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Nathalie Vachiery - One of the best experts on this subject based on the ideXlab platform.
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Characterization of Ehrlichia ruminantium membrane protein, erga-cds- 01230 and its role in adhesion to the host cell
2019Co-Authors: Valérie Pinarello, Nathalie Vachiery, Emmanuel Albina, Damien F. MeyerAbstract:Outer membrane proteins participate to pathogens adhesion to host cells and therefore often mediate cell infection. Such is the case for Ehrlichia ruminantium, an obligate intracellular bacterium that is transmitted by ticks and responsible for cowdriosis, a fatal disease of domestic and wild ruminants. Several experimental vaccines were developed, but the great genetic and presumably antigenic diversity of E. ruminantium make difficult to obtain an effective vaccine against all strains present in the field. In order to propose novel strategies to control cowdriosis, the interaction of E. ruminantium with its host cell, particularly the associated adhesion mechanisms must be first deciphered. A membrane protein of E. ruminantium, ERGA_CDS_01230, a probable iron transporter, initially identified by proteomics approaches in our group, was here studied for its role in host cell adhesion. The recombinant protein was expressed with post-translational glycosylation modifications and tagged GFP/Histidine in Leishmania tarentolae. Using cell biology approaches, we show that recProt01230 is able to adhere to bovine host cells and interacts with proteins from the cell lysate and the "membranes/ organelles” sub-fraction. Furthermore, recProt01230 does not adhere to heparan sulfate but other membrane polysaccharides seem to play a role in E. ruminantium's adhesion to the host cell. Indeed, preliminary experiments have shown that degrading dermatan sulfate and chondroitin sulfate at the cell surface is associated with a reduction of the number of bacteria in the host cells. Moreover, CDS ERGA_CDS_01230 is over expressed at early stages of infection when bacteria begin to attach to their host. So, our results show the implication of ERGA_CDS_01230 in the adhesion of E. ruminantium to host cells. ERGA_CDS_01230 also induces a humoral response in the vaccinated animals. In conclusion, ERGA_CDS_01230 could be a new promising target for vaccine or therapeutics development.
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Genetic diversity of Ehrlichia ruminantium in Mozambique
2018Co-Authors: Laure Bournez, Thierry Lefrançois, Rosalie Aprelon, Valérie Pinarello, Nidia Cangi, Jonathan Gordon, Luis Neves, Nathalie VachieryAbstract:The tick species, Amblyomma hebraeum and A. variegatum, are the main vectors of Heartwater, a tropical infectious bacterial disease of ruminants caused by Ehrlichia ruminantium. In Mozambique, these tick species have a parapatric distribution, with A. variegatum present in the central and northern regions and A. hebraeum in the South. A narrow overlap area between the distributions of the two species occurs around parallel 22o south. In order to determine the prevalence of E. ruminantium in A. hebraeum and A. variegatum and to determine the genetic diversity and structure of isolates from different localities, adult ticks feeding on cattle and wild ruminants were sampled across the south and center of Mozambique as well as in the adjacent Kruger National Park (KNP), South Africa. The prevalence of E. rumimantium in Amblyomma ticks in relation to the tick species and gender, locality and tick abundance was analyzed. Afterwards, Mozambican Ehrlichia isolates were typed using Multi Locus Sequence Typing and the distribution of groups clustering genotypes were analysed. In total, 722 and 388 of A. hebraeum and A. variegatum ticks were collected from 31 localities and screened for E. ruminantium, using pCS20 nested PCR and Sol1TqM qPCR. The prevalence of E. ruminantium in ticks feeding on cattle varied from 0% to 26.7%, with no infected ticks determined in 7 localities. In ticks feeding on wild ruminants, the prevalence was 8.2 % in the KNP and 6.2% in hunting concessions of the Sofala province. After accounting for the effects of tick gender and sampling sites, no significant difference in prevalence was found between tick species. Most MLST genotypes from Mozambique clustered into subgroup 2C and 2E, which were present in similar proportions in 5 of the 19 localities. Interestingly, MLST genotypes from group G1 and G2D were exclusively found in areas of A. variegatum distribution, while subgroup G2C was only detected in A. hebraeum areas. Moreover, genotypes from subgroup G2E were found in both A. hebraeum and A. variegatum areas. These results contribute to a better understanding of spatial distribution of E. ruminantium and will aid in improvement of heartwater monitoring and control strategies in Mozambique.
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Iron Starvation Conditions Upregulate Ehrlichia ruminantium Type IV Secretion System, tr1 Transcription Factor and map1 Genes Family through the Master Regulatory Protein ErxR
Frontiers in Cellular and Infection Microbiology, 2018Co-Authors: Amal Moumène, Nathalie Vachiery, Thierry Lefrançois, Silvina Gonzalez-rizzo, Damien MeyerAbstract:Ehrlichia ruminantium is an obligatory intracellular bacterium that causes heartwater, a fatal disease in ruminants. Due to its intracellular nature, E. ruminantium requires a set of specific virulence factors, such as the type IV secretion system (T4SS), and outer membrane proteins (Map proteins) in order to avoid and subvert the host's immune response. Several studies have been conducted to understand the regulation of the T4SS or outer membrane proteins, in Ehrlichia, but no integrated approach has been used to understand the regulation of Ehrlichia pathogenicity determinants in response to environmental cues. Iron is known to be a key nutrient for bacterial growth both in the environment and within hosts. In this study, we experimentally demonstrated the regulation of virB, map1, and tr1 genes by the newly identified master regulator ErxR (for Ehrlichia ruminantium expression regulator). We also analyzed the effect of iron depletion on the expression of erxR gene, tr1 transcription factor, T4SS and map1 genes clusters in E. ruminantium. We show that exposure of E. ruminantium to iron starvation induces erxR and subsequently tr1, virB, and map1 genes. Our results reveal tight co-regulation of T4SS and map1 genes via the ErxR regulatory protein at the transcriptional level, and, for the first time link map genes to the virulence function sensu stricto, thereby advancing our understanding of Ehrlichia's infection process. These results suggest that Ehrlichia is able to sense changes in iron concentrations in the environment and to regulate the expression of virulence factors accordingly.
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Efficient high-throughput molecular method to detect Ehrlichia ruminantium in ticks
Parasites & vectors, 2017Co-Authors: Nidia Cangi, Thierry Lefrançois, Valérie Pinarello, Laure Bournez, Luis Neves, Emmanuel Albina, Nathalie VachieryAbstract:Ehrlichia ruminantium is the causal agent of heartwater, a fatal tropical disease affecting ruminants with important economic impacts. This bacterium is transmitted by Amblyomma ticks and is present in sub-Saharan Africa, islands in the Indian Ocean and the Caribbean, where it represents a threat to the American mainland. An automated DNA extraction method was adapted for Amblyomma ticks and a new qPCR targeting the pCS20 region was developed to improve E. ruminantium screening capacity and diagnosis. The first step in the preparation of tick samples, before extraction, was not automated but was considerably improved by using a Tissue Lyser. The new pCS20 Sol1 qPCR and a previously published pCS20 Cow qPCR were evaluated with the OIE standard pCS20 nested PCR. pCS20 Sol1 qPCR was found to be more specific than the nested PCR, with a 5-fold increase in sensitivity (3 copies/reaction vs 15 copies/reaction), was less prone to contamination and less time-consuming. As pCS20 Sol1 qPCR did not detect Rickettsia, Anasplasma and Babesia species or closely related species such as Panola Mountain Ehrlichia, E. chaffeensis and E. canis, its specificity was also better than Cow qPCR. In parallel, a tick 16S qPCR was developed for the quality control of DNA extraction that confirmed the good reproducibility of the automated extraction. The whole method, including the automated DNA extraction and pCS20 Sol1 qPCR, was shown to be sensitive, specific and highly reproducible with the same limit of detection as the combined manual DNA extraction and nested PCR, i.e. 6 copies/reaction. Finally, 96 samples can be tested in one day compared to the four days required for manual DNA extraction and nested PCR. The adaptation of an automated DNA extraction using a DNA/RNA viral extraction kit for tick samples and the development of a new qPCR increased the accuracy of E. ruminantium epidemiological studies, as well as the diagnostic capabilities and turn-over time for surveillance of heartwater. This new method paves the way for large-scale screening of other bacteria and viruses in ticks as well as genetic characterization of ticks and tick-pathogen coevolution studies.
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A pilot study for the identification of biomarkers of Ehrlichia ruminantium infection by natural host
2017Co-Authors: Bernard Fernandez, Nathalie Vachiery, Guylaine Miotello, Rosalie Aprelon, Ken Giraud-girard, Jean Armengaud, Philippe HolzmullerAbstract:Heartwater, caused by the Rickettsiales Ehrlichia ruminantium, is one of the most important tick-borne diseases of livestock in Africa, but also in the Caribbean from where it threatens the American mainland. All domestic and wild ruminants can be infected by E. ruminantium, a small pleomorphic gram-negative obligate intracellular bacterium invading the cytoplasm of vascular endothelial cells. Clinical diagnosis of heartwater is based on the presence of Amblyomma sp. ticks, nervous signs, and presence of transudates in the pericardium and thorax on post-mortem examination, and efficient molecular diagnostics allow reliable heartwater confirmation but not early detection. The objective of the present study was to evaluate modifications in serum composition in animals infected by E. ruminantium and define new diagnostic biomarkers. For this, sera from goats experimentally infected by intravenous injection of E. ruminantium cultured in bovine aortic endothelial cells in vitro were sampled. Sera at Day 0 and Day 14 post-infection (occurrence of clinical signs) were pre-fractionated by SDS-PAGE and analysed by shotgun next-generation proteomics by means of high-throughput analysis of peptides with a Q-Exactive HF mass spectrometer incorporating an ultra-high-field Orbitrap analyzer. Preliminary data obtained against the Capra hircus and Ehrlichia ruminantium theoretical molecular databases evidenced 6 and 2 proteins of interest for which abundances were increased or decreased, respectively, at Day 14. These proteins could represent a molecular signature of E. ruminantium infection. Here, we demonstrated the relevance of such proteomic approach to propose candidate diagnostic biomarkers of heartwater. Further investigations will be performed for monitoring more precisely the whole kinetics of E. ruminantium infection for delineating biomarkers for early diagnosis. (Texte integral)
Dominique Martinez - One of the best experts on this subject based on the ideXlab platform.
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Genetic diversity of Amblyomma variegatum (Acari:Ixodidae), the main vector of Ehrlichia ruminantium in Indian Ocean Islands
2012Co-Authors: Stéphanie Jacquet, Nathalie Vachiery, Dominique Martinez, Frédéric Stachurski, Marlène Dupraz, Eric Cardinale, M. Asnaoui, Sébastien Girard, Yannick Grimaud, Karine HuberAbstract:The hard ticks Amblyomma variegatum is the main vector of Ehrlichia ruminantium which is the pathogen responsible for heartwater or cowdriosis, a disease of ruminants. This tick originates from sub-Saharan Africa and is now widely widespread. A. variegatum has been described in 1899 for the first time in Madagascar, but its introduction is probably older and very likely concomitant with livestock introduction from Africa. A. variegatum has also been described in the Comoros, Mayotte, La Reunion and Mauritius islands. The aim of this study was to investigate the genetic and demographic phenomena that have shaped the present distribution and structure of A. variegatum populations in the Indian Ocean area. A first phylogeographic approach has been carried out, by analysing two mitochondrial-DNA genes at an intra-specific level through the analysis of tick samples from Madagascar and from the other Indian Ocean Islands included in this study. These samples have been compared to samples from Africa, where this species is originated from, and samples from the French West Indies where A. variegatum was introduced around the 18th century. This study will help to elucidate A. variegatum introduction history in the different Indian Ocean Islands. A population genetics approach, using microsatellite markers, focused on Madagascar and some other islands (La Reunion, Comoros and Mayotte islands), has given an insight into the present population structure. This study has led to two main lineages identification: one covering all the species distribution and one restricted to East Africa and Indian Ocean area. These two lineages are in sympatry in Madagascar. The results seemed to be in keeping with the historical data concerning the introduction of the tick in the Indian Ocean area. In Madagascar, a high genetic diversity has been described whereas a lower genetic diversity is observed in the other islands. In Madagascar tick populations are clearly structured but in a heterogeneous way. This structure is probably shaped by the complex interaction of geographic, climatic and anthropic factors. (Texte integral)
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Global gene expression profiling of Ehrlichia ruminantium at different stages of development
FEMS immunology and medical microbiology, 2011Co-Authors: Ludovic Pruneau, Thierry Lefrançois, Christian Sheikboudou, Dominique Martinez, Isabel Marcelino, Bernard Mari, Loïc Emboule, Valérie Pinarello, Pryscellia Gely, Damien F. MeyerAbstract:Ehrlichia ruminantium (ER), the causative agent of heartwater on ruminants, is an obligate intracellular bacterium transmitted by ticks of the genus Amblyomma. Previous studies have shown that early stages of development may be critical for Ehrlichia pathogenicity. To gain insights into the biology of intracellular ER, we determined the genome-wide transcriptional profile of ER replicating inside bovine aortic endothelial cells using DNA microarrays. At intermediate and late stages of infection (reticulate and elementary bodies, respectively), a total of 54 genes were differentially expressed. Among them, we measured by q-RTPCR the overexpression of 11 of 14 genes. A number of genes involved in metabolism, nutrient exchange, and defense mechanisms, including those involved in resistance to oxidative stress, were significantly induced in ER reticulate bodies. This is consistent with the oxidative stress condition and nutrient starvation that seem to occur in Ehrlichia-containing vacuoles. During the lysis stage of development, when ER is infectious, we showed the overexpression of a transcription factor, dksA, which is also known to induce virulence in other pathogens such as Salmonella typhimurium. Our results suggest a possible role of these genes in promoting ER development and pathogenicity.
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Proteomic analyses of Ehrlichia ruminantium highlight differential expression of MAP1-family proteins.
Veterinary microbiology, 2011Co-Authors: Isabel Marcelino, Thierry Lefrançois, Christian Sheikboudou, Dominique Martinez, Damien F. Meyer, André Martinho De Almeida, Catarina Brito, Mónica Barreto, Catarina F Franco, Nathalie VachieryAbstract:The Rickettsiales Ehrlichia ruminantium (ER) is the causative agent of heartwater, a fatal tick-borne disease of livestock in sub-Saharan Africa and in the Caribbean, posing strong economical constraints to livestock production. In an attempt to identify the most prominent proteins expressed by this bacterium, especially those encoded by the major antigenic protein 1 (map1) multigene family, a proteome map of ER cultivated in endothelial cells was constructed by using two dimensional gel electrophoresis combined with mass spectrometry. Among the sixty-four spots detected, we could identify only four proteins from the MAP1-family; the other proteins detected were mainly related to energy, amino acid and general metabolism (26%), to protein turnover, chaperones and survival (21%) and to information processes (14%) or classified as hypothetical proteins (23%). Additional studies on MAP1-family protein using immunochemical labeling also revealed that these proteins are differentially expressed along the bacterium life cycle, presenting different structural organization. Interestingly, when infectious elementary bodies (EBs) are released from host cells, MAP1 appears to be organized in SDS and heat-resistant dimers and trimers stabilized by disulfide bridges. Overall, the results presented herein not only reveal the first partial proteome map of ER but provide new insights on the expression ER MAP1-family proteins in host endothelial cells.
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Clonal origin of emerging populations of Ehrlichia ruminantium in Burkina Faso
Infection genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases, 2010Co-Authors: Hassane Adakal, Sébastien Zoungrana, Nathalie Vachiery, Dominique Martinez, Frédéric Stachurski, Laurent Gavotte, Maurice Konkobo, Hélène Henri, Karine Huber, Serge MorandAbstract:Cowdriosis or heartwater is a major tick-borne disease on ruminants in Africa and the Caribbean. The causative agent is Ehrlichia ruminantium, an intracellular bacterium. Development of vaccines against heartwater has been hampered the limited efficiency of vaccine in the field, thought to be a consequence of the high genetic diversity of strains circulating in a same area. A sampling scheme was set to collect ticks over 2 years in a delimited area and well identified flock. Prevalence was low at about 3%. A set of 37 strains was considered for MLST analysis along with two reference strains, i.e. ERGA and ERWO, for which full-length genome was available, using a previously described scheme based on the genes gltA, groEL, lepA, lipA, lipB, secY, sodB and sucA. Two populations were identified both with limited genetic variability but with differing evolutionary patterns. Population 1 is in genomic stasis, in agreement with the paradigm for intracellular bacteria. The two reference strains, one from the Caribbean separated from West African strains three centuries ago and another one isolated in South Africa, belong to Population 1. Population 2 is on expansion following a recent clonal emergence from Population 1. The founder strain was identified as strain 395. Strain 623 displays a particularly high rate of mutations in groEL. Owing to the chaperone function of GroEL, this might indicate another clonal emergence under way. This work brings further insight in the genomic plasticity of E. ruminantium and its impact on vaccine strategy.
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Innovative approach for transcriptomic analysis of obligate intracellular pathogen: selective capture of transcribed sequences of Ehrlichia ruminantium.
BMC molecular biology, 2009Co-Authors: Loïc Emboule, Christian Sheikboudou, Roger Frutos, Alain Viari, Bernard Mari, Damien F. Meyer, Valérie Pinarello, Virginie Magnone, Pascal Barbry, Dominique MartinezAbstract:Background Whole genome transcriptomic analysis is a powerful approach to elucidate the molecular mechanisms controlling the pathogenesis of obligate intracellular bacteria. However, the major hurdle resides in the low quantity of prokaryotic mRNAs extracted from host cells. Our model Ehrlichia ruminantium (ER), the causative agent of heartwater, is transmitted by tick Amblyomma variegatum. This bacterium affects wild and domestic ruminants and is present in Sub-Saharan Africa and the Caribbean islands. Because of its strictly intracellular location, which constitutes a limitation for its extensive study, the molecular mechanisms involved in its pathogenicity are still poorly understood.
Basil A. Allsopp - One of the best experts on this subject based on the ideXlab platform.
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Heartwater--Ehrlichia ruminantium infection.
Revue scientifique et technique (International Office of Epizootics), 2015Co-Authors: Basil A. AllsoppAbstract:Summary Heartwater is a notifiable disease that is listed by the World Organisation for Animal Health. It is caused by Ehrlichia ruminantium, an obligately intracellular Gram-negative bacterium in the order Rickettsiales and the family Anaplasmataceae. The disease is borne byticks in the genus Amblyomma and causes heartwater, or cowdriosis, in wild and domestic ruminants, primarily in Africa, but also in parts of the Caribbean. The disease was recognised in South Africa in the 19th Century and determined to be tick borne in 1900, while the organism was identified in 1925 and first cultured in vitro in 1985. This latter achievement boosted research into the disease at a time when biology was moving into the molecular genetic age. Over the last 20 years, there have been significant improvements in our understanding of E. ruminantium, yielding major advances in diagnosis, epidemiology, genetic characterisation, phylogeny, immunology, and vaccine development. The organism is genetically highly variable; this has important implications for future control measures, and is making it difficult to develop an effective vaccine for protection against tick challenge. Research is continuing into three different types of vaccine, inactivated, attenuated, and recombinant, and the current state of development of each is discussed.
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Identification of Ehrlichia ruminantium proteins that activate cellular immune responses using a reverse vaccinology strategy
Veterinary Immunology and Immunopathology, 2011Co-Authors: Junita Liebenberg, Basil A. Allsopp, A. Pretorius, Nicola E. Collins, F.e. Faber, M. Van KleefAbstract:Abstract Ehrlichia ruminantium is an obligate intracellular bacterial pathogen which causes heartwater, a serious tick-borne disease of ruminants throughout sub-Saharan Africa. The development of promising recombinant vaccines has been reported previously, but none has been as effective as immunisation with live organisms. In this study we have used reverse vaccinology to identify proteins that elicit an in vitro cellular immune response similar to that induced by intact E. ruminantium. The experimental strategy involved four successive steps: (i) in silico selection of the most likely vaccine candidate genes from the annotated genome; (ii) cloning and expression of the selected genes; (iii) in vitro screening of the expressed proteins for their ability to induce interferon-gamma (IFN-γ) production in E. ruminantium–immune lymphocytes; and (iv) further examination of the cytokine response profiles of those lymphocytes which tested positive for IFN-γ induction. Based on their overall cytokine induction profiles the recombinant proteins were divided into four distinct groups. Eleven recombinant proteins induced a cytokine profile that was similar to the recall immune response induced by immune peripheral blood mononuclear cells (PBMC) stimulated with intact E. ruminantium. This response comprised the upregulation of cytokines associated with adaptive cellular immune responses as well as innate immunity. A successful vaccine may therefore need to contain a combination of recombinant proteins which induce both immune pathways to ensure protection against heartwater.
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Studies of a polymorphic Ehrlichia ruminantium gene for use as a component of a recombinant vaccine against heartwater.
Vaccine, 2010Co-Authors: A. Pretorius, Nicola E. Collins, J. Liebenberg, E. Louw, Basil A. AllsoppAbstract:A previously identified polymorphic Ehrlichia ruminantium gene, Erum2510, was investigated to determine its ability to induce protective immunity in ruminants following two different DNA immunisation strategies; DNA-only and a DNA prime/recombinant protein (rprotein) boost immunisation. The DNA-only vaccine was also compared to a cocktail of three polymorphic E. ruminantium (Welgevonden) open reading frames (ORFs) adjacent to Erum2510 in the genome. Weak protection was observed in animals immunised with the pCMViUBs_Erum2510 construct alone, while none of the animals immunised with the DNA cocktail were protected. In contrast, all five animals immunised using a DNA prime/rprotein boost strategy survived challenge, thereby indicating that Erum2510 is a good candidate for inclusion in a recombinant vaccine against heartwater. One drawback of using polymorphic genes is a possible lack of cross-protection between genotypes, therefore the genetic diversity of Erum2510 was investigated to establish the degree of polymorphism among different E. ruminantium stocks. Three distinct genotypes were identified indicating that if this gene is used as a vaccine (prime/boost strategy) the vaccine should include a representative Erum2510 gene from each genotype.
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Natural history of Ehrlichia ruminantium.
Veterinary parasitology, 2009Co-Authors: Basil A. AllsoppAbstract:Ehrlichia ruminantium is an obligately intracellular proteobacterium which causes a disease known as heartwater or cowdriosis in some wild, and all domestic, ruminants. The organism is transmitted by ticks of the genus Amblyomma, and it is of serious economic importance wherever the natural vectors occur, an area which includes all of sub-Saharan Africa, and several islands in the Caribbean. The disease was first recognized in South Africa in the 19th century, where its tick-borne nature was determined in 1900, but the organism itself was not demonstrated until 1925, when it was recognized to be a rickettsia, initially named Rickettsia ruminantium. It was thus the first species of what are now known as Ehrlichia to be discovered, and most of the early work to elucidate the nature of the organisms, and its reservoirs and vectors, was performed in South Africa. The next milestone was the development, in 1945, of an infection and treatment regimen to immunize livestock, and this is still the only commercially available "vaccine" against the disease. Then in 1985, after fruitless attempts over many years, the organism was propagated reliably in tissue culture, opening the way for the first application of the newly developed techniques of molecular genetics. From 1990 onwards the pace of heartwater research accelerated rapidly, with notable advances in phylogeny, diagnosis, epidemiology, immunology, and vaccine development. The complete genome sequence was published in 2005, and during the last two years a new understanding has arisen of the remarkable genetic variability of the organism and new experimental vaccines have been developed. Despite all this the goal of producing an effective vaccine against the disease in the field still remains frustratingly just beyond reach. This article summarises our current understanding of the nature of E. ruminantium, at a time when the prospects for the development of an effective vaccine against the organism seem better than at any time since its discovery 83 years ago.
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Extensive genetic recombination occurs in the field between different genotypes of Ehrlichia ruminantium.
Veterinary microbiology, 2007Co-Authors: M.t.e.p. Allsopp, Basil A. AllsoppAbstract: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.
Thierry Lefrançois - One of the best experts on this subject based on the ideXlab platform.
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Genetic diversity of Ehrlichia ruminantium in Mozambique
2018Co-Authors: Laure Bournez, Thierry Lefrançois, Rosalie Aprelon, Valérie Pinarello, Nidia Cangi, Jonathan Gordon, Luis Neves, Nathalie VachieryAbstract:The tick species, Amblyomma hebraeum and A. variegatum, are the main vectors of Heartwater, a tropical infectious bacterial disease of ruminants caused by Ehrlichia ruminantium. In Mozambique, these tick species have a parapatric distribution, with A. variegatum present in the central and northern regions and A. hebraeum in the South. A narrow overlap area between the distributions of the two species occurs around parallel 22o south. In order to determine the prevalence of E. ruminantium in A. hebraeum and A. variegatum and to determine the genetic diversity and structure of isolates from different localities, adult ticks feeding on cattle and wild ruminants were sampled across the south and center of Mozambique as well as in the adjacent Kruger National Park (KNP), South Africa. The prevalence of E. rumimantium in Amblyomma ticks in relation to the tick species and gender, locality and tick abundance was analyzed. Afterwards, Mozambican Ehrlichia isolates were typed using Multi Locus Sequence Typing and the distribution of groups clustering genotypes were analysed. In total, 722 and 388 of A. hebraeum and A. variegatum ticks were collected from 31 localities and screened for E. ruminantium, using pCS20 nested PCR and Sol1TqM qPCR. The prevalence of E. ruminantium in ticks feeding on cattle varied from 0% to 26.7%, with no infected ticks determined in 7 localities. In ticks feeding on wild ruminants, the prevalence was 8.2 % in the KNP and 6.2% in hunting concessions of the Sofala province. After accounting for the effects of tick gender and sampling sites, no significant difference in prevalence was found between tick species. Most MLST genotypes from Mozambique clustered into subgroup 2C and 2E, which were present in similar proportions in 5 of the 19 localities. Interestingly, MLST genotypes from group G1 and G2D were exclusively found in areas of A. variegatum distribution, while subgroup G2C was only detected in A. hebraeum areas. Moreover, genotypes from subgroup G2E were found in both A. hebraeum and A. variegatum areas. These results contribute to a better understanding of spatial distribution of E. ruminantium and will aid in improvement of heartwater monitoring and control strategies in Mozambique.
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Iron Starvation Conditions Upregulate Ehrlichia ruminantium Type IV Secretion System, tr1 Transcription Factor and map1 Genes Family through the Master Regulatory Protein ErxR
Frontiers in Cellular and Infection Microbiology, 2018Co-Authors: Amal Moumène, Nathalie Vachiery, Thierry Lefrançois, Silvina Gonzalez-rizzo, Damien MeyerAbstract:Ehrlichia ruminantium is an obligatory intracellular bacterium that causes heartwater, a fatal disease in ruminants. Due to its intracellular nature, E. ruminantium requires a set of specific virulence factors, such as the type IV secretion system (T4SS), and outer membrane proteins (Map proteins) in order to avoid and subvert the host's immune response. Several studies have been conducted to understand the regulation of the T4SS or outer membrane proteins, in Ehrlichia, but no integrated approach has been used to understand the regulation of Ehrlichia pathogenicity determinants in response to environmental cues. Iron is known to be a key nutrient for bacterial growth both in the environment and within hosts. In this study, we experimentally demonstrated the regulation of virB, map1, and tr1 genes by the newly identified master regulator ErxR (for Ehrlichia ruminantium expression regulator). We also analyzed the effect of iron depletion on the expression of erxR gene, tr1 transcription factor, T4SS and map1 genes clusters in E. ruminantium. We show that exposure of E. ruminantium to iron starvation induces erxR and subsequently tr1, virB, and map1 genes. Our results reveal tight co-regulation of T4SS and map1 genes via the ErxR regulatory protein at the transcriptional level, and, for the first time link map genes to the virulence function sensu stricto, thereby advancing our understanding of Ehrlichia's infection process. These results suggest that Ehrlichia is able to sense changes in iron concentrations in the environment and to regulate the expression of virulence factors accordingly.
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Efficient high-throughput molecular method to detect Ehrlichia ruminantium in ticks
Parasites & vectors, 2017Co-Authors: Nidia Cangi, Thierry Lefrançois, Valérie Pinarello, Laure Bournez, Luis Neves, Emmanuel Albina, Nathalie VachieryAbstract:Ehrlichia ruminantium is the causal agent of heartwater, a fatal tropical disease affecting ruminants with important economic impacts. This bacterium is transmitted by Amblyomma ticks and is present in sub-Saharan Africa, islands in the Indian Ocean and the Caribbean, where it represents a threat to the American mainland. An automated DNA extraction method was adapted for Amblyomma ticks and a new qPCR targeting the pCS20 region was developed to improve E. ruminantium screening capacity and diagnosis. The first step in the preparation of tick samples, before extraction, was not automated but was considerably improved by using a Tissue Lyser. The new pCS20 Sol1 qPCR and a previously published pCS20 Cow qPCR were evaluated with the OIE standard pCS20 nested PCR. pCS20 Sol1 qPCR was found to be more specific than the nested PCR, with a 5-fold increase in sensitivity (3 copies/reaction vs 15 copies/reaction), was less prone to contamination and less time-consuming. As pCS20 Sol1 qPCR did not detect Rickettsia, Anasplasma and Babesia species or closely related species such as Panola Mountain Ehrlichia, E. chaffeensis and E. canis, its specificity was also better than Cow qPCR. In parallel, a tick 16S qPCR was developed for the quality control of DNA extraction that confirmed the good reproducibility of the automated extraction. The whole method, including the automated DNA extraction and pCS20 Sol1 qPCR, was shown to be sensitive, specific and highly reproducible with the same limit of detection as the combined manual DNA extraction and nested PCR, i.e. 6 copies/reaction. Finally, 96 samples can be tested in one day compared to the four days required for manual DNA extraction and nested PCR. The adaptation of an automated DNA extraction using a DNA/RNA viral extraction kit for tick samples and the development of a new qPCR increased the accuracy of E. ruminantium epidemiological studies, as well as the diagnostic capabilities and turn-over time for surveillance of heartwater. This new method paves the way for large-scale screening of other bacteria and viruses in ticks as well as genetic characterization of ticks and tick-pathogen coevolution studies.
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Recombination is a major driving force of genetic diversity in the Anaplasmataceae Ehrlichia ruminantium
Frontiers in Cellular and Infection Microbiology, 2016Co-Authors: Nidia Cangi, Thierry Lefrançois, Damien Meyer, Rosalie Aprelon, Valérie Pinarello, Laure Bournez, Luis Neves, Karine Huber, Jonathan L. Gordon, Nathalie VachieryAbstract: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.
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A new high-throughput analysis to screen Ehrlichia ruminantium in field ticks
2016Co-Authors: Nathalie Vachiery, Thierry Lefrançois, Valérie Pinarello, Laure Bournez, Nidia Cangi, Emmanuel Albina, Luis NevesAbstract:In order to improve sample screening capacity of Ehrlichia ruminantium in ticks, an automatic DNA extraction method for Amblyomma ticks and a new QPCR targeting E. ruminantium pCS20 region, pCS20 Sol1 QPCR, were developed. A comparison between the new pCS20 Sol1 QPCR, a previously published pCS20 CowTM QPCR and the gold standard nested pCS20 PCR have been carried out. pCS20 Sol1TM QPCR was highly sensitive (up to 2.4 copies per sample) and specific (16 E. ruminantium strains detected and no cross-reaction with close-related species). We showed that Sol1 QPCR and pCS20 nested PCR had similar sensitivity and specificity however there was limited risk of contamination and timeless process for pCS20 Sol1 QPCR. In parallel, a tick 16SSyb rRNA QPCR was successfully developed for DNA quality control. DNA yield and quality from ticks extracted automatically, using 16SSyb rRNA QPCR were high. 16SSyb rRNA QPCR results (n=671 ticks) showed also a good reproducibility of automatic DNA extraction with a mean Ct=23+/-3. The whole method of screening, including automatic DNA extraction and pCS20 Sol1 QPCR, demonstrated a limit of detection between 6 and 0.6 copies per sample. It was also reproducible with restricted standard deviation of Ct (+/-1.3) for samples at the limit of detection. The development of a new automatic DNA extraction and QPCR allows for improving tick sample processing and E. ruminantium diagnostic capacities using high throughput methods. More widely, the automatic DNA extraction based on DNA/RNA virus kit and the new DNA quality control method, 16SSyb rRNA QPCR, can then be used for screening other bacterium and virus and for other tick species. (Texte integral)
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Characterization of Ehrlichia ruminantium membrane protein, erga-cds- 01230 and its role in adhesion to the host cell
2019Co-Authors: Valérie Pinarello, Nathalie Vachiery, Emmanuel Albina, Damien F. MeyerAbstract:Outer membrane proteins participate to pathogens adhesion to host cells and therefore often mediate cell infection. Such is the case for Ehrlichia ruminantium, an obligate intracellular bacterium that is transmitted by ticks and responsible for cowdriosis, a fatal disease of domestic and wild ruminants. Several experimental vaccines were developed, but the great genetic and presumably antigenic diversity of E. ruminantium make difficult to obtain an effective vaccine against all strains present in the field. In order to propose novel strategies to control cowdriosis, the interaction of E. ruminantium with its host cell, particularly the associated adhesion mechanisms must be first deciphered. A membrane protein of E. ruminantium, ERGA_CDS_01230, a probable iron transporter, initially identified by proteomics approaches in our group, was here studied for its role in host cell adhesion. The recombinant protein was expressed with post-translational glycosylation modifications and tagged GFP/Histidine in Leishmania tarentolae. Using cell biology approaches, we show that recProt01230 is able to adhere to bovine host cells and interacts with proteins from the cell lysate and the "membranes/ organelles” sub-fraction. Furthermore, recProt01230 does not adhere to heparan sulfate but other membrane polysaccharides seem to play a role in E. ruminantium's adhesion to the host cell. Indeed, preliminary experiments have shown that degrading dermatan sulfate and chondroitin sulfate at the cell surface is associated with a reduction of the number of bacteria in the host cells. Moreover, CDS ERGA_CDS_01230 is over expressed at early stages of infection when bacteria begin to attach to their host. So, our results show the implication of ERGA_CDS_01230 in the adhesion of E. ruminantium to host cells. ERGA_CDS_01230 also induces a humoral response in the vaccinated animals. In conclusion, ERGA_CDS_01230 could be a new promising target for vaccine or therapeutics development.
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Genetic diversity of Ehrlichia ruminantium in Mozambique
2018Co-Authors: Laure Bournez, Thierry Lefrançois, Rosalie Aprelon, Valérie Pinarello, Nidia Cangi, Jonathan Gordon, Luis Neves, Nathalie VachieryAbstract:The tick species, Amblyomma hebraeum and A. variegatum, are the main vectors of Heartwater, a tropical infectious bacterial disease of ruminants caused by Ehrlichia ruminantium. In Mozambique, these tick species have a parapatric distribution, with A. variegatum present in the central and northern regions and A. hebraeum in the South. A narrow overlap area between the distributions of the two species occurs around parallel 22o south. In order to determine the prevalence of E. ruminantium in A. hebraeum and A. variegatum and to determine the genetic diversity and structure of isolates from different localities, adult ticks feeding on cattle and wild ruminants were sampled across the south and center of Mozambique as well as in the adjacent Kruger National Park (KNP), South Africa. The prevalence of E. rumimantium in Amblyomma ticks in relation to the tick species and gender, locality and tick abundance was analyzed. Afterwards, Mozambican Ehrlichia isolates were typed using Multi Locus Sequence Typing and the distribution of groups clustering genotypes were analysed. In total, 722 and 388 of A. hebraeum and A. variegatum ticks were collected from 31 localities and screened for E. ruminantium, using pCS20 nested PCR and Sol1TqM qPCR. The prevalence of E. ruminantium in ticks feeding on cattle varied from 0% to 26.7%, with no infected ticks determined in 7 localities. In ticks feeding on wild ruminants, the prevalence was 8.2 % in the KNP and 6.2% in hunting concessions of the Sofala province. After accounting for the effects of tick gender and sampling sites, no significant difference in prevalence was found between tick species. Most MLST genotypes from Mozambique clustered into subgroup 2C and 2E, which were present in similar proportions in 5 of the 19 localities. Interestingly, MLST genotypes from group G1 and G2D were exclusively found in areas of A. variegatum distribution, while subgroup G2C was only detected in A. hebraeum areas. Moreover, genotypes from subgroup G2E were found in both A. hebraeum and A. variegatum areas. These results contribute to a better understanding of spatial distribution of E. ruminantium and will aid in improvement of heartwater monitoring and control strategies in Mozambique.
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Efficient high-throughput molecular method to detect Ehrlichia ruminantium in ticks
Parasites & vectors, 2017Co-Authors: Nidia Cangi, Thierry Lefrançois, Valérie Pinarello, Laure Bournez, Luis Neves, Emmanuel Albina, Nathalie VachieryAbstract:Ehrlichia ruminantium is the causal agent of heartwater, a fatal tropical disease affecting ruminants with important economic impacts. This bacterium is transmitted by Amblyomma ticks and is present in sub-Saharan Africa, islands in the Indian Ocean and the Caribbean, where it represents a threat to the American mainland. An automated DNA extraction method was adapted for Amblyomma ticks and a new qPCR targeting the pCS20 region was developed to improve E. ruminantium screening capacity and diagnosis. The first step in the preparation of tick samples, before extraction, was not automated but was considerably improved by using a Tissue Lyser. The new pCS20 Sol1 qPCR and a previously published pCS20 Cow qPCR were evaluated with the OIE standard pCS20 nested PCR. pCS20 Sol1 qPCR was found to be more specific than the nested PCR, with a 5-fold increase in sensitivity (3 copies/reaction vs 15 copies/reaction), was less prone to contamination and less time-consuming. As pCS20 Sol1 qPCR did not detect Rickettsia, Anasplasma and Babesia species or closely related species such as Panola Mountain Ehrlichia, E. chaffeensis and E. canis, its specificity was also better than Cow qPCR. In parallel, a tick 16S qPCR was developed for the quality control of DNA extraction that confirmed the good reproducibility of the automated extraction. The whole method, including the automated DNA extraction and pCS20 Sol1 qPCR, was shown to be sensitive, specific and highly reproducible with the same limit of detection as the combined manual DNA extraction and nested PCR, i.e. 6 copies/reaction. Finally, 96 samples can be tested in one day compared to the four days required for manual DNA extraction and nested PCR. The adaptation of an automated DNA extraction using a DNA/RNA viral extraction kit for tick samples and the development of a new qPCR increased the accuracy of E. ruminantium epidemiological studies, as well as the diagnostic capabilities and turn-over time for surveillance of heartwater. This new method paves the way for large-scale screening of other bacteria and viruses in ticks as well as genetic characterization of ticks and tick-pathogen coevolution studies.
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A new high-throughput analysis to screen Ehrlichia ruminantium in field ticks
2016Co-Authors: Nathalie Vachiery, Thierry Lefrançois, Valérie Pinarello, Laure Bournez, Nidia Cangi, Emmanuel Albina, Luis NevesAbstract:In order to improve sample screening capacity of Ehrlichia ruminantium in ticks, an automatic DNA extraction method for Amblyomma ticks and a new QPCR targeting E. ruminantium pCS20 region, pCS20 Sol1 QPCR, were developed. A comparison between the new pCS20 Sol1 QPCR, a previously published pCS20 CowTM QPCR and the gold standard nested pCS20 PCR have been carried out. pCS20 Sol1TM QPCR was highly sensitive (up to 2.4 copies per sample) and specific (16 E. ruminantium strains detected and no cross-reaction with close-related species). We showed that Sol1 QPCR and pCS20 nested PCR had similar sensitivity and specificity however there was limited risk of contamination and timeless process for pCS20 Sol1 QPCR. In parallel, a tick 16SSyb rRNA QPCR was successfully developed for DNA quality control. DNA yield and quality from ticks extracted automatically, using 16SSyb rRNA QPCR were high. 16SSyb rRNA QPCR results (n=671 ticks) showed also a good reproducibility of automatic DNA extraction with a mean Ct=23+/-3. The whole method of screening, including automatic DNA extraction and pCS20 Sol1 QPCR, demonstrated a limit of detection between 6 and 0.6 copies per sample. It was also reproducible with restricted standard deviation of Ct (+/-1.3) for samples at the limit of detection. The development of a new automatic DNA extraction and QPCR allows for improving tick sample processing and E. ruminantium diagnostic capacities using high throughput methods. More widely, the automatic DNA extraction based on DNA/RNA virus kit and the new DNA quality control method, 16SSyb rRNA QPCR, can then be used for screening other bacterium and virus and for other tick species. (Texte integral)
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Recombination is a major driving force of genetic diversity in the Anaplasmataceae Ehrlichia ruminantium
Frontiers in Cellular and Infection Microbiology, 2016Co-Authors: Nidia Cangi, Thierry Lefrançois, Damien Meyer, Rosalie Aprelon, Valérie Pinarello, Laure Bournez, Luis Neves, Karine Huber, Jonathan L. Gordon, Nathalie VachieryAbstract: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.