Heartwater, a tick-borne disease of domestic and wild ruminants, is caused by the intracellular rickettsia Ehrlichia ruminantium (previously known as Cowdria ruminantium). It is a major constraint to livestock production throughout subSaharan Africa, and it threatens to invade the Americas, yet there is no immediate prospect of an effective vaccine. A shotgun genome sequencing project was undertaken in the expectation that access to the complete protein coding repertoire of the organism will facilitate the search for vaccine candidate genes. We report here the complete 1,516,355-bp sequence of the type strain, the stock derived from the South African Welgevonden isolate. Only 62% of the genome is predicted to be coding sequence, encoding 888 proteins and 41 stable RNA species. The most striking feature is the large number of tandemly repeated and duplicated sequences, some of continuously variable copy number, which contributes to the low proportion of coding sequence. These repeats have mediated numerous translocation and inversion events that have resulted in the duplication and truncation of some genes and have also given rise to new genes. There are 32 predicted pseudogenes, most of which are truncated fragments of genes associated with repeats. Rather then being the result of the reductive evolution seen in other intracellular bacteria, these pseudogenes appear to be the product of ongoing sequence duplication events.gene duplication ͉ bacterial genome ͉ molecular sequence data ͉ intracellular adaptation E hrlichia ruminantium (previously known as Cowdria ruminantium) is an obligate intracellular bacterium in the order Rickettsiales. Species in this order cause serious diseases in man and domestic animals throughout the world. E. ruminantium is transmitted by ticks of the genus Amblyomma and causes heartwater, a fatal and economically important disease of wild and domestic ruminants. The disease occurs throughout subSaharan Africa and on several Caribbean islands, from which it threatens to invade the Americas (1), but the existing immunization procedures are rudimentary and relatively ineffective (2). E. ruminantium is a fragile bacterium with exacting culture requirements in eukaryotic cell lines; genetic manipulation has not been attempted, and little is known about its mechanisms of virulence or pathogenesis. Heartwater affects all domestic ruminants, and 80-95% of naïve animals die within 3 weeks, but those that recover have a T cell-mediated immunity to subsequent homologous challenge (3). In the absence of any directed strategy to identify T cell-stimulatory proteins we sequenced the E. ruminantium genome in the expectation that access to the complete protein-coding repertoire of the organism would facilitate the search for vaccine candidate genes.

Cowdria ruminantium, the causative agent of heartwater disease, expresses an immunodominant and conserved 32-kilodalton protein (MAPl; formerly called Cr32), which is currently in use for serodiagnosis of the disease. The gene encoding this protein, designated mapi, was detected, cloned, and characterized. The gene is conserved between four different stocks of C. ruminantium originating from Senegal, Sudan, South Africa, and Zimbabwe. Homology searches revealed MAP1 to be homologous to the Anaplasma marginale surface protein MSP4, a potential protective antigen. The MAP1 protein, expressed in Escherichia coli fused with glutathione S-transferase, is specifically recognized by sera from animals infected with seven different stocks of C. ruminantium. Heartwater or cowdriosis is a rickettsial disease of wild and domestic ruminants. It is caused by Cowdria ruminantium and transmitted by ticks of the genus Amblyomma. The disease is endemic to sub-Saharan Africa (43) and has also been detected in the Caribbean region (36). The continuing spread of the African tick Amblyomma variegatum in the Caribbean and the presence on the American continent of yet uninfected Amblyomma species capable of spreading the disease pose a serious threat to livestock industries on the American mainland (3). C. ruminantium has developmental stages resembling those of Chlamydia species (25). The deduced developmental cycle consists of an extracellular stage capable of infecting host cells (elementary body) and an intracellular stage capable of multiplication within the host cell (reticulate body). However, phylogenetic studies based on 16S rDNA sequence comparison revealed no significant phylogenetic relationship between C. ruminantium and Chlamydia spp. (9, 45) but a very close relationship among C. ruminantium, several Ehrlichia species (45), and Anaplasma marginale (9, 45). Cloning and expression of major protein antigens of C. ruminantium would make it possible to test whether these antigens are protective, as has been described for Anaplasma marginale (1, 33, 34, 41) and Ehrlichia risticii (14, 39), or could facilitate the use of recombinant antigen in serological tests. Studies on immunodominant protein antigens of C. ruminantium identified a 32-kDa protein (20) and a 27-kDa protein (37). The 32-kDa protein, which was designated Cr32, is conserved in all isolates tested (17, 20, 22). It has also been reported to have a molecular weight of 31,000 (37). Actually, the Cr32 protein varies in molecular weight depending on the