Molecular detection and genetic characterisation of pathogenic Theileria, Anaplasma and Ehrlichia species among apparently healthy sheep in central and western Kenya

Abstract

Tick-borne diseases (TBDs) caused by Theileria, Babesia, Anaplasma and Ehrlichia species are common in tropical and subtropical regions. In this study, we investigated the presence and genetic diversity of Theileria spp., Anaplasma ovis, B. ovis, E. ruminantium and Anaplasma spp. in sheep from the Machakos and Homa Bay counties of Kenya. In order to improve the diagnosis and control of ovine TBDs, a total of 76 blood samples from apparently healthy sheep were screened using a polymerase chain reaction (PCR). The assays were conducted using primers based on Theileria spp. 18S rRNA, Anaplasma ovis Major surface protein-4 ( AoMSP4 ), B. ovis 18S rRNA, E. ruminantium pCS20 and Anaplasma spp. 16S rRNA . The overall infection rates for Theileria spp., A. ovis, E. ruminantium and Anaplasma spp. were 39/76 (51.3%), 26/76 (34.2%), 6/76 (7.9%) and 31/76 (40.8%), respectively. The overall co-infection was 47/76 (61.8%). All Theileria spp. positive samples were confirmed to be of Theileria ovis on sequencing. A phylogenetic analysis of the 18S rRNA gene sequences of T. ovis revealed that all isolates of this study clustered with T. ovis sequences extracted from the GenBank suggesting this gene is highly conserved. E. ruminantium pCS20 sequences were in the same clade on the phylogenetic tree. However, three AoMSP4 sequences from this study appeared in the same clade, while one sequence formed a separate branch revealing genetic divergence. The 16S rRNA sequencing revealed uncharacterised Anaplasma spp. and A. ovis . The phylogenetic analyses of the uncharacterised Anaplasma spp. revealed that the two sequences from this study appear in an independent clade from other sequences extracted from the GenBank. This study provides important information regarding the occurrence of tick-borne pathogens and their degree of genetic diversity among sheep in Kenya, which is useful for the diagnosis and control of TBDs.

Molecular epidemiology of anaplasmosis in small ruminants along a human-livestock-wildlife interface in Uganda

“…This molecular based study has demonstrated a high-level of Anaplasmataceae infection in small ruminants within western Uganda. This was the first molecular study in small ruminants of Uganda and the prevalence reported in this study was much higher than reports from Kenya (40.8%) ( Ringo et al., 2019 ) and Sudan (60.1%) ( Lee et al., 2018 ). Here we targeted only one species of Anaplasma ( A. ovis ).…”

“…PCR targeting the Anaplasmataceae 16S rRNA gene was used to detect Anaplasma spp ( Ringo et al., 2019 ) with minor modifications. A 10 μL reaction containing 1x My Taq mix (Bioline®), about 1 μg of genomic DNA 0.2 μM each of the forward and reverse primers.…”

“…A 10 μL reaction containing 1x My Taq mix (Bioline®), about 1 μg of genomic DNA 0.2 μM each of the forward and reverse primers. Primer nucleotide sequences used were: forward primers, 5′GGTTTAATTCGATGCAACGCGA-3′, and reverse primers 5′CGTATTCACCGTGGCATG 3’ ( Bekker et al., 2002 ; Ringo et al., 2019 ). Anaplasma centrale DNA was used as a positive control ( Ikwap et al., 2010 ).…”

“…Recent surveys show increasing evidence of Anaplasma spp in small ruminants. The prevalence of Anaplasma spp was found to be 40.8% in ovines from central and western Kenya by PCR ( Ringo et al., 2019 ). In northeastern Uganda, a prevalence of 19.5% for Anaplasma spp in small ruminants was reported by microscopy ( Lolli et al., 2016 ).…”

“…Molecular diagnosis of Anaplasma bacteria is by detection of the 16S rRNA gene ( Ringo et al., 2019 ; Shi et al., 2020 ; Yang et al., 2016 ). This forms the basis of phylogenetic analysis in the Anaplasmataceae ( Seo et al., 2018 ) for insight into genotypic variation and diversity.…”