Detection of Crimean-Congo haemorrhagic fever virus in Hyalomma marginatum ticks, southern France, May 2022 and April 2023.

Crimean-Congo haemorrhagic fever (CCHF), a potentially severe zoonotic viral disease causing fever and haemorrhagic manifestations in humans. As the Crimean-Congo haemorrhagic fever virus (CCHFV) has been detected in ticks in Spain and antibodies against the virus in ruminant sera in Corsica, it was necessary to know more about the situation in France. In 2022-2023, CCHFV was detected in 155 ticks collected from horses and cattle in southern France.

Searching algorithm for Type IV effector proteins (S4TE) 2.0: Improved tools for Type IV effector prediction, analysis and comparison in proteobacteria.

Bacterial pathogens have evolved numerous strategies to corrupt, hijack or mimic cellular processes in order to survive and proliferate. Among those strategies, Type IV effectors (T4Es) are proteins secreted by pathogenic bacteria to manipulate host cell processes during infection. They are delivered into eukaryotic cells in an ATP-dependent manner via the type IV secretion system, a specialized multiprotein complex. T4Es contain a wide spectrum of features including eukaryotic-like domains, localization signals or a C-terminal translocation signal. A combination of these features enables prediction of T4Es in a given bacterial genome. In this study, we developed a web-based comprehensive suite of tools with a user-friendly graphical interface. This version 2.0 of S4TE (Searching Algorithm for Type IV Effector Proteins; http://sate.cirad.fr) enables accurate prediction and comparison of T4Es. Search parameters and threshold can be customized by the user to work with any genome sequence, whether publicly available or not. Applications range from characterizing effector features and identifying potential T4Es to analyzing the effectors based on the genome G+C composition and local gene density. S4TE 2.0 allows the comparison of putative T4E repertoires of up to four bacterial strains at the same time. The software identifies T4E orthologs among strains and provides a Venn diagram and lists of genes for each intersection. New interactive features offer the best visualization of the location of candidate T4Es and hyperlinks to NCBI and Pfam databases. S4TE 2.0 is designed to evolve rapidly with the publication of new experimentally validated T4Es, which will reinforce the predictive power of the algorithm. The computational methodology can be used to identify a wide spectrum of candidate bacterial effectors that lack sequence conservation but have similar amino acid characteristics. This approach will provide very valuable information about bacterial host-specificity and virulence factors and help identify host targets for the development of new anti-bacterial molecules.

Comparative Transcriptome Profiling of Virulent and Attenuated Ehrlichia ruminantium Strains Highlighted Strong Regulation of map1- and Metabolism Related Genes.

The obligate intracellular pathogenic bacterium, Ehrlichia ruminantium, is the causal agent of heartwater, a fatal disease in ruminants transmitted by Amblyomma ticks. So far, three strains have been attenuated by successive passages in mammalian cells. The attenuated strains have improved capacity for growth in vitro, whereas they induced limited clinical signs in vivo and conferred strong protection against homologous challenge. However, the mechanisms of pathogenesis and attenuation remain unknown. In order to improve knowledge of E. ruminantium pathogenesis, we performed a comparative transcriptomic analysis of two distant strains of E. ruminantium, Gardel and Senegal, and their corresponding attenuated strains. Overall, our results showed an upregulation of gene expression encoding for the metabolism pathway in the attenuated strains compared to the virulent strains, which can probably be associated with higher in vitro replicative activity and a better fitness to the host cells. We also observed a significant differential expression of membrane protein-encoding genes between the virulent and attenuated strains. A major downregulation of map1-related genes was observed for the two attenuated strains, whereas upregulation of genes encoding for hypothetical membrane proteins was observed for the four strains. Moreover, CDS_05140, which encodes for a putative porin, displays the highest gene expression in both attenuated strains. For the attenuated strains, the significant downregulation of map1-related gene expression and upregulation of genes encoding other membrane proteins could be important in the implementation of efficient immune responses after vaccination with attenuated vaccines. Moreover, this study revealed an upregulation of gene expression for 8 genes encoding components of Type IV secretion system and 3 potential effectors, mainly in the virulent Gardel strain. Our transcriptomic study, supported by previous proteomic studies, provides and also confirms new information regarding the characterization of genes involved in E. ruminantium virulence and attenuation mechanisms.

Efficient high-throughput molecular method to detect Ehrlichia ruminantium in ticks.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Iron Starvation Conditions Upregulate Ehrlichia ruminantium Type IV Secretion System, tr1 Transcription Factor and map1 Genes Family through the Master Regulatory Protein ErxR.

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.

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.

Comparative Proteomic Profiling of Ehrlichia ruminantium Pathogenic Strain and Its High-Passaged Attenuated Strain Reveals Virulence and Attenuation-Associated Proteins.

The obligate intracellular bacterium Ehrlichia ruminantium (ER) causes heartwater, a fatal tick-borne disease in livestock. In the field, ER strains present different levels of virulence, limiting vaccine efficacy, for which the molecular basis remains unknown. Moreover, there are no genetic tools currently available for ER manipulation, thus limiting the knowledge of the genes/proteins that are essential for ER pathogenesis and biology. As such, to identify proteins and/or mechanisms involved in ER virulence, we performed the first exhaustive comparative proteomic analysis between a virulent strain (ERGvir) and its high-passaged attenuated strain (ERGatt). Despite their different behaviors in vivo and in vitro, our results from 1DE-nanoLC-MS/MS showed that ERGvir and ERGatt share 80% of their proteins; this core proteome includes chaperones, proteins involved in metabolism, protein-DNA-RNA biosynthesis and processing, and bacterial effectors. Conventional 2DE revealed that 85% of the identified proteins are proteoforms, suggesting that post-translational modifications (namely glycosylation) are important in ER biology. Strain-specific proteins were also identified: while ERGatt has an increased number and overexpression of proteins involved in cell division, metabolism, transport and protein processing, ERGvir shows an overexpression of proteins and proteoforms (DIGE experiments) involved in pathogenesis such as Lpd, AnkA, VirB9 and B10, providing molecular evidence for its increased virulence in vivo and in vitro. Overall, our work reveals that ERGvir and ERGatt proteomes are streamlined to fulfill their biological function (maximum virulence for ERGvir and replicative capacity for ERGatt), and we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.

An in vitro model to assess the immunosuppressive effect of tick saliva on the mobilization of inflammatory monocyte-derived cells.

Tick-borne pathogens cause potent infections. These pathogens benefit from molecules contained in tick saliva that have evolved to modulate host innate and adaptive immune responses. This is called "saliva-activated transmission" and enables tick-borne pathogens to evade host immune responses. Ticks feed on their host for relatively long periods; thus, mechanisms counteracting the inflammation-driven recruitment and activation of innate effector cells at the bite site, are an effective strategy to escape the immune response. Here, we developed an original in vitro model to evaluate and to characterize the immunomodulatory effects of tick saliva that prevent the establishment of a local inflammatory immune response. This model mimics the tick bite and enables the assessment of the effect of saliva on the inflammatory-associated dynamic recruitment of cells from the mononuclear phagocyte system. Using this model, we were able to recapitulate the dual effect of tick saliva on the mobilization of inflammatory monocyte-derived cells, i.e. (i) impaired recruitment of monocytes from the blood to the bite wound; and (ii) poor mobilization of monocyte-derived cells from the skin to the draining lymph node. This simple tool reconstitutes the effect of tick saliva in vivo, which we characterized in the mouse, and should enable the identification of important factors facilitating pathogen infection. Furthermore, this model may be applied to the characterization of any pathogen-derived immunosuppressive molecule affecting the establishment of the inflammatory immune response.

Proteomic profiling of the outer membrane fraction of the obligate intracellular bacterial pathogen Ehrlichia ruminantium.

The outer membrane proteins (OMPs) of Gram-negative bacteria play a crucial role in virulence and pathogenesis. Identification of these proteins represents an important goal for bacterial proteomics, because it aids in vaccine development. Here, we have developed such an approach for Ehrlichia ruminantium, the obligate intracellular bacterium that causes heartwater. A preliminary whole proteome analysis of elementary bodies, the extracellular infectious form of the bacterium, had been performed previously, but information is limited about OMPs in this organism and about their role in the protective immune response. Identification of OMPs is also essential for understanding Ehrlichia's OM architecture, and how the bacterium interacts with the host cell environment. First, we developed an OMP extraction method using the ionic detergent sarkosyl, which enriched the OM fraction. Second, proteins were separated via one-dimensional electrophoresis, and digested peptides were analyzed via nano-liquid chromatographic separation coupled with mass spectrometry (LC-MALDI-TOF/TOF). Of 46 unique proteins identified in the OM fraction, 18 (39%) were OMPs, including 8 proteins involved in cell structure and biogenesis, 4 in transport/virulence, 1 porin, and 5 proteins of unknown function. These experimental data were compared to the predicted subcellular localization of the entire E. ruminantium proteome, using three different algorithms. This work represents the most complete proteome characterization of the OM fraction in Ehrlichia spp. The study indicates that suitable subcellular fractionation experiments combined with straightforward computational analysis approaches are powerful for determining the predominant subcellular localization of the experimentally observed proteins. We identified proteins potentially involved in E. ruminantium pathogenesis, which are good novel targets for candidate vaccines. Thus, combining bioinformatics and proteomics, we discovered new OMPs for E. ruminantium that are valuable data for those investigating new vaccines against this organism. In summary, we provide both pioneering data and novel insights into the pathogenesis of this obligate intracellular bacterium.

A user-friendly and scalable process to prepare a ready-to-use inactivated vaccine: the example of heartwater in ruminants under tropical conditions.

The use of cheap and thermoresistant vaccines in poor tropical countries for the control of animal diseases is a key issue. Our work aimed at designing and validating a process for the large-scale production of a ready-to-use inactivated vaccine for ruminants. Our model was heartwater caused by the obligate intracellular bacterium Ehrlichia ruminantium (ER). The conventional inactivated vaccine against heartwater (based on whole bacteria inactivated with sodium azide) is prepared immediately before injection, using a syringe-extrusion method with Montanide ISA50. This is a fastidious time-consuming process and it limits the number of vaccine doses available. To overcome these issues, we tested three different techniques (syringe, vortex and homogenizer) and three Montanide ISA adjuvants (50, 70 and 70M). High-speed homogenizer was the optimal method to emulsify ER antigens with both ISA70 and 70M adjuvants. The emulsions displayed a good homogeneity (particle size below 1 µm and low phase separation), conductivity below 10 µS/cm and low antigen degradation at 4 °C for up to 1 year. The efficacy of the different formulations was then evaluated during vaccination trials on goats. The inactivated ER antigens emulsified with ISA70 and ISA70M in a homogenizer resulted in 80% and 100% survival rates, respectively. A cold-chain rupture assay using ISA70M+ER was performed to mimic possible field conditions exposing the vaccine at 37 °C for 4 days before delivery. Surprisingly, the animal survival rate was still high (80%). We also observed that the MAP-1B antibody response was very similar between animals vaccinated with ISA70+ER and ISA70M+ER emulsions, suggesting a more homogenous antigen distribution and presentation in these emulsions. Our work demonstrated that the combination of ISA70 or ISA70M and homogenizer is optimal for the production of an effective ready-to-use inactivated vaccine against heartwater, which could easily be produced on an industrial scale.

Understanding Anaplasmataceae pathogenesis using "Omics" approaches.

This paper examines how "Omics" approaches improve our understanding of Anaplasmataceae pathogenesis, through a global and integrative strategy to identify genes and proteins involved in biochemical pathways key for pathogen-host-vector interactions. The Anaplasmataceae family comprises obligate intracellular bacteria mainly transmitted by arthropods. These bacteria are responsible for major human and animal endemic and emerging infectious diseases with important economic and public health impacts. In order to improve disease control strategies, it is essential to better understand their pathogenesis. Our work focused on four Anaplasmataceae, which cause important animal, human and zoonotic diseases: Anaplasma marginale, A. phagocytophilum, Ehrlichia chaffeensis, and E. ruminantium. Wolbachia spp. an endosymbiont of arthropods was also included in this review as a model of a non-pathogenic Anaplasmataceae. A gap analysis on "Omics" approaches on Anaplasmataceae was performed, which highlighted a lack of studies on the genes and proteins involved in the infection of hosts and vectors. Furthermore, most of the studies have been done on the pathogen itself, mainly on infectious free-living forms and rarely on intracellular forms. In order to perform a transcriptomic analysis of the intracellular stage of development, researchers developed methods to enrich bacterial transcripts from infected cells. These methods are described in this paper. Bacterial genes encoding outer membrane proteins, post-translational modifications, eukaryotic repeated motif proteins, proteins involved in osmotic and oxidative stress and hypothetical proteins have been identified to play a key role in Anaplasmataceae pathogenesis. Further investigations on the function of these outer membrane proteins and hypothetical proteins will be essential to confirm their role in the pathogenesis. Our work underlines the need for further studies in this domain and on host and vector responses to infection.

Using species distribution models to optimize vector control in the framework of the tsetse eradication campaign in Senegal.

Tsetse flies are vectors of human and animal trypanosomoses in sub-Saharan Africa and are the target of the Pan African Tsetse and Trypanosomiasis Eradication Campaign (PATTEC). Glossina palpalis gambiensis (Diptera: Glossinidae) is a riverine species that is still present as an isolated metapopulation in the Niayes area of Senegal. It is targeted by a national eradication campaign combining a population reduction phase based on insecticide-treated targets (ITTs) and cattle and an eradication phase based on the sterile insect technique. In this study, we used species distribution models to optimize control operations. We compared the probability of the presence of G. p. gambiensis and habitat suitability using a regularized logistic regression and Maxent, respectively. Both models performed well, with an area under the curve of 0.89 and 0.92, respectively. Only the Maxent model predicted an expert-based classification of landscapes correctly. Maxent predictions were therefore used throughout the eradication campaign in the Niayes to make control operations more efficient in terms of deployment of ITTs, release density of sterile males, and location of monitoring traps used to assess program progress. We discuss how the models' results informed about the particular ecology of tsetse in the target area. Maxent predictions allowed optimizing efficiency and cost within our project, and might be useful for other tsetse control campaigns in the framework of the PATTEC and, more generally, other vector or insect pest control programs.

Boosting the sterile insect technique to control mosquitoes.

Mosquitoes are vectors of major diseases. Auto-dissemination recently proved very efficient to control Aedes species, using adult females contaminated with dissemination stations of juvenile hormone to treat breeding habitats, but cannot be used at large scales. Here we propose to combine it to the Sterile Insect Technique (SIT) to create a new control concept, named 'boosted SIT' that might enable the area-wide eradication of mosquitoes and many other vectors and insect pests.

West Nile virus transmission in sentinel chickens and potential mosquito vectors, Senegal River Delta, 2008-2009.

West Nile virus (WNV) is an arthropod-borne Flavivirus usually transmitted to wild birds by Culex mosquitoes. Humans and horses are susceptible to WNV but are dead-end hosts. WNV is endemic in Senegal, particularly in the Senegal River Delta. To assess transmission patterns and potential vectors, entomological and sentinel serological was done in Ross Bethio along the River Senegal. Three sentinel henhouses (also used as chicken-baited traps) were set at 100 m, 800 m, and 1,300 m from the river, the latter close to a horse-baited trap. Blood samples were taken from sentinel chickens at 2-week intervals. Seroconversions were observed in sentinel chickens in November and December. Overall, the serological incidence rate was 4.6% with 95% confidence interval (0.9; 8.4) in the sentinel chickens monitored for this study. Based on abundance pattern, Culex neavei was the most likely mosquito vector involved in WNV transmission to sentinel chickens, and a potential bridge vector between birds and mammals.

West Nile virus surveillance, Brazil, 2008-2010.

BACKGROUND: West Nile virus (WNV) is an emergent pathogen that is widely distributed in North and Central America. The recent introduction in South America has focused attention on the spread of WNV across Southern American countries. The transmission network involves mosquitoes, birds, horses and humans. METHODS: The serological evaluation of sera from 678 equids and 478 birds was performed using a WNV-specific blocking ELISA, and only the positive results were confirmed by plaque reduction neutralisation tests (PRNTs). Molecular analysis was performed on sera from 992 healthy equids and on 63 macerates of brains from equids that died of encephalitis and had previously tested negative for other pathogens. We also tested swabs from 928 birds. The samples analysed were collected in different biomes of Brazil. RESULTS: We identified WNV antibodies by ELISA in thirteen equids and five birds, and PRNT90 confirmed WNV positivity in four equid samples collected in 2009 in an area between the Amazon and the Pantanal. None of the ELISA positive bird samples were confirmed by PRNT90, and all samples tested by RT-PCR were negative. CONCLUSION: WNV circulation is confirmed by this large scale survey even in the absence of detection of clinical cases.

Prevalence of and examination of exposure factors for Salmonella on commercial egg-laying farms in Barbados.

During January-March 2009, an outbreak of Salmonella was diagnosed in four commercial egg-laying farms in Barbados. During the outbreak, 511 layers died while 3257 layers were voluntarily culled from one affected farm. Salmonella ser Enteritidis was identified in avian tissues (ovaries, liver, and uterus), and egg samples submitted to the Ministry of Agriculture, Food, Fisheries, and Water Resource Management (MAFFW) Veterinary Services (VS) Laboratory by managers of the affected farms. As a result of this outbreak, MAFFW VS conducted a survey to investigate the prevalence of Salmonella on commercial egg-laying farms in Barbados. In addition, the relationships between farm-level exposure factors and diagnosis of Salmonella were examined. This paper describes the results of this survey. Nineteen of 26 farms (73%; 95% CI=56-90%) were classified as positive for Salmonella. The odds of testing positive for Salmonella were 10 times higher in large farms, compared to small farms (OR=10.80; 95% CI=1.01, 115.10; p=0.04). More small farms (8/11) cleaned and disinfected poultry facilities quarterly or more often than large farms did (1/10) (p<0.01). Prevalence of Salmonella in commercial egg-laying farms in Barbados is high. Salmonella ser Enteritidis was the most common serotype identified in study farms, and this is a public health issue of concern. This is the first prevalence survey of Salmonella in egg-laying farms in Barbados. Study results can be used as a baseline for future control programmes aimed at reducing the prevalence of Salmonella in egg-laying farms in the country.

Tick-borne diseases in cattle: applications of proteomics to develop new generation vaccines.

Tick-borne diseases (TBDs) affect 80% of the world's cattle population, hampering livestock production throughout the world. Livestock industry is important to rural populations not only as food supply, but also as a source of income. Tick control is usually achieved by using acaricides which are expensive, deleterious to the environment and can induce chemical resistance of vectors; the development of more effective and sustainable control methods is therefore required. Theileriosis, babesiosis, anaplasmosis and heartwater are the most important TBDs in cattle. Immunization strategies are currently available but with variable efficacy. To develop a new generation of vaccines which are more efficient, cheaper and safer, it is first necessary to better understand the mechanisms by which these parasites are transmitted, multiply and cause disease; this becomes especially difficult due to their complex life cycles, in vitro culture conditions and the lack of genetic tools to manipulate them. Proteomics and other complementary post-genomic tools such as transcriptomics and metabolomics in a systems biology context are becoming key tools to increase knowledge on the biology of infectious diseases. Herein, we present an overview of the so called "Omics" studies currently available on these tick-borne pathogens, giving emphasis to proteomics and how it may help to discover new vaccine candidates to control TBDs.