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Pour éviter les risques de dérives méthodologiques ou d’hétérogénéité au fil du temps ou des opérateurs, il est nécessaire pour une bonne qualité des travaux réalisés en écologie comme en dynamique des populations de disposer de protocoles d’acquisition de données formalisés suffisamment détaillés. Dans le cadre de l’analyse du fonctionnement de la population de grande alose Alosa alosa du bassin versant Gironde-Garonne-Dordogne (France), nous avons mené un travail de standardisation des différentes étapes de l’acquisition des données d’échantillonnage. Après une analyse détaillée et exhaustive de la démarche d’acquisition, nous avons identifié trois parties : la procédure d’échantillonnage, le mode opératoire des prélèvements sur le terrain et le mode opératoire de la préparation et du montage des écailles au laboratoire. Chacune de ces parties est présentée chronologiquement, le cheminement de l’information est précisé au long de la chaîne des transcripteurs (poissons, écailles, données) et certaines étapes clés sont précisées à titre d’illustration. Les contraintes supplémentaires engendrées par cette formalisation sont essentiellement dues au temps requis pour la rédaction des protocoles selon les normes de l’assurance qualité ; en revanche au cours de la réalisation de l’étude, cette démarche apporte un gain de temps et une amélioration de fiabilité des données en la sécurisant et en la standardisant. C’est également une étape indispensable pour envisager l’accréditation de laboratoire.

The epidemiology of animal trypanosomosis around Bobo-Dioulasso (Burkina Faso, West Africa) benefited a lot in the last years from the progress of molecular tools. The two most used molecular techniques were the polymerase chain reaction for the diagnosis of the disease in cattle and the characterization of the trypanosomes in the host and the vector on one hand, and the microsatellite DNA polymorphism in tsetse flies to study the intraspecific genetic variability of the vector on the other hand. The results obtained in the Sideradougou area during a recent two year survey with these techniques, associated with many other georeferenced informations concerning vector and cattle distribution, natural environment, landuse, ground occupation, livestock management, were combined in a Geographical Information System. This new approach of a complex pathogenic system led to a better evaluation of the risk of trypanosome transmission.

En mars 2021, Montpellier Université d’excellence (MUSE) et Agropolis International ont réuni des décideurs, acteurs opérationnels, représentants d’organisations internationales et scientifiques pour partager leurs expériences des approches intégrées en santé dans le cadre du concept One Health. Ces échanges intersectoriels fructueux ont croisé les points de vue et ont pointé les obstacles et les enjeux de la mise en œuvre concrète de ces approches. Les participants ont abouti à des propositions pour rendre plus efficace cette mise en œuvre et faire face aux menaces sanitaires émergentes et aux futures pandémies : coconstruire des projets intégrés avec tous les acteurs concernés (citoyens, décideurs politiques, chercheurs, services locaux) ; développer des méthodes d’évaluation de leurs impacts ; former les acteurs ; institutionnaliser et coordonner les actions du niveau local au niveau mondial.

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.

Competing Interests: Declaration of interests All authors are members of the French COVID-19 Scientific Council. J-FD is the President of the Council.

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., Competing Interests: The authors have declared that no competing interests exist.

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.

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 e x pression r egulator). 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.

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.

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.

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.

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.

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.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Introduction: Congenital portosystemic shunts (CPS) are rare vascular malformations that lead to liver metabolic modifications. The aim of this study was to describe the management and outcome of CPS in children who were followed in the western region of France and to compare our experience to a literature review., Patients and Methods: This retrospective study reports nine children with CPS who were followed in the western region of France between 2008 and 2013., Results: Among these nine children, six were detected by prenatal ultrasonography, one had cholestatic jaundice during the postoperative period, one had cholestatic jaundice during the first days of life, and one had dyspnea and fatigue. Two children had liver tumors. The CPS was intrahepatic for four children and extrahepatic for two children. Three others had a patent ductus venosus. Among the intrahepatic shunts that were discovered before the age of 2 years, three closed spontaneously. The five other shunts were closed surgically or by interventional radiology techniques, after angiography for four children. After closure of eight shunts, the outcome was favorable for six children. Two children presented complications after surgery: portal hypertension and portal thrombus., Conclusion: When CPS is suspected, the diagnosis should be confirmed and complications should be investigated. Shunt closure restores portal flow and prevents complications., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)