Phylogenetic Position of Cowdria ruminantium (Rickettsiales) Determined by Analysis of Amplified 16S Ribosomal DNA Sequences

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Abstract
The 165 ribosomal DNA sequence of Cowdriu ruminantium, the causative agent of heartwater disease in ruminants, was determined. An analysis of this sequence showed that C. rumincurtiurn forms a tight phylogenetic cluster with the canine pathogen Ehrlichia canis and the human pathogen Ehrlichia chrtgeenssis. Although a close relationship between the genus Cowdtia and several members of the tribe Ehdichieae has been suspected previously, the tight phylogenetic cluster with E. cunis and E. ch&2enssis is surprising in view of known differences in host preference and target cells. The rickettsia Cowdria ruminantium is the causative agent of heartwater, a tick-borne disease of wild and domestic ruminants. This disease is endemic in sub-Saharan Africa (28). Recently, Cowdria ruminantium has also been detected in the Caribbean region (28). In view of the continuing spread of the African tick Amblyomma variegatum in the Caribbean (29), heartwater may become a serious threat to livestock on the American mainland (2). The genus Cowdria is currently classified in the tribe Ehrlichieae in the order Rickettsiales, together with the genera Ehrlichia and Neorickettsia (33). The members of the genus Cowdria share antigenic determinants with several members of the genus Ehrlichia (5, 13, 17) and also with members of the genus Chlamydia (11); A recent electron microscopic study of the multiplication of Cowdria cells in cultured bovine umbilical endothelial cells showed that the genus Cowdria has developmental stages that resemble those of chlamydia1 species (14). It has been assumed that the developmental cycle consists of an extracellular stage (extracellular bodies) and an intracellular stage (reticulate bodies). A phylogenetic study of the 16s ribosomal DNA (rDNA) of Cowdria ruminantium was initiated to determine the taxonomic relationship of the genus Cowdria with Ehr- Zichia and Chlamydia species more precisely. The Senegal stock (12) of Cowdria ruminantium was cultured in bovine umbilical endothelial cells as described previously (10). Escherichia coli K-12 strain PC2495, an hsdS red derivative of strain JMlOl (36), was used for the propagation of pBS( -) phagemid and derived clones (Strat- agene, La Jolla, Calif.) and was grown overnight at 37°C in Terrific Broth (27) supplemented with 100 pg of ampicillin per ml. Genomic DNA for polymerase chain reaction amplifica- tion was extracted from the extracellular body-containing supernatant of an infected 162-cm2 culture (infection score, 2+) (10). The supernatant was centrifuged at 15,000 x g for 15 min, the extracellular bodies were suspended in 1 ml of TEG buffer (25 mM Tris-HC1 (pH 8.01, 10 mM EDTA, 50 mM glucose) supplemented with 4 mg of lysozyme per ml, and the preparation was incubated for 15 min at room temperature. Sodium dodecyl sulfate and proteinase K were

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