Molecular Identification and Characterization of Anaplasma Haemoparasites Isolated From Cattle and Sheep in Homabay County, Kenya

Abstract

Anaplasmosis is a cosmopolitan tick-borne disease of great importance in tropical and subtropical regions of sub-Saharan Africa and is caused by Anapalsma haemoparasites. The parasite infects a variety of hosts including cattle, sheep, dogs, humans as well as wildlife and affects the health of livestock and human population. The disease causes economic losses in livestock production. Some studies have been carried out on characterization of Anaplasma heamoparasites by using molecular techniques in Kenya. However, these studies covered limited regions in Kenya including Homabay County in Western Kenya where the disease is endemic. In addition, previous diagnostic techniques used in Homabay for identification included history, clinical signs, microscopy and serology. These were inadequate for identification of the exact species of Anaplasma currently present in Homabay County. Therefore the present study was conducted to identify and characterize of Anaplasma heamoparasites infecting cattle and sheep in Homabay County, Kenya. Anaplasma marginale 16S rRNA (Am-16SrRNA), Anaplasma marginale msp1b (Am-msp1b), Anaplasma centrale msp2 (Ac-msp2) and Anaplasma centrale 16S rRNA (Ac-16S rRNA) genes were analyzed using bioinformatics tools. The information obtained was used to design specific gene primers by using Primer Quest software of the Integrated DNA Technology (California, USA). The primers were tested using conventional and multiplex PCR reactions with positive and negative control DNA samples. For screening, whole blood samples were collected in vacutainer tubes containing EDTA from 180 animals which included 157 cattle and 23 sheep. The samples were transported to the laboratory facility located at the Department of Public Health, Pharmacology and Toxicology of the University of Nairobi. DNA was extracted using Qiagen‘s QIAamp DNA Mini kit following the manufacturer's instructions and stored at -20oC. xiv PCR was conducted and the products were subsequently purified using ZR-96 DNA Clean-up KitTM and sequenced using the Sanger method to characterize the Anaplasma species. The generated sequences were analysed by bioinformatics and were used to construct a phylogenetic tree to determine the genetic diversity of the Anaplasma species. As predicted, these primers yielded PCR amplicons of 835 bp for Am-16S rRNA, 436 bp for Ac-16S rRNA, and 576 bp for Ac-msp2. The amplification of Am-16S rRNA, Ac-16S rRNA and Ac- msp2 in cattle revealed 9(5.7%), 11(7%) and 3(1.91%) were positive, while in sheep it was 1(4.3%), 9(39.1%) and 2(8.7%) respectively. The overall prevalence from both cattle and sheep was 10 (5.55%), 20 (11.1%) and 5 (2.7%) for the genes Am-16S rRNA, Ac-16S rRNA and Ac-msp2 respectively. The Ac-msp2 and Ac-16S rRNA genes were detected in five different isolates from samples labelled; 30A, 108A, 58A, 88A, and 171A. Blast analysis of the Ac-16S rRNA amplicons revealed that the gene was homologous to Anaplasma phagoctophylum having sequence identities of 99% for cattle and 99% identity to Anaplasma ovis in sheep. Three samples revealed homologous sequences to Anaplasma ovis in cattle with 99% nucleotide identity. The phylogenetic analysis revealed that the sequences of Anaplasma centrale 16S rRNA of Homabay samples clustered together, suggesting that they were genetically related. The Kenyan isolates were also grouped with other isolates from China and USA. The findings indicated that the novel primer for Ac-16S rRNA could amplify specific conserved region of the 16S rRNA of the Anaplasma species. Primers Ac-msp2 yielded non-specific bands and needed further PCR optimization before use. The results also provided information on possible infection of cattle with Anaplasma ovis although further studies are required to confirm this. There was also a possible occurrence of Anaplasma phagoctophylum in cattle, necessitating further studies to confirm the role of this zoonotic pathogen in cattle in Homabay Counties.