Mining the genetic diversity of Ehrlichia ruminantium using map genes family.

Understanding bacterial genetic diversity is crucial to comprehend pathogenesis. Ehrlichia ruminantium (E. ruminantium), a tick-transmitted intracellular bacterial pathogen, causes heartwater disease in ruminants. This model rickettsia, whose genome has been recently sequenced, is restricted to neutrophils and reticulo-endothelial cells of its mammalian host and to the midgut and salivary glands of its vector tick. E. ruminantium harbors a multigene family encoding for 16 outer membrane proteins including MAP1, a major antigenic protein. All the 16 map paralogs are expressed in bovine endothelial cells and some are specifically translated in the tick or in the mammalian host. In this study, we carried out phylogenetic analyses of E. ruminantium using sequences of 6 MAP proteins, MAP1, MAP1-2, MAP1-6, MAP1-5, MAP1+1 and MAP1-14, localized either in the center or at the borders of the map genes cluster. We show that (i) map1 gene is a good tool to characterize the genetic diversity among Africa, Caribbean islands and Madagascar strains including new emerging isolates of E. ruminantium; (ii) the different map paralogs define different genotypes showing divergent evolution; (iii) there is no correlation between all MAP genotypes and the geographic origins of the strains; (iv) The genetic diversity revealed by MAP proteins is conserved whatever is the scale of strains sampling (village, region, continent) and thus was not related to the different timing of strains introduction, i.e. continuous introduction of strains versus punctual introduction (Africa versus Caribbean islands). These results provide therefore a significant advance towards the management of E. ruminantium diversity. The differential evolution of these paralogs suggests specific roles of these proteins in host-vector-pathogen interactions that could be crucial for developing broad-spectrum vaccines.

Differential strain-specific diagnosis of the heartwater agent: Ehrlichia ruminantium.

Ehrlichia ruminantium is the causative agent of heartwater, a major tick-borne disease of livestock in Africa introduced in the Caribbean and threatening to emerge and spread in the American mainland. Complete genome sequencing was done for two isolates of E. ruminantium of differing phenotype, isolates Gardel (Erga) from Guadeloupe Island and Welgevonden (Erwe) originating from South Africa and maintained in Guadeloupe. The type strain of E. ruminantium (Erwo), previously isolated and sequenced in South Africa; is identical to Erwe with respect to target genes. They make the Erwe/Erwo complex. Comparative analysis of the genomes shows the presence of 49 unique CDS and 28 truncated CDS differentiating Erga from Erwe/Erwo. Three regions of accumulated differences (RAD) acting as mutational hot spots were identified in E. ruminantium. Ten CDS, six unique CDS and four truncated CDS corresponding to major genomic changes (deletions or extensive mutations) were considered as targets for differential diagnosis on four isolates of E. ruminantium: Erga, Erwe/Erwo, Senegal and Umpala. Pairs of PCR primers were developed for each target gene. PCR analysis of the target genes generated strain-specific patterns on Erga and Erwe/Erwo as predicted by comparative genomics, but also for isolates Senegal and Umpala. The target genes identified by bacterial comparative genomics are shown to be highly efficient for strain-specific PCR diagnosis of E. ruminantium and further vaccine management tools.

Optimisation of the inactivated vaccine dose against heartwater and in vitro quantification of Ehrlichia ruminantium challenge material.

The aim of this work was to evaluate the minimal protective dose of the inactivated vaccine against heartwater. In order to conduct a reliable vaccination trial, an in vitro quantification method of Ehrlichia ruminantium (ER) challenge doses was developed. In experimental conditions, homologous Gardel challenges with 2.1 x 10(4) to 9.4 x 10(4) live ER elementary bodies were reproducible and mimicked a natural challenge. Similar results were obtained when animals were challenged with 3 x 10(4) live elementary bodies from five different ER strains. A 28-fold reduction of the vaccine dose did not decrease protection when compared to the conventional dose of inactivated vaccine. Two injections of 35 microg of ER antigen induced good protection against heartwater. Moreover, we found that the amount of IFNgamma secreted in blood from vaccinated animals after in vitro stimulation with ER antigen was not a reliable predictor of survival and could not be used to test the potency of the inactivated vaccine.

Comparative genomic analysis of three strains of Ehrlichia ruminantium reveals an active process of genome size plasticity.

Ehrlichia ruminantium is the causative agent of heartwater, a major tick-borne disease of livestock in Africa that has been introduced in the Caribbean and is threatening to emerge and spread on the American mainland. We sequenced the complete genomes of two strains of E. ruminantium of differing phenotypes, strains Gardel (Erga; 1,499,920 bp), from the island of Guadeloupe, and Welgevonden (Erwe; 1,512,977 bp), originating in South Africa and maintained in Guadeloupe in a different cell environment. Comparative genomic analysis of these two strains was performed with the recently published parent strain of Erwe (Erwo) and other Rickettsiales (Anaplasma, Wolbachia, and Rickettsia spp.). Gene order is highly conserved between the E. ruminantium strains and with A. marginale. In contrast, there is very little conservation of gene order with members of the Rickettsiaceae. However, gene order may be locally conserved, as illustrated by the tuf operons. Eighteen truncated protein-encoding sequences (CDSs) differentiate Erga from Erwe/Erwo, whereas four other truncated CDSs differentiate Erwe from Erwo. Moreover, E. ruminantium displays the lowest coding ratio observed among bacteria due to unusually long intergenic regions. This is related to an active process of genome expansion/contraction targeted at tandem repeats in noncoding regions and based on the addition or removal of ca. 150-bp tandem units. This process seems to be specific to E. ruminantium and is not observed in the other Rickettsiales.

Nested PCR for detection and genotyping of Ehrlichia ruminantium: use in genetic diversity analysis.

Ehrlichia ruminantium, the agent of cowdriosis transmitted by Amblyomma ticks, presents an extensive genetic and antigenic diversity of key importance for vaccine formulation. Two means of nested polymerase chain reaction (PCR) targeting were developed to conduct molecular epidemiology studies in the Caribbean and Africa. The first used a conserved DNA fragment for detection of the pathogen in animals and vectors, and the second relied on the polymorphic map1 gene for genotyping. As compared to a PCR, the nested PCR showed a 2-Log10 improvement of sensitivity and allowed amplification from ticks, blood, brain, and lungs from infected animals, providing a more accurate picture of the tick infection rate. In Guadeloupe, this rate reached 36% (N = 212) instead of 1.7% (N = 224), as previously estimated. Genetic typing was done by restriction fragment length polymorphism or sequencing of map1 amplification products. Molecular epidemiology studies conducted in field sites selected for vaccination trials with inactivated vaccine, revealed the circulation of genetically divergent strains in limited geographical areas. It is known, then, that genetic clustering based on map1 has no predictive value regarding the protective value of a given strain against a new strain. However, tracing the strains by this technique revealed the extent of E. ruminantium diversity that one can expect in a given region, and the method allows differentiation between an inadequate immune response and the challenge by a breakthrough strain on animals dying despite vaccination. Up to now, genetic typing does not avoid cross-protection studies, which were conducted in parallel, although on a more limited scale. The importance of pathogen diversity studies for optimization of vaccine design is discussed as well as the research for new polymorphic genes. These genes may allow better predictions on cross-protection, given the recent completion of the sequence of the full genome of two E. ruminantium strains.