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This assessment was conducted by the ad hoc working group ‘ASF vectors’ of the French Agency for Food, Environmental and Occupational Health & Safety (Anses).; International audience; African swine fever (ASF) represents a global threat with huge economic consequences for the swine industry. Even though direct contact is likely to be the main transmission route from infected to susceptible hosts, recent epidemiological investigations have raised questions regarding the role of haematophagous arthropods, in particular the stable fly (Stomoxys calcitrans). In this study, we developed a mechanistic vector-borne transmission model for ASF virus (ASFV) within an outdoor domestic pig farm in order to assess the relative contribution of stable flies to the spread of the virus. The model was fitted to the ecology of the vector, its blood-feeding behaviour and pig-to-pig transmission dynamic. Model outputs suggested that in a context of low abundance (

This assessment was conducted by the ad hoc working group ‘ASF vectors' of the French Agency for Food, Environmental and Occupational Health & Safety (Anses).; International audience; To deal with the limited literature data on the vectorial capacity of blood-feeding arthropods (BFAs) and their role in the transmission of African swine fever virus (ASFV) in Metropolitan France, a dedicated working group of the French Agency for Food, Environmental and Occupational Health & Safety performed an expert knowledge elicitation. In total, 15 different BFAs were selected as potential vectors by the ad hoc working group involved. Ten criteria were considered to define the vectorial capacity: vectorial competence, current abundance, expected temporal abundance, spatial distribution, longevity, biting rate, active dispersal capacity, trophic preferences forSuidae, probability of contact with domestic pigs and probability of contact with wild boar. Fourteen experts participated to the elicitation. For each BFA, experts proposed a score (between 0 and 3) for each of the above criteria with an index of uncertainty (between 1 and 4). Overall, all experts gave a weight for all criteria (by distributing 100 marbles). A global weighted sum of score per BFA was calculated permitting to rank the different BFAs in decreasing order. Finally, a regression tree analysis was used to group those BFAs with comparable likelihood to play a role in ASF transmission. Out of the ten considered criteria, the experts indicated vectorial competence, abundance and biting rate as the most important criteria. In the context of Metropolitan France, the stable fly (Stomoxys calcitrans) was ranked as the most probable BFA to be a vector of ASFV, followed by lice (Haematopinus suis), mosquitoes (Aedes, Culex and Anopheles),Culicoides and Tabanidea. Since scientific knowledge on their vectorial competence for ASF is scarce and associated uncertainty on expert elicitation moderate to high, more studies are however requested to investigate the potential vector role of these BFAs could have in ASFV spread, starting with Stomoxys calcitrans.

International audience; The recent development of high-throughput NGS technologies, (ie, next-generation sequencing) has highlighted the complexity of tick microbial communities—which include pathogens, symbionts, and commensals—and also their dynamic variability. Symbionts and commensals can confer crucial and diverse benefits to their hosts, playing nutritional roles or affecting fitness, development, nutrition, reproduction, defence against environmental stress and immunity. Nonpathogenic tick bacteria may also play a role in modifying tick-borne pathogen colonization and transmission, as relationships between microorganisms existing together in one environment can be competitive, exclusive, facilitating or absent, with many potential implications for both human and animal health. Consequently, ticks represent a compelling yet challenging system in which to investigate the composition and both the functional and ecological implications of tick bacterial communities, and thus merits greater attention. Ultimately, deciphering the relationships between microorganisms carried by ticks as well as symbiont‑tick interactions will garner invaluable information, which may aid in some future arthropod‑pest and vector-borne pathogen transmission control strategies. This review outlines recent research on tick microbiome composition and dynamics, highlights elements favouring the reciprocal influence of the tick microbiome and tick-borne agents and finally discusses how ticks and tick-borne diseases might potentially be controlled through tick microbiome manipulation in the future.

Background. Assessment of the age-dependent cancer risk conferred by germline predicted pathogenic variants (PPV) in cancer susceptibility genes is often hampered by the way the data are collected. Cohort-based data frequently contain an overrepresentation of patients carrying a gene variant of interest and an underrepresentation of cancer-free gene variant carriers. In order to overcome this problem, penetrance estimates can be grounded on family-based study designs, through the evaluation of index patients (IP) and their family relatives. The purpose of the TUMOSPEC study is to estimate the penetrance of PPV in genes whose literature data are currently inaccurate or limited and to determine their associated tumour spectrum. This will lead to an appropriate assessment of the clinical utility of testing these genes. Methods. IP are enrolled consecutively among patients who are being offered a germline genetic test in a hereditary breast and ovary cancer (HBOC) context in a participating cancer genetics clinics. A panel of 24 genes (ATM, BAP1, BARD1, BRIP1, CHEK2, FANCM, FAM175A, MRE11A, NBN, STK11, RINT1, XRCC2, PALB2, MLH1, MSH2, MSH6, PMS2, CDH1, PTEN, RAD50, RAD51B, RAD51C, RAD51D, TP53) is tested in parallel of BRCA1 and BRCA2. If a PPV is identified, the IP is asked to give her/his first- and second-degree relatives and cousins address, regardless of their health status. Each relative is then contacted by the study coordinating centre (CC) to be invited. Each participant completes an epidemiological questionnaire addressing personal medical history and exposure to various risk factors, and provides a saliva sample to determine if he/she carries the familial PPV. The CC collects questionnaires, family history, clinical and genetic data. Results. Enrolment for the feasibility study takes place between Sep. 2017 and Dec. 2019 at each of the 46 participating french clinics. In June 2019, the study included 3,298 IP. So far, the CC received 1,241 TUMOSPEC panel results (37.6%) with a mean delay of 5 months [0.7 -17.5]. Among them, 169 carried a PPV in at least one of the genes (totalizing 183 PPV). Additionally, 44 IP with PPV identified beforehand were enrolled (table 1). Relatives’ invitation of these 213 IP began in June 2018. Among the relatives of the first 29 index cases contacted so far, 4.6 relatives per family consented to participate. Conclusions. The feasibility study showed that inclusion process is well adapted and that the communication between the various partners (clinicians, biologists, investigators and study participants) is quite smooth. Rates of inclusion for invited relatives (50%), for IP questionnaire completion (45%) and relatives biological sample collection (50%) are also very satisfactory and yet underestimated due to the recent start of relatives inclusions. Overall, this study shows that it is feasible to conduct a large-scale study to gather sufficient number of positive families for each gene included in the panel in a reasonable interval of time. This on-going national effort will allow to appropriately assess cancer risks cancer in families with a PPV in a gene often included in HBOC multi-gene panels. This is an essential step to optimize clinical management guidelines specific to each gene, and will represent a valuable resource for future research on the genetics of breast and ovary cancers. Number of PPV identified in IPGeneATMCHEK2PALB2RAD51CBRIP1RAD51DMSH6RAD50PMS2MRE11ANBNMSH2MLH1CDH1BARD1TP53STK11XRCC2RINT1BAP1RAD51BFAM175AFANCMPTENNumber of PPV512019161111111010999865544322110 Citation Format: Olivier Caron, Séverine Eon-Marchais, Sarah Bonnet-Boissinot, Juana Beauvallet, Marie-Gabrielle Dondon, Chrystelle Colas, Florence Coulet, Capucine Delnatte, Claude Houdayer, Christine Lasset, Jérôme Lemonnier, Michel Longy, Catherine Nogues, Dominique Stoppa-Lyonnet, Dominique Vaur, Fabienne Lesueur, Nadine Andrieu, TUMOSPEC Investigators Group, UNICANCER Groupe Génétique et Cancer. Feasibility of a nation-wide family-based study to assess cancer risks in families with a predicted pathogenic variant identified through hereditary breast and ovary multi-gene panel testing: The TUMOSPEC study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-08-12.

Ticks are the most important vectors of pathogens affecting both domestic and wild animals worldwide. Hard tick feeding is a slow process&mdash, taking up to several days&mdash, and necessitates extended control over the host response. The success of the feeding process depends upon injection of tick saliva, which not only controls host hemostasis and wound healing, but also subverts the host immune response to avoid tick rejection that creates a favorable niche for the survival and propagation of diverse tick-borne pathogens. Here, we report on the molecular and biochemical features and functions of an Ixodes ricinus serine protease inhibitor (IrSPI). We characterize IrSPI as a Kunitz elastase inhibitor that is overexpressed in several tick organs&mdash, especially salivary glands&mdash, during blood-feeding. We also demonstrated that when IrSPI is injected into the host through saliva, it had no impact on tissue factor pathway-induced coagulation, fibrinolysis, endothelial cell angiogenesis or apoptosis, but the protein exhibits immunomodulatory activity. In particular, IrSPI represses proliferation of CD4+ T lymphocytes and proinflammatory cytokine secretion from both splenocytes and macrophages. Our study contributes valuable knowledge to tick-host interactions and provides insights that could be further exploited to design anti-tick vaccines targeting this immunomodulator implicated in I. ricinus feeding.

Objectives Antibiotics for febrile neutropenia (FN) in acute myeloid leukaemia (AML) patients undergoing intensive chemotherapy are usually maintained until neutropenia resolution, because of the risk of uncontrolled sepsis in this vulnerable population. This leads to unnecessarily prolonged antimicrobial therapy. Methods Based on ECIL-4 recommendations, we modified our management strategy and discontinued antibiotics after a pre-established duration in patients treated for a first episode of FN between August 2014 and October 2017. Results Antibiotics were stopped during 62 FN episodes, and maintained in the control group (n = 13). Median age of patients was 54 years. A total of 39 (63%) patients received induction and 23 (37%) consolidation chemotherapy; 36 (58%) patients had fever of unknown origin. Median neutropenia length was 26 days (IQR 24–30). Antibiotics were started at day 9 (IQR 5–13). Most patients received piperacillin-tazobactam (56%) or cefepime (32%). Antimicrobial therapy was longer in the control group than in the policy compliant group, 10 (IQR 7–16) vs. 19 days (IQR 15–23), P = 0.0001. After antibiotics discontinuation, 20% patients experienced fever recurrence, within 5.5 days (IQR 3–7.5). None of these febrile episodes were severe and 80% patients remained afebrile, with neutrophil recovery occurring within 5 days (IQR 2–8.5). Overall, 287 antibiotics days were spared; this represents 49% of all days with antibiotics. No patient had died at day 30 from intervention; six died during late follow-up, two from graft-versus-host disease and four from relapsed or refractory leukaemia. Conclusions Discontinuing antibiotics in neutropenic AML patients treated for a first episode of FN is safe, and results in significant antibiotic sparing.

International audience; Ticks and the pathogens they transmit constitute a growing burden for human and animal health worldwide. In the last years, high-throughput detection and sequencing technologies (HTT) have revealed that individual ticks carry a high diversity of microorganisms, including pathogenic and non-pathogenic bacteria. Despite several studies have contributed to the availability of a catalog of microorganisms associated to different tick species, major limitations and challenges remain ahead HTT studies to acquire further insights on the microbial complexity associated to ticks. Currently, using next generation sequencing (NGS), bacteria genera (or higher taxonomic levels) can be recorded; however, species identification remains problematic which in turn affects pathogen detection using NGS. Microfluidic PCR, a high-throughput detection technology, can detect up to 96 different pathogen species, and its combination with NGS might render interesting insights into pathogen-microbiota co-occurrence patterns. Microfluidic PCR, however, is also limited because detection of pathogen strains has not been implemented, and therefore, putative associations among bacterial genotypes are currently unknown. Combining NGS and microfluidic PCR data may prove challenging. Here, we review the impact of some HTT applied to tick microbiology research and propose network analysis as an integrative data analysis benchmark to unravel the structure and significance of microbial communities associated to ticks in different ecosystems.

Etude des mécanismes moléculaires de la compétence vectorielle d’I. ricinus pour 2 flavivirus. Journées du Réseau "Tiques et Maladies à Tiques"

International audience

Ticks are major vectors of pathogens affecting both humans and animals worldwide and surpass all other arthropods in the variety of microorganisms—including viruses, bacteria, and parasites (protozoa and helminths)—they are able to transmit. They are strict hematophagous arthropods and pool feeders, ingesting all the fluids that are exuded into the wound generated by the bite. For most tick-borne pathogens (TBP), transmission occurs via the saliva injected alternately with blood uptake during the tick bite. This saliva is secreted by the tick salivary glands, which are implicated in several other functions that are essential to tick biology and development. Saliva is an essential bio-fluid for ticks, which dampens host defenses and facilitates the flow of blood to assure adequate feeding. It exhibits cytolytic, vasodilator, anticoagulant, anti-inflammatory, and immunosuppressive activities that, in addition, to underpinning blood feeding, provide a favorable context for transmission, survival, and propagation of TBP.

International audience; The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the laboratory. In ticks, all developmental stages (except egg) are hematophagous, and acquiring a blood-meal when attached to their vertebrate hosts is essential for the successful completion of their life cycle. Here we demonstrate a simple method that uses easily openable capsules for feeding of hard ticks on rabbits. The advantages of the proposed method include its simplicity, short duration and most importantly versatile adjustment to the needs of specific experimental requirements. The method makes possible the use of multiple chambers (of various sizes) on the same animal, which permits feeding of multiple stages or different experimental groups while reducing the overall animal requirement. The non-irritating and easily accessible materials used minimizes discomfort to the animals, which can be easily recovered from an experiment and offered for adoption or reused if the ethical protocol allows it.

Broad-spectrum vaccine against tick-borne pathogens. Ticks, and in particular Ixodes ricinus, transmit diverse pathogens of major concern to human and animal health and represent the most important vectors in Europe. Current strategies of control and prevention are essentially based on the deployment of chemical acaricides, which cause harm to off-target species and contaminate the environment and foodstuffs, and generate selection of resistant strains of ticks. Vaccines are available for only a handful of tick-borne pathogens and are thus largely inadequate in view of the vast diversity of pathogenic agents that may be transmitted by ticks, and the risk that new tick-borne pathogens will emerge. Thus, new strategies compatible with values of sustainable development and capable of addressing pathogen diversity and even anticipating pathogen emergence must be sought. The development of vaccines that target the tick itself is thus an appealing means of seeking broad-spectrum protection against tick-borne diseases., En Europe, les tiques représentent les premiers vecteurs de maladies animales et humaines. À l’heure actuelle, la stratégie de lutte contre les tiques est essentiellement fondée sur l’utilisation d’acaricides qui sont polluants pour l’environnement et les produits issus de l’élevage, atteignent la faune non ciblée, et dont l’utilisation génère des résistances. Très peu de vaccins existent contre les agents pathogènes transmis par les tiques. De plus, ils ne représentent pas une solution réaliste pour protéger hommes et animaux compte tenu de la très grande diversité des agents transmis par ces vecteurs et de la possibilité d’émergence de nouveaux micro-organismes pathogènes. Il est donc urgent de développer de nouvelles stratégies de lutte contre les tiques. Dans cette optique, la vaccination visant le vecteur, et non pas chaque micro-organisme individuellement, représente une alternative prometteuse., Bonnet Sarah, Richardson Jennifer. La vaccination anti-tiques comme outil de prévention contre les multiples agents qu’elles transmettent. In: Bulletin de l'Académie Vétérinaire de France tome 171 n°1, 2018. pp. 14-19.

Ticks and the pathogens they transmit constitute a growing burden for human and animal health worldwide. Vector competence is a component of vectorial capacity and depends on genetic determinants affecting the ability of a vector to transmit a pathogen. These determinants affect traits such as tick-host-pathogen and susceptibility to pathogen infection. Therefore, the elucidation of the mechanisms involved in tick-pathogen interactions that affect vector competence is essential for the identification of molecular drivers for tick-borne diseases. In this review, we provide a comprehensive overview of tick-pathogen molecular interactions for bacteria, viruses, and protozoa affecting human and animal health. Additionally, the impact of tick microbiome on these interactions was considered. Results show that different pathogens evolved similar strategies such as manipulation of the immune response to infect vectors and facilitate multiplication and transmission. Furthermore, some of these strategies may be used by pathogens to infect both tick and mammalian hosts. Identification of interactions that promote tick survival, spread, and pathogen transmission provides the opportunity to disrupt these interactions and lead to a reduction in tick burden and the prevalence of tick-borne diseases. Targeting some of the similar mechanisms used by the pathogens for infection and transmission by ticks may assist in development of preventative strategies against multiple tick-borne diseases., Part of the research included in this review was supported by the Ministerio de Economia y Competitividad (Spain) grant BFU2016-79892-P and the European Union (EU) Seventh Framework Programme (FP7) ANTIGONE project number 278976. SA and AD would like to acknowledge FCT for funds to GHTM - UID/Multi/04413/2013. MV was supported by the Research Plan of the University of Castilla-La Mancha (UCLM), Spain.

National audience

Insular environments provide ideal natural conditions to study disease ecology, especially emerging diseases, due to clear differentiation between local and long-distance transmission. Such environments are of particular interest regarding tick-borne pathogens (TBP), since animal exchange with the mainland (along with any ticks they carry) is limited, and because such locations could lie on migratory routes for birds carrying ticks. Therefore both tick species and TBP may display different prevalence than those observed on the continent. As such, an epidemiological survey was performed on Belle-Ile-en-Mer, an island off the coast of Western France, in order to estimate the prevalence of tick species and the microorganisms they carried. Three tick species, Dermacentor marginatus, D. reticulatus, and Haemaphysalis punctata were collected at five different sites in 2010 and 2011. All ticks were tested for pathogen's and symbiont's DNA by (i) PCR for Anaplasma spp., Borrelia spp., Rickettsia spp.; (ii) real-time PCR for Francisella tularensis, Francisella-like endosymbionts (FLE) and Coxiella spp. and (iii) PCR-RLB for Babesia-Theileria spp. Pathogen DNA detected in D. marginatus including Borrelia spp. (18%), Rickettsia spp. (13%) which was identified as R. slovaca, Babesia spp. (8%), and Theileria spp. (1%). Pathogens detected in D. reticulatus including Rickettsia spp. (31%) identified as R. raoulti, Francisella-like endosymbiont (86%), and Babesia spp (21%). Pathogens detected in H. punctata including Rickettsia spp. (1%) identified as R. aeschlimannii, FLE (0.4%), Babesia spp. (18%), and Theileria spp. (7%). Anaplasma spp., F. tularensis, or Coxiella spp. were not detected in any of the collected ticks. This study represents the first epidemiological survey of the insular Belle-Ile-en-Mer environment. It demonstrated the presence of expected pathogens, consistent with reports from island veterinarians or physicians, as well as unexpected pathogens, raising questions about their potential introduction through infected animals and/or their dispersion by migratory birds.

International audience; In Europe, Ixodes ricinus ticks are vectors of many emerging pathogens, including Borrelia burgdorferi sensu lato (sl), Anaplasma phagocytophilum, spotted fever group Rickettsia sp., Babesia sp., and very likely Bartonella sp. In this study, we looked for the presence of DNA of these microorganisms in 572 ticks from two forests in the west of France. DNA extraction and polymerase chain reaction (PCR) amplification were performed on individual nymphal, male, and female I. ricinus ticks. Amplification from 1 tick among the 572 samples (0.2%) resulted in PCR products with Bartonella-specific primers. Sequence analysis of the amplified fragment did not lead to species identification. Two ticks (0.3%) carried A. phagocytophilum–specific DNA. Eight ticks (1.4%) were positive with spotted fever group Rickettsia–specific primers, and all PCR fragments were related to Rickettsia helvetica. Thirty-five ticks (6.1%) were positive with B. burgdorferi sl–specific primers; the sequences were all related to Borrelia garinii or Borrelia afzelii, except one that was related to Borrelia carolinensis, a newly described species never reported in Europe so far. Thirty-five ticks (6.1%) carried Babesia sp. DNA. Female adults were more infected by B. burgdorferi sl than male adults. The prevalence of B. burgdorferi sl and Babesia sp. was significantly different between the two forests, with a higher prevalence of B. burgdorferi sl in ticks from the forest of Princé and a higher prevalence of Babesia sp. in ticks from the forest of Gâvre. To our knowledge, this is the first study that has detected all five pathogens in questing I. ricinus in the west of France and the first report of B. carolinensis DNA in ticks in Europe.

Background Worldwide changes in socio-economic and environmental factors and the global climate are recognised causes of variation in tick distribution and density. Thus it is of great importance that new studies address the changing risk of infection for exposed populations. In Europe, Ixodes ricinus ticks are the most common vectors of several pathogens impacting veterinary and public health that have colonised suburban habitats. Methods This study aimed to evaluate longitudinal I. ricinus questing densities and infection rates over 7 years in a French suburban forested area with high human population density. Ticks were collected in spring yearly between 2008 and 2014 and, out of a total of 8594 collected I. ricinus, a representative subset of adult females (n = 259) were individually examined for the presence of several pathogens via PCR. Results Nymph densities peaked in 2009–2011, and then declined in 2012–2014. Changes in monthly temperature only had a modest impact on this variation. In contrast, analysis revealed a complex intra-annual relationship between mean nymph density and both concurrent and lagged mean monthly temperatures. The following pathogens were detected in the studied area: Anaplasma phagocytophilum, Rickettsia helvetica, Babesia venatorum and B. divergens, Francisella tularensis, Borrelia miyamotoi, B. afzelii/valaisiana, B. garinii/lusitaniae and Bartonella spp. Conclusion Our findings reinforce the conclusion that ticks are important vectors of pathogenic microorganisms in suburban forests and suggest that despite complex intra-annual relationships between tick densities and temperature, there is no evidence for a climate-associated increase in infection risk over the 7-year period. Rather, tick densities are likely to be strongly influenced by population density fluctuations in vertebrate host species and wildlife management. Further detailed studies on the impact of climate change on tick population densities are required. Electronic supplementary material The online version of this article (doi:10.1186/s13071-016-1591-5) contains supplementary material, which is available to authorized users.

International audience

AlthoughBabesia divergensis the the principal confirmed zoonoticBabesia sp. in Europe, there are gaps in our knowledge of its biology and transmission by the tickIxodes ricinus. In order to reproduce the part of the parasite cycle that occurs in the vector, anin vitroanimal skin feeding technique on blood containingin vitrocultivatedB. divergenswas developed. Parasite DNA was detected in all samples of salivary glands of nymphs and adults that had fed on parasitized blood as larvae and nymphs, respectively, indicating acquisition as well as a transtadial persistence ofB. divergens. PCR performed on eggs and larvae produced by females that had fed on parasitized blood demonstrated the existence of a transovarial transmission of the parasite. GorgingB. divergensinfected larvae on non-infected gerbils showed persistance of the parasite over moulting into the resulting nymphs. These results indicate that the parasitic stages infective for the vector (i.e. the sexual stages) can be producedin vitro. To our knowledge, this is the first report of artificial feeding ofI. ricinusvia membrane as well asin vitrotransmission ofB. divergensto its vector. The opportunities offered by the use of such a transmission model of a pathogen byI. ricinusare discussed.

International audience; Recombinant homologues of the Plasmodium merozoite surface protein 1 C-terminus are leading blood stage malaria vaccine candidates. MSP1 is anchored to the merozoite plasma membrane in vivo by a glycosyl-phosphatidyl-inositol (GPI) moiety, implicated in malaria pathology. Two types of recombinant Plasmodium falciparum MSP1p19 (PfMSP1p19) expressed in baculovirus/insect cells are described here: (1) a soluble, secreted form (PfMSP1p19S) and (2) detergent soluble cellular form(s) (PfMSP1p19 + A), released from the infected cell surface by treatment with GPI specific phosphatidyl-inositol phospholipase C (PI-PLC). Soluble and cellular PfMSP1p19 were purified and characterized using SDS-PAGE, mass spectrometry (MS), N-terminal amino acid sequencing, gel filtration and glycan analyses. Quantitative inositol dosage suggested that surface GPI processed entities constituted only 14% of the purified cellular PfMSP1p19 + A, with GPI unprocessed forms likely recovered in the endoplasmic reticulum. Nevertheless, this preparation has dramatic immuno-stimulatory activity to be described elsewhere. The interest of these results for both malaria specific and generic vaccine development are discussed.

International audience; Tick-borne diseases represent major public and animal health issues worldwide. Ixodes ricinus, primarily associated with deciduous and mixed forests, is the principal vector of causative agents of viral, bacterial, and protozoan zoonotic diseases in Europe. Recently, abundant tick populations have been observed in European urban green areas, which are of public health relevance due to the exposure of humans and domesticated animals to potentially infected ticks. In urban habitats, small and medium-sized mammals, birds, companion animals (dogs and cats), and larger mammals (roe deer and wild boar) play a role in maintenance of tick populations and as reservoirs of tick-borne pathogens. Presence of ticks infected with tick-borne encephalitis virus and high prevalence of ticks infected with Borrelia burgdorferi s.l., causing Lyme borreliosis, have been reported from urbanized areas in Europe. Emerging pathogens, including bacteria of the order Rickettsiales (Anaplasma phagocytophilum, "Candidatus Neoehrlichia mikurensis," Rickettsia helvetica, and R. monacensis), Borrelia miyamotoi, and protozoans (Babesia divergens, B. venatorum, and B. microti) have also been detected in urban tick populations. Understanding the ecology of ticks and their associations with hosts in a European urbanized environment is crucial to quantify parameters necessary for risk pre-assessment and identification of public health strategies for control and prevention of tick-borne diseases.

International audience; Ticks are the most common arthropod vector, after mosquitoes, and are capable of transmitting the greatest variety of pathogens. For both humans and animals, the worldwide emergence or re-emergence of tick-borne disease is becoming increasingly problematic. Despite being such an important issue, our knowledge of pathogen transmission by ticks is incomplete. Several recent studies, reviewed here, have reported that the expression of some tick factors can be modulated in response to pathogen infection, and that some of these factors can impact on the pathogenic life cycle. Delineating the specific tick factors required for tick-borne pathogen transmission should lead to new strategies in the disruption of pathogen life cycles to combat emerging tick-borne disease.

Background Risk assessment of tick-borne and zoonotic disease emergence necessitates sound knowledge of the particular microorganisms circulating within the communities of these major vectors. Assessment of pathogens carried by wild ticks must be performed without a priori, to allow for the detection of new or unexpected agents. Methodology/Principal Findings We evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce an inventory of parasites carried by questing ticks. Sequences corresponding to parasites from two distinct genera were recovered in Ixodes ricinus ticks collected in Eastern France: Babesia spp. and Theileria spp. Four Babesia species were identified, three of which were zoonotic: B. divergens, Babesia sp. EU1 and B. microti; and one which infects cattle, B. major. This is the first time that these last two species have been identified in France. This approach also identified new sequences corresponding to as-yet unknown organisms similar to tropical Theileria species. Conclusions/Significance Our findings demonstrate the capability of NGS to produce an inventory of live tick-borne parasites, which could potentially be transmitted by the ticks, and uncovers unexpected parasites in Western Europe., Author Summary Diseases transmitted by ticks have diverse etiology (viral, bacterial, parasitic) and are responsible for high morbidity and mortality rates around the world, both in humans and animals. The emergence or re-emergence of tick-borne diseases is increasingly becoming a problem as the geographical distribution of several tick species is expanding, as well as the numbers of potential or known tick-borne pathogens are constantly evolving. It is thus necessary to know which microorganisms circulate within communities of this major vector to ensure adequate epidemiological surveillance. In this study, we evaluated the potential of Next-Generation Sequencing techniques (NGS) to produce, without a priori, an inventory of both predicted and non-expected parasites carried by Ixodes ricinus, the most prevalent human biting tick in France. Our findings suggest that NGS strategies could be used to produce an inventory of live parasites residing in ticks from a selected area, thereby expanding our knowledge base of tick-associated parasites.

International audience; Artificial membrane feeding systems are effective tools for both tick rearing and studying tick-borne pathogen transmission. In order to compare the effects of the type of feeding system on tick engorgement, Ixodes ricinus ticks were either fed on an artificial membrane feeding system or on live mice. Sheep and chicken blood were used with the membrane system to assess the effects of blood origin on tick engorgement. To investigate the effects of blood meal infection on tick engorgement, ticks were either fed with Bartonella-infected or uninfected blood, both via membrane feeding and on mice. The proportion of engorged ticks, the duration of tick feeding, and the weight of engorged ticks were assessed. Feeding on the artificial system led to a longer duration of tick feeding and a lower proportion of engorged ticks than when fed on mice, however, the weight of engorged ticks was unaffected. The proportion and weight of engorged ticks, as well as the duration of feeding were not affected by blood origin. Feeding on an infected blood meal or on infected mice decreased the proportion and the weight of engorged ticks, but did not affect tick feeding duration. (C) 2014 Elsevier GmbH. All rights reserved.

International audience; Francisella tularensis, the causative agent of tularemia, is commonly transmitted by ticks. To ensure accurate F. tularensis reporting rates in epidemiological surveys, specific discrimination between F. tularensis and Francisella-like tick endosymbionts (FLEs) is absolutely critical. Four molecular available techniques capable of distinguishing Francisella spp. were compared here for the first time in French Dermacentor reticulatus ticks in order to estimate their specificity as well as their ease and speed of use. Results showed that tul4 and fopA real-time PCR assays can easily and effectively discriminate between F. tularensis and FLEs in D. reticulatus. In addition, a high prevalence of FLEs in D. reticulatus collected in France was reported by the use of fopA real-time PCR assay (79%). Finally, phylogenetic analysis showed that FLEs isolated from D. reticulatus correspond to a well-defined group compared to FLEs originating from various tick species. (C) 2013 Elsevier B.V. All rights reserved.

International audience; Each Bartonella species appears to be highly adapted to one or a limited number of reservoir hosts, in which it establishes long-lasting intraerythrocytic bacteremia as the hallmark of infection. Recently, we identified Trw as the bacterial system involved in recognition of erythrocytes according to their animal origin. The T4SS Trw is characterized by a multiprotein complex that spans the inner and outer bacterial membranes, and possesses a hypothetical pilus structure. TrwJ, I, H and trwL are present in variable copy numbers in different species and the multiple copies of trwL and trwJ in the Bartonella trw locus are considered to encode variant forms of surface-exposed pilus components. We therefore aimed to identify which of the candidate Trw pilus components were located on the bacterial surface and involved in adhesion to erythrocytes, together with their erythrocytic receptor. Using different technologies (electron microscopy, phage display, invasion inhibition assay, far western blot), we found that only TrwJ1 and TrwJ2 were expressed and localized at the cell surface of B. birtlesii and had the ability to bind to mouse erythrocytes, and that their receptor was band3, one of the major outer-membrane glycoproteins of erythrocytes, (anion exchanger). According to these results, we propose that the interaction between TrwJ1, TrwJ2 and band 3 leads to the critical host-specific adherence of Bartonella to its host cells, erythrocytes.

Despite its importance, our knowledge of pathogen transmission by ticks is incomplete. Detailed studies on the transmission, maintenance, infectivity, virulence, and pathogenicity of tick-borne microparasites all require the use of large numbers of live ticks raised under controlled conditions and difficulties in rearing ticks in the laboratory could partly explain the current lack of data. The most complex part in maintaining tick colonies doubtlessly lies in their engorgement, as ticks are strict haematophagous arthropods. Indeed, relatively few research teams have worked on artificial feeding systems for ticks due to the long, complex, and poorly understood feeding patterns of these arthropods. It is nonetheless essential to investigate the mechanisms underlying tick infection and infectiousness in order to better understand parasite-host-vector relationships and elaborate new control strategies for transmitted pathogens. The various methods used to date to feed ticks and infect them with their associated pathogens are reviewed here and their advantages and inconveniences are discussed.

Contains fulltext : 108727.pdf (Publisher’s version ) (Open Access) INTRODUCTION: In the era of malaria elimination and eradication, drug-based and vaccine-based approaches to reduce malaria transmission are receiving greater attention. Such interventions require assays that reliably measure the transmission of Plasmodium from humans to Anopheles mosquitoes. METHODS: WE COMPARED TWO COMMONLY USED MOSQUITO FEEDING ASSAY PROCEDURES: direct skin feeding assays and membrane feeding assays. Three conditions under which membrane feeding assays are performed were examined: assays with i) whole blood, ii) blood pellets resuspended with autologous plasma of the gametocyte carrier, and iii) blood pellets resuspended with heterologous control serum. RESULTS: 930 transmission experiments from Cameroon, The Gambia, Mali and Senegal were included in the analyses. Direct skin feeding assays resulted in higher mosquito infection rates compared to membrane feeding assays (odds ratio 2.39, 95% confidence interval 1.94-2.95) with evident heterogeneity between studies. Mosquito infection rates in membrane feeding assays and direct skin feeding assays were strongly correlated (p

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International audience; The role of Ixodes ricinus ticks in the transmission of pathogens of public health importance such as Borrelia burgdorferi s.l. is widely recognized and is suspected in several emerging vector-borne pathogens in Europe. Here, we assess prevalence rates of several endemic and emerging zoonotic pathogens in tick populations in an area of high human population density in France, to contribute to a risk assessment for potential transmission to humans. Pathogen prevalence rates were evaluated by polymerase chain reaction detection and sequencing in questing ticks, individually for adults and in pools of 10 for nymphs. In addition to finding micro-organisms corresponding to symbionts, we found high prevalence rates of B. burgdorferi s.l. (32% of adult females and 10% of nymphs) and low to moderate ones of Anaplasma phagocytophilum (*1%), spotted fever group Rickettsia spp.(*6%), Babesia sp. EU1 (*1%), Bartonella birtlesii (0.1%), and Francisella tularensis (*1%). Our findings extend the knowledge of the geographical distribution of these endemic and emergent pathogens and support the conclusion that ticks are important vectors of pathogenic micro-organisms in suburban forests. Moreover, tick coinfection with multiple pathogens was found to occur frequently, which poses a serious challenge for diagnosis and appropriate treatment. The incrimination of these pathogens in potentially severe pathologies requires widespread surveillance to assess the risk of infection, thereby facilitating diagnosis and treatment, as well as raising local awareness of tick-borne diseases.

International audience; Babesia divergens, a tick-borne protozoan parasite of red blood cells, is the main agent of bovine and human babesiosis in Europe. Very few data are available concerning its life cycle and sexual reproduction inside the tick vector, Ixodes ricinus. The aim of this study was to define some markers of the B. divergens sexual stage. An in silico post-genomic approach was used to analyze genomic, transcriptomic and proteomic data and to select specific sexual stage proteins of the related apicomplexan genus Plasmodium. Three proteins, based on sequence identity between the available genomes of Plasmodium and Babesia spp., were chosen, as members of a highly conserved and apicomplexan sexual stages specific protein family (CCp) potentially involved in adhesive functions. Degenerate primers were used to amplify and clone three B. divergens orthologs (bdccp1, bdccp2, and bdccp3) corresponding to newly identified genes in this parasite. The opportunities offered by such markers to study parasite development in its vector are discussed. (C) 2010 Elsevier B.V. All rights reserved.

The genus Bartonella comprises a unique group of emerging gram-negative, intracellular bacteria that can cause a long-lasting intraerythrocytic bacteremia in their reservoir hosts. In recent years, the widespread occurrence and diversity of these bacteria has been increasingly recognized. This has resulted in a dramatic expansion of the genus Bartonella to 24 currently described species or subspecies, among which at least half have been associated with human disease. Bartonella infections have been observed in virtually all species examined, extending from humans to carnivores, ungulates, rodents, lagomorphs, insectivores, and bats. Adaptation by Bartonellae to such a diverse range of mammals has resulted in host specificity, and all validated Bartonella species described to date are capable of parasitizing only a limited number of animal species. In this review, the possible mechanisms explaining the specificity of each Bartonella species for its reservoir host are discussed.

Anopheles gambiae is the major African vector of Plasmodium falciparum , the most deadly species of human malaria parasite and the most prevalent in Africa. Several strategies are being developed to limit the global impact of malaria via reducing transmission rates, among which are transmission-blocking vaccines (TBVs), which induce in the vertebrate host the production of antibodies that inhibit parasite development in the mosquito midgut. So far, the most promising components of a TBV are parasite-derived antigens, although targeting critical mosquito components might also successfully block development of the parasite in its vector. We previously identified A. gambiae genes whose expression was modified in P. falciparum -infected mosquitoes, including one midgut carboxypeptidase gene, cpbAg1. Here we show that P. falciparum up-regulates the expression of cpbAg1 and of a second midgut carboxypeptidase gene, cpbAg2 , and that this up-regulation correlates with an increased carboxypeptidase B (CPB) activity at a time when parasites establish infection in the mosquito midgut. The addition of antibodies directed against CPBAg1 to a P. falciparum -containing blood meal inhibited CPB activity and blocked parasite development in the mosquito midgut. Furthermore, the development of the rodent parasite Plasmodium berghei was significantly reduced in mosquitoes fed on infected mice that had been immunized with recombinant CPBAg1. Lastly, mosquitoes fed on anti-CPBAg1 antibodies exhibited reduced reproductive capacity, a secondary effect of a CPB-based TBV that could likely contribute to reducing Plasmodium transmission. These results indicate that A. gambiae CPBs could constitute targets for a TBV that is based upon mosquito molecules.

Background Despite a long history of attempts to model malaria epidemiology, the over-riding conclusion is that a detailed understanding of host-parasite interactions leading to immunity is required. It is still not known what governs the duration of an infection and how within-human parasite dynamics relate to malaria epidemiology. Presentation of the hypothesis Immunity to Plasmodium falciparum develops slowly and requires repeated exposure to the parasite, which thus generates age-structure in the host-parasite interaction. An age-structured degree of immunity would present the parasite with humans of highly variable quality. Evolutionary theory suggests that natural selection will mould adaptive phenotypes that are more precise (less variant) in "high quality" habitats, where lifetime reproductive success is best. Variability in malaria parasite gametocyte density is predicted to be less variable in those age groups who best infect mosquitoes. Thus, the extent to which variation in gametocyte density is a simple parasite phenotype reflecting the complex within-host parasite dynamics is addressed. Testing the hypothesis Gametocyte densities and corresponding infectiousness to mosquitoes from published data sets and studies in both rural and urban Cameroon are analysed. The mean and variation in gametocyte density according to age group are considered and compared with transmission success (proportion of mosquitoes infected). Across a wide range of settings endemic for malaria, the age group that infected most mosquitoes had the least variation in gametocyte density, i.e. there was a significant relationship between the variance rather than the mean gametocyte density and age-specific parasite transmission success. In these settings, the acquisition of immunity over time was evident as a decrease in asexual parasite densities with age. By contrast, in an urban setting, there were no such age-structured relationships either with variation in gametocyte density or asexual parasite density. Implications of the hypothesis Gametocyte production is seemingly predicted by evolutionary theory, insofar as a reproductive phenotype (gametocyte density) is most precisely expressed (i.e. is most invariant) in the most infectious human age group. This human age group would thus be expected to be the habitat most suitable for the parasite. Comprehension of the immuno-epidemiology of malaria, a requisite for any vaccine strategies, remains poor. Immunological characterization of the human population stratified by parasite gametocyte allocation would be a step forward in identifying the salient immunological pathways of what makes a human a good habitat.

International audience; We previously used differential display to identify several Anopheles gambiae genes, whose expression in the mosquito midgut was regulated upon ingestion of Plasmodium falciparum. Here, we report the characterization of one of these genes, cpbAg1 , which codes for the first zinc-carboxypeptidase B identified in An. gambiae and in any insect. Expression of cpbAg1 in baculovirus gave rise to an active enzyme, and determination of the N-terminal amino acids confirmed that CPBAg1 contains a signal peptide and a pro-peptide, typical features of digestive zinc carboxypeptidases. cpbAg1 mRNA was mainly produced in the mosquito midgut, where it accumulated in unfed females and was rapidly down-regulated upon blood feeding. Annotation of the An. gambiae genome predicts twentythree sequences coding for zinc-carboxypeptidases of which only two (cpbAg1 and cpbAg2) are expressed at a significant level in the mosquito midgut.

To the Editor: The Ixodes ricinus tick is a predominant vector of a large variety of pathogens of veterinary and medical consequence in Europe (1). The most prevalent I. ricinus–borne infection of persons in Europe is Lyme borreliosis, a multisystemic disorder caused by spirochetes of the Borrelia burgdorferi sensu lato complex (2). Persons bitten by ticks can also become infected with many other pathogens, such as bacteria (Anaplasma spp., Francisella spp., Coxiella burnetii, Bartonella spp., Rickettsiae spp., and Neoerhlichia mikurensis); parasites (Babesia spp., Theileria spp.); and arboviruses (tick-borne encephalitis virus, Crimean-Congo hemorrhagic fever virus, and Eyach virus) (1). Symptoms induced by such pathogens are often diverse and nonspecific, complicating accurate diagnosis of the disease. In Romania, cases of Lyme disease and tick-borne encephalitis (caused by tick-borne encephalitis virus) have been identified (3). However, little is known about the public health impact of these diseases, and none of the other tick-borne pathogens present in Europe have been reported as causes of infection in Romania. Although I. ricinus is the most abundant and widespread tick in Romania (3), the public health impact of I. ricinus–borne disease is likely to be underestimated. Therefore, the first step in evaluating the distribution of these potential pathogens is to establish their presence in ticks from previously unexplored areas. We conducted a study to identify the main tick-borne bacterial and parasitic human pathogens known to be present in Europe but not previously detected in Romania. We tested for the presence of DNA from spotted fever group Rickettsia spp., Anaplasma spp., Francisella tularensis, and Babesia spp. in 147 I. ricinus ticks collected from roe deer and goats at 2 sites in eastern Romania: Bacau (46°35′0″N, 26°55′0″E) and Galati (45°26′22″N, 28°2′4″E). Specimens were tested by PCR, using specific primers for each pathogen or group of pathogens, as described (4). Sequences obtained from Eurofins MWG Operon (Ebersberg, Germany) were identified by using BLAST (www.ncbi.nlm.nih.gov/BLAST) and compared with sequences available in GenBank. DNA from Rickettsia spp. was detected in 20 (13.6%) ticks. Sequence analyses revealed that 9 (6.1%) sequences were related to the R. monacensis strain IRd/Serbia glta gene (99%–100% nt similarity) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GQ925820","term_id":"262181579","term_text":"GQ925820"}}GQ925820) and 11 (7.48%) were related to R. helvetica gltA gene (99%–100% nt similarity) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AM418450","term_id":"118918333","term_text":"AM418450"}}AM418450). DNA from Anaplasma spp. was identified in 33 (22.4%) ticks. Analysis revealed that 30 of the 33 amplified fragments showed 100% identity to the 16S rDNA gene of a symbiont in the family Anaplasmataceae, Candidatus Midichloria mitochondrii (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU780455","term_id":"190607147","term_text":"EU780455"}}EU780455), and the remaining 3 were related to known pathogenic species identified in Romania: 2 (1.4%) exhibited 100% identity to Anaplasma phagocytophilum (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"EU982548","term_id":"197360744","term_text":"EU982548"}}EU982548), and 1 (0.7%) showed 99% similarity to Ehrlichia muris (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU358691","term_id":"284975291","term_text":"GU358691"}}GU358691). Francisella tularensis–specific DNA was amplified from 4 DNA extracts (2.7%). All 4 sequences were identical and shared 99% similarity with the F. tularensis peptidyl-propyl cis-trans isomerase gene (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"CP003048","term_id":"377828067","term_text":"CP003048"}}CP003048). Babesia spp.–specific DNA was amplified in 1 DNA extract (0.7%), and it shared 99% sequence identity with the Babesia sp. EU1 18S rDNA gene (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"HQ830266","term_id":"317383279","term_text":"HQ830266"}}HQ830266). This is a report on the identification of the human pathogens R. monacensis, R. helvetica, A. phagocytophilum, E. muris, and Babesia EU1 in Romania. R. monacensis (also known as the Cadiz agent or Rickettsia IRS3 and IRS4) was first identified in I. ricinus tick samples from Germany (5) and was recently recognized as the cause of Mediterranean spotted fever-like rickettsiosis in Spain (6). R. helvetica was isolated from I. ricinus ticks in Switzerland in 1979, and, since then, it has been isolated in many other European countries (7). R. helvetica was associated with human infections in the late 1990s (8). The role of animals as reservoirs for these pathogens is unknown. A. phagocytophilum is the causative agent of granulocytic anaplasmosis in humans, cattle, horses, and dogs, and is widespread throughout Europe (8). E. muris has been detected in ticks from Finland and European Russia and from ticks and rodents from Slovakia. In 2009, E. muris was also detected in patients with febrile illness in the United States (9). Babesia sp. EU1 has been detected in roe deer and ticks in several countries, and in 2003 was associated with human disease in Italy and Austria (4,10). Tularemia is known to be present in Romania and is thought to be exclusively and directly transmitted by hares. However, detection of F. tularensis DNA in I. ricinus ticks suggests that this bacterium might also be tick-borne. In conclusion, the detection of DNA of various human pathogens in ticks in Romania strongly suggests that these microorganisms circulate in the country. Because all of these pathogens affect humans, our study highlights the urgent requirement for further research to assess their impact on public health in Romania.

International audience; Understanding the interactions between the most deadly malaria parasite, Plasmodium falciparum, and its main vector, Anopheles gambiae, would be of great help in developing new malaria control strategies. The malaria parasite undergoes several developmental transitions in the mosquito midgut and suffers population losses to which mosquito factors presumably contribute. To identify such factors, we analysed An. gambiae midgut transcripts whose expression is regulated upon ingestion of invasive or non-invasive forms of P. falciparum using a differential display approach. Sixteen cDNA were studied in detail; 12 represent novel genes of An. gambiae including a gene encoding profilin. Four transcripts were specifically regulated by P. falciparum gametocytes (invasive forms), whereas the others were regulated by either non-invasive or both non-invasive and invasive forms of the parasite. This differential regulation of some genes may reflect the adaptation of P. falciparum to its natural vector. These genes may be involved in the development of P. falciparum in An. gambiae or in the defence reaction of the mosquito midgut towards the parasite.

A new method for specific reamplification of DDRT-PCR products is presented. After transient ligation of the primary DDRT-PCR fragments into a T-vector, the cDNAs of interest were reamplified by hemi-nested PCR and thermally asymmetric cycles. In contrast to the originally described protocol, this method of reamplification is specific, sensitive, reproducibly gives a high yield of DNA and allows direct sequencing of the reamplified product without purification or cloning. We used the differential display technique to investigate the effect of a blood meal on the expression of Anopheles gambiae midgutspecific genes and thereby identify genes potentially involved in the sporogonic cycle of the human malaria parasite Plasmodium falciparum. The differential display technique (DDRT-PCR) (1‐3) is a method that can be used for analysis of changes in gene expression by detecting differential production of mRNA and which requires a small amount of RNA. This method has been used successfully to identify a variety of differentially expressed genes (2,4‐6). However, it has some drawbacks such as the large number of false positives generated, and different modifications of the original protocol have been proposed to limit the production of false positives or to facilitate the identification of putative positives (2,7‐10). Another disadvantage is the difficulty in selectively re-amplifying the target cDNA, which is a necessary step in verifying the expression pattern of the DDRT-PCR band ( 8,11). The original procedure for reamplification resulted, in our hands, in poor yields even after two rounds of re-amplification which in most cases generated non-specific PCR products. Furthermore, such direct reamplification often led to the production of fragments which contained the same arbitrary upstream primer at both ends ( 8,9 and unpublished results). These products have no T-tail and may correspond either to mRNA or to contaminating DNA. To circumvent these problems, we developed a procedure for reamplification at high annealing temperature using a transient ligation step (11) and a modified oligo-dT anchored primer (DDT3). To increase the specificity and the yield of the reaction, three hemi-nested reactions are performed with thermal asymmetric cycles (12). Total RNA was isolated from mosquito midguts dissected prior to

We report in vitro culture of zoonotic Babesia sp. EU1 from blood samples of roe deer in France. This study provides evidence of transovarial and transstadial transmission of the parasite within Ixodes ricinus, which suggests that this tick could be a vector and reservoir of EU1.