Recombination and purifying and balancing selection determine the evolution of major antigenic protein 1 (map 1) family genes in Ehrlichia ruminantium.

Heartwater is an economically important disease of ruminants caused by the tick-borne bacterium Ehrlichia ruminantium. The disease is present throughout sub-Saharan Africa as well as on several islands in the Caribbean, where it poses a risk of spreading onto the American mainland. The dominant immune response of infected animals is directed against the variable outer membrane proteins of E. ruminantium encoded by a polymorphic multigene family. Here, we examined the full-length sequence of the major antigenic protein 1 (map1) family genes in multiple E. ruminantium isolates from different African countries and the Caribbean, collected at different time points to infer the possible role of recombination breakpoint and natural selection. A high level of recombination was found particularly in map1 and map1-2. Evidence of strong negative purifying selection in map1 and balancing selection to maintain genetic variation across these samples from geographically distinct countries suggests host-pathogen co-evolution. This co-evolution between the host and pathogen results in balancing selection by maintaining genetic diversity that could be explained by the demographic history of long-term pathogen pressure. This signifies the adaptive role and the molecular evolutionary forces underpinning E. ruminantium map1 multigene family antigenicity.

Recombination Is a Major Driving Force of Genetic Diversity in the Anaplasmataceae Ehrlichia ruminantium.

The disease, Heartwater, caused by the Anaplasmataceae E. ruminantium, represents a major problem for tropical livestock and wild ruminants. Up to now, no effective vaccine has been available due to a limited cross protection of vaccinal strains on field strains and a high genetic diversity of Ehrlichia ruminantium within geographical locations. To address this issue, we inferred the genetic diversity and population structure of 194 E. ruminantium isolates circulating worldwide using Multilocus Sequence Typing based on lipA, lipB, secY, sodB, and sucA genes. Phylogenetic trees and networks were generated using BEAST and SplitsTree, respectively, and recombination between the different genetic groups was tested using the PHI test for recombination. Our study reveals the repeated occurrence of recombination between E. ruminantium strains, suggesting that it may occur frequently in the genome and has likely played an important role in the maintenance of genetic diversity and the evolution of E. ruminantium. Despite the unclear phylogeny and phylogeography, E. ruminantium isolates are clustered into two main groups: Group 1 (West Africa) and a Group 2 (worldwide) which is represented by West, East, and Southern Africa, Indian Ocean, and Caribbean strains. Some sequence types are common between West Africa and Caribbean and between Southern Africa and Indian Ocean strains. These common sequence types highlight two main introduction events due to the movement of cattle: from West Africa to Caribbean and from Southern Africa to the Indian Ocean islands. Due to the long branch lengths between Group 1 and Group 2, and the propensity for recombination between these groups, it seems that the West African clusters of Subgroup 2 arrived there more recently than the original divergence of the two groups, possibly with the original waves of domesticated ruminants that spread across the African continent several thousand years ago.

A new typing technique for the Rickettsiales Ehrlichia ruminantium: multiple-locus variable number tandem repeat analysis.

Ehrlichia ruminantium (ER) is a member of the order Rickettsiales transmitted by Amblyomma ticks. This obligatory intracellular bacterium is the causative agent of a fatal disease in ruminants, named heartwater. It represents a constraint on breeding development in sub-Saharan Africa and in the Caribbean. The genetic diversity of the strains of ER, which could be a limiting factor to obtain effective vaccines, needs to be better characterized. For this purpose, we developed a molecular typing technique based on the polymorphism of variable number tandem repeat (VNTR) sequences, MLVA (multiple locus VNTR analysis). Eight (out of 21) VNTR candidates were validated using 17 samples representing a panel of ER strains from different geographical origins from West, South Africa, and Caribbean areas and in ER infected ticks and goat tissues. This result demonstrated the ability of these VNTRs to type a wide range of strains. The stability of the selected VNTR markers was very good, at the time scale needed for epidemiological purposes: in particular, no difference in the VNTR profiles was observed between virulent and attenuated strains (for Gardel and Senegal strains) and between strains (Gardel and Blonde strains) isolated in the same area 19years apart. We validated the strong discriminatory power of MLVA for ER and found a high level of polymorphism between the available strains, with 10 different profiles out of 13 ER strains. The MLVA scheme described in this study is a rapid and efficient molecular typing tool for ER, which allows rapid and direct typing of this intracellular pathogen without preliminary culture and gives reliable results that can be used for further epidemiological studies.

Multi-locus sequence typing of Ehrlichia ruminantium strains from geographically diverse origins and collected in Amblyomma variegatum from Uganda.

BACKGROUND: The rickettsial bacterium Ehrlichia ruminantium is the causative agent of heartwater in ruminants. A better understanding of the population genetics of its different strains is, however, needed for the development of novel diagnostic tools, therapeutics and prevention strategies. Specifically, the development of effective vaccination policies relies on the proper genotyping and characterisation of field isolates. Although multi-locus sequence typing (MLST) has been recently developed, only strains from geographically restricted collections have been analysed so far. The expansion of the MLST database to include global strains with different geographic origins is therefore essential. In this study, we used a panel of reference strains from geographically diverse origins and field samples of E. ruminantium detected from its vector, Amblyomma variegatum, in heartwater-endemic areas in Uganda. RESULTS: A total of 31 novel alleles (six, four, six, three, two, five, three, and two for gltA, groEL, lepA, lipA, lipB, secY, sodB, and sucA loci, respectively) and 19 novel sequence types (STs) were identified. Both neighbour-joining and minimum spanning tree analyses indicated a high degree of genetic heterogeneity among these strains. No association was observed between genotypes and geographic origins, except for four STs from West African countries. When we performed six different tests for recombination (GeneConv, Bootscan, MaxChi, Chimaera, SiScan, and 3Seq) on concatenated sequences, four possible recombination events were identified in six different STs. All the recombination breakpoints were located near gene borders, indicating the occurrence of intergenic recombination. All four STs that localized to a distinct group in clustering analysis showed evidence of identical recombination events, suggesting that recombination may play a significant role in the diversification of E. ruminantium. CONCLUSIONS: The compilation of MLST data set across the African continent will be particularly valuable for the understanding of the existing genetic diversity of field isolates in African countries. Comprehensive information on the degree of cross-protection between strains and further understanding of possible relationships between genotypes and phenotypes such as vaccine efficacy are expected to lead to the development of region-specific vaccination strategies.

An alternative approach to multiple genome comparison.

Genome comparison is now a crucial step for genome annotation and identification of regulatory motifs. Genome comparison aims for instance at finding genomic regions either specific to or in one-to-one correspondence between individuals/strains/species. It serves e.g. to pre-annotate a new genome by automatically transferring annotations from a known one. However, efficiency, flexibility and objectives of current methods do not suit the whole spectrum of applications, genome sizes and organizations. Innovative approaches are still needed. Hence, we propose an alternative way of comparing multiple genomes based on segmentation by similarity. In this framework, rather than being formulated as a complex optimization problem, genome comparison is seen as a segmentation question for which a single optimal solution can be found in almost linear time. We apply our method to analyse three strains of a virulent pathogenic bacteria, Ehrlichia ruminantium, and identify 92 new genes. We also find out that a substantial number of genes thought to be strain specific have potential orthologs in the other strains. Our solution is implemented in an efficient program, qod, equipped with a user-friendly interface, and enables the automatic transfer of annotations between compared genomes or contigs (Video in Supplementary Data). Because it somehow disregards the relative order of genomic blocks, qod can handle unfinished genomes, which due to the difficulty of sequencing completion may become an interesting characteristic for the future. Availabilty: http://www.atgc-montpellier.fr/qod.

Efficiency of inactivated vaccines against heartwater in Burkina Faso: impact of Ehrlichia ruminantium genetic diversity.

In order to identify the appropriate strains to use in vaccination trials against heartwater in Burkina Faso, the protective effect of Gardel and Welgevonden strains was assessed against local strains on sheep vaccinated by infection-and-treatment method: Gardel protected significantly against Burkina Faso strains tested (survival rate 59% for immunised sheep vs 13% for control sheep) while Welgevonden did not (survival rate 45% for immunised sheep vs 25% for control sheep). The efficacy of the ISA50 inactivated vaccine, produced under industrial process, was evaluated in sheep during field challenges in two successive years. During year 1, there was a limited protective effect of the Gardel vaccine with 65% of survival rate for the vaccinated group compared to 49% for the control group (N=153, p=0.053). During year 2, the vaccine containing Gardel and a local strain gave an increased protective effect compared to the first trial: 72% of the vaccinated animals survived compared to 47% of the naïve animals (N=173, p<0.001). There was an important genetic diversity of strains in the field with detection of 11 different map1 genotypes in brains from control and vaccinated sheep post mortem. Map1 genotyping of strains detected in brains from control sheep showed that genotype distribution varied according to time and study areas, which could explain the difference in efficacy of the vaccine.

Extensive genetic recombination occurs in the field between different genotypes of Ehrlichia ruminantium.

The intracellular bacterium Ehrlichia ruminantium is the causative agent of heartwater throughout sub-Saharan Africa, Madagascar, and some islands of the Caribbean. The disease is tick-borne and causes substantial livestock losses, threatening food security and productivity in both the commercial and small-scale farming sectors in endemic areas. Immunization by infection and treatment is currently practised in South Africa, and it is known that a variety of immunotypes of the organism occur in the field, and that cross-protection between them varies widely from total to minimal. Future vaccines may therefore need to incorporate components from different genotypes so it is essential to have information on the extent of genetic variation among isolates. To obtain this information we amplified and sequenced a panel of eight core function genes from 12 different cultured stocks originally isolated in different areas of Africa and the Caribbean. Phylogenetic trees inferred from the sequences yielded different branching orders for different genes, and the reason for this inconsistency appears to be that extensive recombination takes place between different genotypes in the field. It is possible that recombination occurs during the period when the organisms are extracellular within the tick, immediately after feeding and before intracellular infection is established, although detection of more than one genotype in DNA from single ticks is encountered infrequently. The results of the analysis show that the phylogenetic variation is greatest among the isolates of southern African origin, suggesting that this is the region where the parasite first evolved. It also appears likely that the Gardel genotype, isolated in the Caribbean, originally came from west central Africa, not from west Africa as had long been assumed.

Restriction and expansion of Ehrlichia strain diversity.

Ehrlichia are tick-borne gram negative, obligately intracellular bacteria. The 16S rRNA gene DNA sequences are highly conserved among strains of each Ehrlichia species. The 28-kDa/Map-1 outer membrane protein genes are highly diversified among strains of Ehrlichia chaffeensis and E. ruminantium, but are highly conserved among E. canis isolates. The diversity of the immunodominant proteins of E. chaffeensis and E. ruminantium in contrast with the conservation of the immunodominant proteins of E. canis suggests that E. chaffeensis and E. ruminantium face more host immune pressure than E. canis or that E. chaffeensis and E. ruminantium evolved earlier than E. canis and have diverged.

Homologous recombination and the pattern of nucleotide substitution in Ehrlichia ruminantium.

Patterns of nucleotide substitution at orthologous loci were examined between three genomes of Ehrlichia ruminantium, the causative agent of heartwater disease of ruminants. The most recent common ancestor of two genomes (Erwe and Erwo) belonging to the Welgevonden strain was estimated to have occurred 26,500-57,000 years ago, while the most recent common ancestor of these two genomes and the Erga genome (Gardel strain) was estimated to have occurred 2.1-4.7 million years ago. The search for genes showing extremely high values of the number of synonymous substitutions per site was used to identify genes involved in past homologous recombination. The most striking case involved the map1 gene, encoding major antigenic protein-1; evidence for homologous recombination is consistent with previous phylogenetic analysis of map1 alleles. At this and certain other loci, homologous recombination may have contributed to the evolution of host-pathogen interactions. In addition, comparison of the patterns of synonymous and nonsynonymous substitution provided evidence for positive selection favoring a high level of amino acid change between the Welgevonden and Gardel strains at a locus of unknown function (designated Erum4340 in the Erwo genome).

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.

Characterization of the pCS20 region of different Ehrlichia ruminantium isolates.

Heartwater is a serious tick-borne disease of ruminants caused by the rickettsial organism Ehrlichia (Cowdria) ruminantium. A diagnostic test, targeting the pCS20 genomic region and using PCR amplification and probe hybridization, detects E. ruminantium infection in ticks and animals. However, only the pCS20 sequence of the Crystal Springs E. ruminantium isolate is available and the existence of sequence variation amongst different E. ruminantium isolates has not been determined. Primers were designed from the published pCS20 sequence to obtain sequences of the pCS20 region of various E. ruminantium isolates. These primers were unable to amplify the pCS20 region from genomic Welgevonden DNA and genome walking was used to characterize the pCS20 region. This technique showed that the published pCS20 sequence is from a chimeric clone. Sequences of the pCS20 region of 14 different E. ruminantium isolates were determined after amplification with newly designed primers. Sequencing data indicated that West African E. ruminantium isolates are highly conserved, whereas more variation occurs amongst the southern African isolates. These results facilitated the design of a short pCS20 probe and a large PCR target that improved the sensitivity of the E. ruminantium detection assay.

Phylogenetic relationships among Ehrlichia ruminantium isolates.

Ehrlichia ruminantium, the causative agent of heartwater, is a tick-borne pathogen infecting ruminants throughout sub-Saharan Africa and on some Caribbean islands. The most reliable test for E. ruminantium is PCR-based, but this gives positive results in some areas free of clinical heartwater and of the known Amblyomma spp. tick vectors. To investigate the molecular basis for this finding we have sequenced and carried out phylogenetic analysis of a range of genes from a number of E. ruminantium isolates. The genes include ribonuclease III and cytochrome c oxidase assembly protein genes (the pCS20 region), groESL, citrate synthase (gltA), and 16S ribosomal RNA. Relationships among major antigenic protein (map1) genes have been exhaustively investigated in a previous study that showed that the genes are variable in length, have non-synonymous mutations, and show no geographical specificity among isolates. The 16S sequences are highly conserved, except in the V1 loop region. The pCS20, groESL, and gltA genes show only single nucleotide polymorphisms (SNPs) dispersed throughout the sequenced regions. Phylogenetic analysis using pCS20 data differentiates the western African isolates into a single clade, which also includes a southern African isolate. All other southern African isolates and a Caribbean isolate fall into a further clade, which is subdivided into two groups. Sequence variation within this clade is greater than that within the western African clade, suggesting that E. ruminantium originated in southern Africa.

The Kümm isolate of Ehrlichia ruminantium: in vitro isolation, propagation and characterization.

An effective culture system for Ehrlichia (Cowdria) ruminantium comb. nov. was first established in 1985 and many stocks were subsequently isolated and propagated in vitro. A notable exception, however, was the Kümm isolate that resisted all attempts at in vitro culture until the successful experiment described here. In one experiment white blood cells were harvested from heparinized blood derived from a sheep infected with the Kümm isolate. The cells were added to DH 82 cells and incubated at 37 degrees C. The high metabolic activity of the DH 82 cells necessitated that cell growth be retarded by the addition of cycloheximide. Colonies were first detected 19 days after culture initiation and, once the cultures were established, they could be passaged every 3 days. Bovine and sheep endothelial cells were readily infected with culture supernatant obtained from the infected DH 82 cells. In a further experiment another sheep was infected, using a higher dose of the same batch of Kümm stabilate, and we attempted to infect several different cell lines: these were DH 82 cells, bovine aorta (BA 886) cells, sheep brain endothelial (SBE 189) cells and sheep fibroblastoid cells (E2). Ten days after culture initiation only the E2 cells had become positive for E. ruminantium. Culture supernatant from the first cultured isolate (Kümm-1) was less virulent for mice than that of the second cultured isolate (Kümm-2) which killed all mice. Upon molecular characterization with E. ruminantium 16S probes we found that Kümm-1 hybridized with a Senegal 16S genotype probe, whereas Kümm-2 hybridized only with an Omatjenne 16S genotype probe. The original stabilate used to infect the sheep hybridized with both probes. These results clearly indicate that two different stocks had been isolated in culture.

Host-symbiont conflicts: positive selection on an outer membrane protein of parasitic but not mutualistic Rickettsiaceae.

The Rickettsiaceae is a family of intracellular bacterial symbionts that includes both vertically transmitted parasites that spread by manipulating the reproduction of their host (Wolbachia in arthropods) and horizontally transmitted parasites (represented by Cowdria ruminantium), and mutualists (Wolbachia pipientis in nematode worms). We have investigated the nature of natural selection acting on an outer membrane protein, the wsp gene in Wolbachia and its homologue map1 in Cowdria, thought likely to be involved in host-parasite interactions in these bacteria. The ratio of nonsynonymous to synonymous substitution rates (d(N)/d(S)) at individual amino acid sites or at lineages within the gene's phylogeny was estimated using maximum likelihood models of codon substitution. The first hypothesis we tested was that this protein is under positive selection in the parasitic but not in the mutualistic Rickettsiaceae. This hypothesis was supported as positive selection and was detected in Cowdria and arthropod Wolbachia sequence evolution but not in the evolution of Wolbachia sequences from nematodes. Furthermore, this selection was concentrated outside the transmembrane region of the protein and, therefore, in the regions of the protein that may interact with the host. The second hypothesis tested was that positive selection would be stronger in the strains of arthropod Wolbachia that distort the host sex ratio than in those that induce cytoplasmic incompatibility. However, we found no support for this hypothesis. In conclusion, our results are consistent with the hypothesis that antagonistic coevolution causes faster evolution of surface protein sequences in parasites than in mutualists. Confirmation of this conclusion awaits the replication of these results both in additional genes and across more bacterial taxa. The regions of the wsp and map1 genes we identified as likely to be involved in host-parasite arms races should be examined in future studies of parasite virulence and host immune responses, and during the design of vaccines.

Phylogenetic relationships of Anaplasma marginale and 'Ehrlichia platys' to other Ehrlichia species determined by GroEL amino acid sequences.

The heat-shock protein (GroEL) genes of Anaplasma marginale, Ehrlichia muris and 'Ehrlichia platys' were sequenced and compared with the GroEL of other species of Ehrlichia. The GroEL amino acid sequences of A. marginale and 'E. platys' were most similar to the GroEL sequence of Ehrlichia phagocytophila, with which they formed one group with 6-10% divergence. The E. muris GroEL was most closely related to the GroEL of two unclassified strains (HF-565 and Anan), then to Ehrlichia chaffeensis, Ehrlichia canis, Ehrlichia ewingii and Cowdria ruminantium, forming a second distinct group (0.3-8.6% divergence). The GroELs of Ehrlichia risticii and Ehrlichia sennetsu were very similar to one another (only 2% divergence), forming the third group. The first two groups were relatively closely related (17-20% divergence), while the third group was only distantly related to the first two groups (62-73% divergence).

Reorganization of genera in the families Rickettsiaceae and Anaplasmataceae in the order Rickettsiales: unification of some species of Ehrlichia with Anaplasma, Cowdria with Ehrlichia and Ehrlichia with Neorickettsia, descriptions of six new species combinations and designation of Ehrlichia equi and 'HGE agent' as subjective synonyms of Ehrlichia phagocytophila.

The genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolbachia encompass a group of obligate intracellular bacteria that reside in vacuoles of eukaryotic cells and were previously placed in taxa based upon morphological, ecological, epidemiological and clinical characteristics. Recent genetic analyses of 16S rRNA genes, groESL and surface protein genes have indicated that the existing taxa designations are flawed. All 16S rRNA gene and groESL sequences deposited in GenBank prior to 2000 and selected sequences deposited thereafter were aligned and phylogenetic trees and bootstrap values were calculated using the neighbour-joining method and compared with trees generated with maximum-probability, maximum-likelihood, majority-rule consensus and parsimony methods. Supported by bootstrap probabilities of at least 54%, 16S rRNA gene comparisons consistently clustered to yield four distinct clades characterized roughly as Anaplasma (including the Ehrlichia phagocytophila group, Ehrlichia platys and Ehrlichia bovis) with a minimum of 96.1% similarity, Ehrlichia (including Cowdria ruminantium) with a minimum of 97.7% similarity, Wolbachia with a minimum of 95.6% similarity and Neorickettsia (including Ehrlichia sennetsu and Ehrlichia risticii) with a minimum of 94.9% similarity. Maximum similarity between clades ranged from 87.1 to 94.9%. Insufficient differences existed among E. phagocytophila, Ehrlichia equi and the human granulocytic ehrlichiosis (HGE) agent to support separate species designations, and this group was at least 98.2% similar to any Anaplasma species. These 16S rRNA gene analyses are strongly supported by similar groESL clades, as well as biological and antigenic characteristics. It is proposed that all members of the tribes Ehrlichieae and Wolbachieae be transferred to the family Anaplasmataceae and that the tribe structure of the family Rickettsiaceae be eliminated. The genus Anaplasma should be emended to include Anaplasma (Ehrlichia) phagocytophila comb. nov. (which also encompasses the former E. equi and the HGE agent), Anaplasma (Ehrlichia) bovis comb. nov. and Anaplasma (Ehrlichia) platys comb. nov., the genus Ehrlichia should be emended to include Ehrlichia (Cowdria) ruminantium comb. nov. and the genus Neorickettsia should be emended to include Neorickettsia (Ehrlichia) risticii comb. nov. and Neorickettsia (Ehrlichia) sennetsu comb. nov.

Macrorestriction fragment profiles reveal genetic variation of Cowdria ruminantium isolates.

Macrorestriction profile analysis by pulsed-field gel electrophoresis (PFGE) was used to distinguish between seven isolates of Cowdria ruminantium from geographically different areas. Characteristic profiles were generated for each isolate by using the restriction endonucleases KspI, SalI, and SmaI with chromosomal sizes ranging between 1,546 and 1,692 kb. Statistical analysis of the macrorestriction profiles indicated that all the isolates were distinct from each other; these data contribute to a better understanding of the epidemiology of this pathogen and may be exploited for the identification of genotype-specific DNA probes.

Evaluation of 16S, map1 and pCS20 probes for detection of Cowdria and Ehrlichia species.

A panel of 16S ribosomal RNA gene probes has been developed for the study of the epidemiology of heartwater; five of these detect different cowdria genotypes, one detects five distinct genotypes; one detects any Group III Ehrlichia species other than Cowdria and one detects any Group II Ehrlichia species. These probes have been used on PCR-amplified rickettsial 16S rRNA genes from over 200 Amblyomma hebraeum ticks. Control ticks were laboratory-reared and either uninfected or fed on sheep experimentally infected with different cowdria isolates, field ticks were collected from animals in heartwater-endemic areas. All tick-derived DNA samples were also examined by PCR amplification and probing for two other cowdria genes (map1 and pCS20) which have previously been used for heartwater epidemiology. This paper describes the first direct comparison of all currently available DNA probes for heartwater-associated organisms.