinces except the Free State.A total of 280 whole blood samples were collected from cattle in all provinces withexception of the Free State. Twenty six districts and municipalities were included in thissampling. Anaplasma marginale genomic DNA was then extracted from the bloodsample using ZR Genomic DNA 1M Tissue Miniprep (Zymo Research, CA, USA). Apolymerase chain reaction (PCR) was done with primers targeting msp1a and msp4genes and the PCR products were sequenced using genetic analyser (ABI, Lifetechnologies, CA, USA). The generated sequences were analysed by bioinformaticsand their phylogeny as well as genetic diversity index (GDI) was determined based onthe sequences of msp1a and msp4 genes.Overall, the prevalence of A. marginale infection in cattle was 76% in all provincesexcept for Northern Cape Province where the prevalence was zero. The prevalence perprovince 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 lowvariable 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 orlowest GDI.The analysis of msp4 gene sequences, which provided evolutionary information aboutgeographically distinct A. marginale strains, was used in the present study forphylogenetic 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 firstclade (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 combinationwith isolates from other countries outside South Africa, important phylogeographicinformation was observed. The South African strains had 100% identity with isola tesfrom Kenya, Zimbabwe and Australia. Good representation of the Southern andNorthern Hemispheres was observed and demonstrated that the msp4 gene was agood phylogeographic marker. These results indicated that A. marginale is widespreadin South Africa, and suggested that the analysis of msp1a and msp4 gene sequencesprovided an understanding of the phylogeny and epidemiology of A. marginale in SouthAfrica.

[发布机构] University of the Free State