DIFFERENT ORGANISMS ASSOCIATED WITH HEARTWATER AS SHOWN BY ANALYSIS OF 16S RIBOSOMAL-RNA GENE-SEQUENCES

Cowdria ruminantium is a rickettsial parasite which causes heartwater, an economically important disease of domestic and wild ruminants in t ropical and subtropical Africa and parts of the Caribbean. Because exi sting diagnostic methods are unreliable, we investigated the small-sub unit ribosomal RNA (srRNA) gene from heartwater-infected material to c haracterise the organisms present and to develop specific oligonucleot ide probes for polymerase chain reaction (PCR) based diagnosis. DNA wa s obtained from ticks and ruminants from heartwater-free and heartwate r-endemic areas and from Cowdria in tissue culture. PCR was carried ou t using primers designed to amplify only rickettsial srRNA genes, the target region being the highly variable V1 loop. Amplicons were cloned and sequenced; 51% were C. ruminantium sequences corresponding to fou r genotypes, two of which were identical to previously reported C. rum inantium sequences while the other two were new. The four different Co wdria genotypes can be correlated with different phenotypes. Tissue-cu lture samples yielded only Cowdria genotype sequences, but an extraord inary heterogeneity of 16S sequences was obtained from field samples. In addition to Cowdria genotypes we found sequences from previously un known Ehrlichia spp., sequences showing homology to other Rickettsiale s and a variety of Pseudomonadaceae. One Ehrlichia sequence was phylog enetically closely related to Ehrlichia platys (Group II Ehrlichia) an d one to Ehrlichia canis (Group III Ehrlichia). This latter sequence w as from an isolate (Germishuys) made from a naturally infected sheep w hich, from brain smear examination and pathology, appeared to be suffe ring from heartwater; nevertheless no Cowdria genotype sequences were found in this isolate. In addition no Cowdria sequences were obtained from uninfected ticks. Complete 16S rRNA gene sequences were determine d for two C. ruminantium genotypes and for two previously uncharacteri sed heartwater-associated Ehrlichia spp. Sequence differences within t he V1 loop were sufficient for the derivation of four Cowdria genotype -specific oligonucleotide probes. Four further probes were designed: o ne for the detection of any Cowdria genotype, one for the detection of any Group II Ehrlichia sp., one for any Group III Ehrlichia sp. and o ne for all Pseudomonadaceae. All the probes were specific except that for the Cowdria (Ball 3) genotype. The high prevalence (96%) in field samples of pseudomonad-like 16S sequences was the result of environmen tal contamination. The probes were used to screen DNA from gears in an area free of both Amblyomma ticks and clinical heartwater. A substant ial proportion (42%) gave positive reactions for the apparently apatho genic Cowdria in (Omatjenne), indicating that this genotype is relativ ely common. (C) 1997 Elsevier Science B.V.