Prevalence and genotyping of Echinococcus granulosus in sheep in Narok County, Kenya.

Cystic echinococcosis (CE) is a zoonotic disease caused by the larval stage of Echinococcus granulosus species (sensu lato, s.l.). In East Africa, several species/strains occur in livestock, wildlife, and humans, but there is limited information on frequencies of infection by different genotypes in the various mammalian hosts. We have obtained data on E. granulosus infection prevalence in sheep sampled from abattoirs in Narok County, southern Kenya. We inspected carcasses for the presence of hydatid cysts in 180 sheep randomly selected in five sub-locations. The overall prevalence was 16.0% (144/900 animals), with the majority of cysts (50.7%) found in the liver, followed by the lungs (36.8%), while infections involving the liver and lungs were detected in 12.5% of the sheep. PCR-RFLP genotyping of the mitochondrial nad-1 gene in all the 343 cysts identified E. granulosus G1-G3 (sensu stricto, s.s.) as the only genotype. The majority of the cysts (62.1%) were fertile, and 35.2% were sterile, while 2.7% were calcified. Considering cyst fertility, 73.02% of lung cysts were fertile compared to 53.4% in liver cysts. Our data extends previous CE studies in livestock and indicates a high level of CE infection of sheep in Narok, with a predominance of E. granulosus s.s., which is highly pathogenic and commonly infects humans. Given the high fertility rates observed in the cysts, there is an urgent need to determine whether there is a significant incidence of human infection in Narok, and initiate "One Health" control measures.

Differential transcription of two highly divergent gut-expressed Bm86 antigen gene homologues in the tick Rhipicephalus appendiculatus (Acari: Ixodida).

The transcriptional control of gene expression is not well documented in the Arthropoda. We describe transcriptional analysis of two exceptionally divergent homologues (Ra86) of the Bm86 gut antigen from Rhipicephalus appendiculatus. Bm86 forms the basis of a commercial vaccine for the control of Rhipicephalus (Boophilus) microplus. The R. appendiculatus Ra86 proteins contain 654 and 693 amino acids, with only 80% amino acid sequence identity. Reverse-transcription PCR of gut cDNA showed transcription of only one genotype in individual female ticks. PCR amplification of 3' untranslated sequences from genomic DNA indicated that both variants could be encoded within a single genome. When both variants were present, one of the two Ra86 genotypes was transcriptionally dominant.

Linkage disequilibrium between alleles at highly polymorphic mini- and micro-satellite loci of Theileria parva isolated from cattle in three regions of Kenya.

Theileria parva schizont-infected lymphocyte culture isolates from western, central and coastal Kenya were analysed for size polymorphism at 30 T. parva-specific variable number tandem repeat (VNTR) loci using a panel of mini- and micro-satellite markers. The mean number of alleles ranged from 3 to 11 at individual loci and 183 distinct alleles were observed in total, indicating high genetic diversity within the T. parva gene pool in Kenyan cattle. The frequency distribution of the length variation of specific alleles among isolates ranged from normal to markedly discontinuous. Genetic relationships between isolates were analysed using standard indices of genetic distance. Genetic distances and dendrograms derived from these using neighbour-joining algorithms did not indicate significant clustering on a geographical basis. Analysis of molecular variance demonstrated that the genetic variation between individual isolates was 72%, but only 2.3% when isolates from different regions were pooled. Both these observations suggest minimal genetic sub-structuring relative to geographical origin. Linkage disequilibrium was observed between pairs of loci within populations, as in certain Ugandan T. parva populations. A novel observation was that disequilibrium was also detected between alleles at three individual pairs of VNTR loci when isolates from the three regional meta-populations were pooled for analysis.

The African buffalo parasite Theileria. sp. (buffalo) can infect and immortalize cattle leukocytes and encodes divergent orthologues of Theileria parva antigen genes.

African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. 'Deep 454 pyrosequencing' of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva.

A panel of microsatellite and minisatellite markers for the characterisation of field isolates of Theileria parva.

Mini- and microsatellite sequences show high levels of variation and therefore provide excellent tools for both the genotyping and population genetic analysis of parasites. Herein we describe the identification of a panel of 11 polymorphic microsatellites and 49 polymorphic minisatellites of the protozoan haemoparasite Theileria parva. The PCR products were run on high resolution Spreadex gels on which the alleles were identified and sized. The sequences of the mini- and microsatellites were distributed across the four chromosomes with 16 on chromosome 1, 12 on chromosome 2, 14 on chromosome 3 and 18 on chromosome 4. The primers from the 60 sequences were tested against all the Theileria species that co-infect cattle in East and Southern Africa and were found to be specific for T. parva. In order to demonstrate the utility of these markers, we characterised eight tissue culture isolates of T. parva isolated from cattle in widely separated regions of Eastern and Southern Africa (one from Zambia, one from Uganda, two from Zimbabwe, four from Kenya) and one Kenyan tissue culture isolate from Cape buffalo (Syncerus caffer). The numbers of alleles per locus range from three to eight indicating a high level of diversity between these geographically distinct isolates. We also analysed five isolates from cattle on a single farm at Kakuzi in the central highlands of Kenya and identified a range of one to four alleles per locus. Four of the Kakuzi isolates represented distinct multilocus genotypes while two exhibited identical multilocus genotypes. This indicates a high level of diversity in a single population of T. parva. Cluster analysis of multilocus genotypes from the 14 isolates (using a neighbour joining algorithm) revealed that genetic similarity between isolates was not obviously related to their geographical origin.