The genome of the heartwater agent Ehrlichia ruminantium contains multiple tandem repeats of actively variable copy number

Abstract

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.

A quantitative real-time PCR assay for Ehrlichia ruminantium using pCS20

“…Because each plasmid contains one copy of pCS20 one can assume that each copy of plasmid correlates to one copy of pCS20. Furthermore, there is one copy of pCS20 per genome equivalent of E. ruminantium (Collins et al, 2005). Therefore, it can be assumed that each copy of pCS20, measured by the real-time PCR assay, is equivalent to one genome copy of E. ruminantium.…”

Immunisation of sheep against heartwater in The Gambia using inactivated and attenuated Ehrlichia ruminantium vaccines

“…In the present study, real-time PCR [24] was applied to estimate the number of organisms per dose of live attenuated inoculum used to induce immunity in sheep. The technique quantifies the map1-1 gene, which is a single copy gene [42] and therefore allows a direct estimation of the number of E. ruminantium organisms per sample. In the on-station laboratory trial, the quantification showed that sheep were vaccinated with approximately 153,000 attenuated organisms, which caused transient temperature reaction in some animals indicating a need for further reduction in number of attenuated organisms in the immunising inoculum.…”

Heartwater : an abridged historical account : historical article

“…In 2004 the genome project was successfully completed and the fully annotated sequence published, the first bacterial genome to be sequenced in Africa 3 . The genetic information thus obtained allowed a more rational selection of genes with potential for vaccine production.…”