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

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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.

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International collaborative research: significance of tick-borne hemoparasitic diseases to world animal health.

Gerrit Uilenberg (1995)

A general review is given of the tick-borne hemoparasitic diseases of greatest economic importance in ruminants, babesiosis, anaplasmosis, theileriosis and cowdriosis, each caused by one or more species of hemoparasites. Most affected are cattle and small ruminants, buffalo are more resistant and little is known regarding camels. The situation varies from one continent or region to another. Innate and breed susceptibility to these diseases are of tremendous importance. Disease in the field cannot be considered separated from the whole complex of tick-borne diseases and from the ticks themselves, particularly if the aim is to attain endemic stability. International coordination is needed now that research funds are scarce. An appendix contains tables with hemoparasites of various domestic animals and notes with background details.

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Geographic distribution and frequency of a taurine Bos taurus and an indicine Bos indicus Y specific allele amongst sub-saharan African cattle breeds.

O Hanotte, C L Tawah, D Bradley … (2000)

We report for the first time, and for the whole of sub-Saharan Africa, the geographical distribution and the frequency of an indicine and a taurine Y specific allele amongst African cattle breeds. A total of 984 males from 69 indigenous African populations from 22 countries were analysed at the microsatellite locus INRA 124. The taurine allele is probably the oldest one on the continent. However, the taurine and the indicine alleles were present in 291 males (30%), and 693 males (70%), respectively. More particularly, 96% of zebu males (n = 470), 50% of taurine males (n = 263), 29% of sanga males (crossbreed Bos taurus x Bos indicus, n = 263) and 95% of zebu x sanga crossbred males (n = 56) had the indicine allele. The Borgou, a breed classified as zebu x taurine cross showed only the zebu allele (n = 12). The indicine allele dominates today in the Abyssinian region, a large part of the Lake Victoria region and the sahelian belt of West Africa. All the sanga males (n = 64) but only one from the Abyssinian region had the indicine allele. The taurine allele is the commonest only among the sanga breeds of the southern African region and the trypanotolerant taurine breeds of West Africa. In West Africa and in the southern Africa regions, zones of introgression were detected with breeds showing both Y chromosome alleles. Our data also reveal a pattern of male zebu introgression in Mozambique and Zimbabwe, probably originating from the Mozambique coast. The sanga cattle from the Lake Victoria region and the Kuri cattle of Lake Chad, cattle populations surrounded by zebu breeds were, surprisingly, completely devoid of the indicine allele. Human migration, phenotypic preferences by the pastoralists, adaptation to specific habitats and to specific diseases are the main factors explaining the present-day distribution of the alleles in sub-Saharan Africa.

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Simultaneous detection of Anaplasma and Ehrlichia species in ruminants and detection of Ehrlichia ruminantium in Amblyomma variegatum ticks by reverse line blot hybridization.

Cornelis Bekker, Sander de Vos, Amar Taoufik … (2002)

The detection of Anaplasma and Ehrlichia species is usually based on species-specific PCR assays, since no assay is yet available which can detect and identify these species simultaneously. To this end, we developed a reverse line blot (RLB) assay for simultaneous detection and identification of Anaplasma and Ehrlichia species in domestic ruminants and ticks. In a PCR the hypervariable V1 region of the 16S ribosomal RNA (rRNA) gene was amplified with a set of primers unique for members of the genera Anaplasma and Ehrlichia [Int. J. Syst. Evol. Microbiol. 51 (2001) 2145]. Amplified PCR products from blood of domestic ruminants or Amblyomma variegatum tick samples were hybridized onto a membrane to which eight species-specific oligonucleotide probes and one Ehrlichia and Anaplasma catch-all oligonucleotide probe were covalently linked. No DNA was amplified from uninfected blood, nor from other hemoparasites such as Theileria annulata, or Babesia bigemina. The species-specific probes did not cross-react with DNA amplified from other species. E. ruminantium, A. ovis and another Ehrlichia were identified by RLB in blood samples collected from small ruminants in Mozambique. Finally, A. variegatum ticks were tested after feeding on E. ruminantium infected sheep. E. ruminantium could be detected in adult ticks even if feeding of nymphs was carried out 3.5 years post-infection. In conclusion, the developed species-specific oligonucleotide probes used in an RLB assay can simultaneously detect and identify several Ehrlichia and Anaplasma species. However, as no quantitative data for the detection limit are available yet, only positive results are interpretable at this stage.