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A panel of 16S ribosomal RNA gene probes has been developed for the study of the
epidemiology of heartwater; five of these detect different cowdria genotypes, one detects five
distinct genotypes; one detects any Group III Ehrlichia
species other than Cowdria and one
detects any Group II Ehrlichia species. These probes have been used on PCR-amplified
rickettsial 16S rRNA genes from over 200 Amblyomma hebraeum ticks. Control ticks were
laboratory-reared and either uninfected or fed on sheep experimentally infected with different
cowdria isolates, field ticks were collected from animals in heartwater-endemic areas. All tick-derived DNA samples were also examined by PCR amplification and probing for two other
cowdria genes (map1 and pCS20) which have previously been used for heartwater
epidemiology. This paper describes the first direct comparison of all currently available DNA
probes for heartwater-associated organisms.
Small subunit ribosomal RNA (srRNA) genes of three Theileria species, one Cytauxzoon and four Babesia species were amplified using the polymerase chain reaction (PCR), cloned and sequenced. Our sequences were aligned with srRNA sequences previously published for eight species of Apicomplexa, one ciliate and one dinoflagellate, the last two being included as free-living outgroup species. Phylogenetic relationships between the organisms were inferred by four in-dependent methods of phylogenetic tree construction using the ciliate Oxytricha nova to root the trees. Our trees fail to show a consensus branching order. They do, however, clearly indicate that the theilerias form a monophyletic taxon derived from a paraphyletic group which includes the species B. equi, C. felis and B. rodhaini. The distance trees indicate that the babesias sensu stricto (B. canis, B. caballi, B. bigemina and B. bovis) form another monophyletic taxon which diverged before the theilerias separated from the above-mentioned paraphyletic group. The parsimony and maximum likelihood trees suggest that the babesias and theilerias are sister taxa, both of which were derived from the paraphyletic group.
The complete small subunit ribosomal RNA (srRNA) gene of Theileria parva was cloned and sequenced. Two primers were designed which permitted the specific amplification of part of the Theileria srRNA gene from Theileria-infected cell line samples which were predominantly (> 95%) bovine DNA. The sequence of the central (variable) region of the srRNA genes of T. annulata, T. taurotragi, T. mutans and two unidentified parasites referred to as Theileria sp. (buffalo) and Theileria sp. (Marula) were obtained. An alignment of the sequences was generated from which 6 oligonucleotide probes, corresponding to species-specific regions, were designed. These probes were demonstrated to provide unequivocal identification of each of the 6 species either by direct detection of parasite srRNA or by hybridization to amplified parasite srRNA genes. The probes were not able to distinguish buffalo-derived T. parva, the causal agent of Corridor disease, from cattle-derived T. parva, the causal agent of East Coast fever.
Taenia cestodes were obtained from 5 different definitive host species in Kenya and 175 different samples were examined by classical morphological methods and by isoenzyme analysis using isoelectric focusing in agarose. Gels were stained for 17 different enzymes and 3 of these were used in the construction of isoenzyme profiles. The samples fell into 25 zymodemes, and no zymodeme contained more than 1 species of Taenia, indicating that isoenzyme analysis can reliably be used for the identification of species of this genus.
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