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When studying a vector-borne disease, an eco-epidemiological approach is vital for a comprehensive understanding of how the pathogen circulates amongst populations. Equine piroplasmosis (EP), a tick-borne disease caused by the protozoans Babesia caballi and Theileria equi, is endemic in the Mediterranean basin of Europe and causes both animal health and economic issues for the equine sector. With no vaccine available, defining the episystem of the disease can help to identify which components of the host-pathogen-vector-environment system to target to improve preventive measures. In this systematic literature review, we collected relevant data on the eco-epidemiology of EP in Europe. The 62 studies remaining after the selection procedure explored potential vectors, indicators of parasite circulation and putative risk factors of EP. Eight hard tick species were identified as potential vectors of one or both piroplasm species. Meta-analyses were then conducted on prevalence and seroprevalence data in equids in European countries, demonstrating an estimated seroprevalence of 30% and 8% and prevalence of 25% and 2% for T. equi and B. caballi, respectively. Finally, herd management practices and environmental risk factors analysed in studies showed no real consensus between studies, but revealed a general trend highlighting age and exposure to ticks as risk factors, and vaccination as a protective factor. Through this study, we point out that only a few studies have focused on disease management practices and even fewer have studied the effect of environmental parameters on equid infections. Further investigation in these areas is required to better characterise the eco-epidemiology of EP and risk factors associated with this disease. This article is protected by copyright. All rights reserved.

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.

This review was conducted by the ad hoc working expert group “ASF vectors” of the French Agency for Food, Environmental, and Occupational Health and Safety (Anses). Opinion 2019-SA-0028.; International audience; African swine fever (ASF) is one of the most important diseases in Suidae due to its significant health and socioeconomic consequences and represents a major threat to the European pig industry, especially in the absence of any available treatment or vaccine. In fact, with its high mortality rate and the subsequent trade restrictions imposed on affected countries, ASF can dramatically disrupt the pig industry in afflicted countries. In September 2018, ASF was unexpectedly identified in wild boars from southern Belgium in the province of Luxembourg, not far from the Franco-Belgian border. The French authorities rapidly commissioned an expert opinion on the risk of ASF introduction and dissemination into metropolitan France. In Europe, the main transmission routes of the virus comprise direct contact between infected and susceptible animals and indirect transmission through contaminated material or feed. However, the seasonality of the disease in some pig farms in Baltic countries, including outbreaks in farms with high biosecurity levels, have led to questions on the possible involvement of arthropods in the transmission of the virus. This review explores the current body of knowledge on the most common arthropod families present in metropolitan France. We examine their potential role in spreading ASF—by active biological or mechanical transmission or by passive transport or ingestion—in relation to their bio-ecological properties. It also highlights the existence of significant gaps in our knowledge on vector ecology in domestic and wild boar environments and in vector competence for ASFV transmission. Filling these gaps is essential to further understanding ASF transmission in order to thus implement appropriate management measures.

Purpose: One of the main limitations to anticancer radiotherapy lies in irreversible damage to healthy tissues located within the radiation field. “FLASH” irradiation at very high dose-rate is a new treatment modality that has been reported to specifically spare normal tissue from late radiation-induced toxicity in animal models and therefore could be a promising strategy to reduce treatment toxicity. Experimental Design: Lung responses to FLASH irradiation were investigated by qPCR, single-cell RNA sequencing (sc-RNA-Seq), and histologic methods during the acute wound healing phase as well as at late stages using C57BL/6J wild-type and Terc−/− mice exposed to bilateral thorax irradiation as well as human lung cells grown in vitro. Results: In vitro studies gave evidence of a reduced level of DNA damage and induced lethality at the advantage of FLASH. In mouse lung, sc-RNA-seq and the monitoring of proliferating cells revealed that FLASH minimized the induction of proinflammatory genes and reduced the proliferation of progenitor cells after injury. At late stages, FLASH-irradiated lungs presented less persistent DNA damage and senescent cells than after CONV exposure, suggesting a higher potential for lung regeneration with FLASH. Consistent with this hypothesis, the beneficial effect of FLASH was lost in Terc−/− mice harboring critically short telomeres and lack of telomerase activity. Conclusions: The results suggest that, compared with conventional radiotherapy, FLASH minimizes DNA damage in normal cells, spares lung progenitor cells from excessive damage, and reduces the risk of replicative senescence.

Ticks are important vectors of pathogens affecting human and animal health around the world. They act as vectors of several pathogens causing diseases of major concern including Lyme borreliosis, anaplasmosis, rickettsiosis, ehrlichiosis, tularemia, and tick-borne encephalitis (TBE) in humans, and babesiosis, theileriosis, and anaplasmosis in livestock. Therefore, a One Health approach is important for the effective prevention and control of tick-borne diseases (TBDs)., Editorial on the Research Topic.

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.

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.

International audience; Metallic nanoparticles have great potential in cancer radiotherapy as theranostic drugs since, they serve simultaneously as contrast agents for medical imaging and as radio-therapy sensitizers. As with other anticancer drugs, intratumoral diffusion is one of the main limiting factors for therapeutic efficiency. To date, a few reports have investigated the intratumoral distribution of metallic nanoparticles. The aim of this study was to determine the quantitative distribution of gadolinium (Gd) nanoparticles after direct intratumoral injection within U87 human glioblastoma tumors grafted in mice, using micro-PIXE (Particle Induced X-ray Emission) imaging. AGuIX (Activation and Guiding of Irradiation by X-ray) 3 nm particles composed of a polysiloxane network surrounded by gadolinium chelates were used. PIXE results indicate that the direct injection of Gd nanoparticles in tumors results in their heterogeneous diffusion, probably related to variations in tumor density. All tumor regions contain Gd, but with markedly different concentrations, with a more than 250-fold difference. Also Gd can diffuse to the healthy adjacent tissue. This study highlights the usefulness of mapping the distribution of metallic nanoparticles at the intratumoral level, and proposes PIXE as an imaging modality to probe the quantitative distribution of metallic nanoparticles in tumors from experimental animal models with micrometer resolution.

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.

International audience; Gametocytes are responsible for the transmission of malaria parasites, Plasmodium spp., from man to mosquito. Although transmission success, as measured by the proportion of mosquitoes infected, generally increases with gametocyte density, the proportion of parasites that are gametocytes is always paradoxically only a few percent of the asexual blood parasites. To address this paradox, we analyse transmission data sets from an urban and an adjacent rural setting in Cameroon to elucidate whether there are discernable lower and upper limits to Plasmodium falciparum gametocyte density that are linked to transmission success. We find that there exists a lower gametocyte density at which mosquito infection rates considerably increase. In addition, we identify upper gametocyte densities at which mosquito infection rates level off. Greatest increases in infection rates occur at low gametocyte densities and coincide with maximum oocyst aggregation within the infected mosquito population. This aggregated oocyst distribution remains despite increases in gametocyte density and ever-decreasing gains in mosquito infection rates. There is increasing suggestion that malaria parasites have evolved sex allocation strategies to ensure transmission in response to a changing, transmission-blocking environment. Here transmission-blocking immunity is proposed not only to ensure low density gametocyte transmission success but also to impose upper limits on transmission success.

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; Reservoir competence is a key parameter in understanding the role of host species in the epidemiology of multi-host-especially vector-borne-pathogens. With this aim in view, we studied the reservoir competence of the Siberian chipmunk (Tamias sibiricus barberi) recently introduced into Europe, for the multi-host tick-borne bacteria, Borrelia burgdorferi sl, the agent of Lyme borreliosis. T. sibiricus were experimentally exposed to bites from Ixodes ricinus ticks infected with Borrelia burgdorferi sensu stricto and Borrelia afzelii, with subsequent assessment of bacteremia and antibody responses. Borrelia was detected in chipmunk blood samples, ear biopsies and organ necropsies, and in nymphs used for xenodiagnosis (at one and six months after the initial chipmunk infection) via both serological and molecular methods. In total, eight out of twelve chipmunks showed evidence of infection by Borrelia sp., either by ELISA or PCR. Five chipmunks developed an immune response against the bacteria one month after infection. Borrelia infection in at least one organ was observed in seven animals at 14, 38, 93 or 178 days post-infection. Xenodiagnosis was positive for one chipmunk at 38 days, but no longer at 178 days post-infection. Four chipmunks remained uninfected, despite similar infection pressures to those observed in the field. Taken together, these results suggest that chipmunks can be infected through Borrelia-infected tick bites, and can transmit Borrelia to nymphs, but do not remain persistently infected.

Background Reliable information on host use by arthropod vectors is required to study pathogen transmission ecology and to predict disease risk. Direct observation of host use is often difficult or impossible and indirect methods are therefore necessary. However, the reliability of currently available methods to identify the last host of blood-feeding arthropods has not been evaluated, and may be particularly problematic for ticks because host blood has been digested at capture. Biases in host detection may lead to erroneous conclusions on both vector ecology and pathogen circulation. Methods Here, we experimentally tested for biases in host detection using the generalist three-host tick Ixodes ricinus as a model system. We fed ticks using an artificial feeding system and amplified blood meal traces post-moult (i.e., in the succeeding unfed life stage) via both a quantitative real-time polymerase chain reaction assay and a reverse line blotting method. We then experimentally tested for three types of biases in host detection: 1) time post-moult, 2) tick life stage and 3) host type (non-nucleated mammal blood versus nucleated avian blood), and compared these biases between the two molecular methods. Results Our results show that all three factors can influence host detection in ticks but not necessarily in the expected way. Although host detection rates decreased with time post-moult, mammal blood tended to be more readily detected than bird blood. Tick life stage was also an important factor; detection was higher in nymphs than in adults and, in some cases, remnants from both larval and nymphal blood meals could be detected in the adult stage. These biases were similar for the two detection techniques. Conclusions We show that different factors associated with questing ticks may influence our ability to correctly infer previous host use and that these factors may bias inferences from field-based studies. As these biases may be common to other vector-borne disease systems, their implications for our understanding of vector ecology and disease transmission require more explicit consideration. Electronic supplementary material The online version of this article (doi:10.1186/s13071-015-1043-7) contains supplementary material, which is available to authorized users.

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.

Plasmodium falciparum transmission blocking immunity (TBI) was investigated in 3 different endemic areas. Reared Anopheles gambiae s.s. were experimentally infected with the blood of gametocyte carriers, either in the presence of autologous plasma (OWN) or after replacement of the OWN plasma with a nonimmune serum of AB blood group (control). Transmission reduction was defined by a lower level of mosquito infection in the OWN batch compared with the control. After controlling for the effect of gametocytemia, the proportion of "transmission reducers" was lower in the town of Yaounde in Cameroon (UC), (14%, N = 75) than in the two rural areas of South Cameroon (RC) (29%, N = 31) and Sénégal (RS) (44%, N = 32). The contribution of TBI relative to the total inhibition of the parasite development (including human, parasite, and mosquito factors) was higher in RS (49.6%) than in RC (12.6%) and UC (9.5%).

Contains fulltext : 58663.pdf (Publisher’s version ) (Closed access) Transmission blocking immunity (TBI) was studied in relation to age, gametocyte density and transmission intensity. subjects with high gametocytaemias were selected in a hypo-endemic urban district and a hyper-endemic rural area in South Cameroon. TBI was determined in blood from gametocyte carriers in a bioassay (Direct Membrane Feeding Assay), with either autologous plasma (OWN) or control serum (AB). Mosquito infection rates (IR) were compared. infection rates correlated positively with gametocyte and oocyst densities. Three TBI indicators were analysed: the proportion of transmission reducers (IRAB > IROWN, P < 0.01), the mean intensity of TBI (IRAB - IROWN), and the contribution of TBI to total inhibition [(IRAB-IROWN)/(100-IROWN)]. we could not discriminate between areas with regard to either the proportion of transmission reducers (urban 15% and rural 29%) or the mean levels of TBI (urban 10% and rural 9%), or contribution of TBI to total inhibition (urban 10% and rural 13%). there was no relationship between TBI indicators and age, but a trend of increasing values was observed with rising gametocytaemia, which was considered as a confusing factor. a multivariable analysis showed that the probability of being a reducer was 4.6 fold higher in the rural area than in the urban district.

Tick-borne rickettsiae are considered to be emerging, but data about their presence in western Europe are scarce. Ixodes ricinus ticks, the most abundant and widespread tick species in western Europe, were collected and tested for the presence of several tick-borne pathogens in western France, a region never previously explored in this context. There was a high tick abundance with a mean of 4 females, 4.5 males, and 23.3 nymphs collected per hour per collector. Out of 622 tested ticks, specific PCR amplification showed the presence of tick symbionts as well as low prevalence of Borrelia burgdorferi (0.8%), Bartonella spp. (0.17%), and Anaplasma phagocytophilum (0.09%). The most prevalent pathogen was Rickettsia helvetica (4.17%). This is the first time that this bacteria has been detected in ticks in this region, and this result raises the possibility that bacteria other than those classically implicated may be involved in rickettsial diseases in western France., Author summary Due to socio-economical and meteorological changes, the geographical distribution of several tick species is changing. As ticks are the most important vectors of pathogens in northern latitudes, performing epidemiological studies is essential to assess the extent of tick activity as well as the risk of pathogens transmission. The detection of the bacteria Rickettsia helvetica in questing ticks in western France, for the first time, raises the possibility that bacteria other than those classically implicated may be involved in rickettsial diseases in this region.

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.

Experimental infections of laboratory-reared anopheline mosquitoes were carried out with 57 Plasmodium falciparum gametocyte carriers from Cameroon. Prevalence of infected mosquitoes and oocyst intensity were determined by two independent methods. Young P. falciparum oocysts were detected on day 2 after feeding using an immunofluorescent assay, and the results were compared with direct microscopic examination of midgut oocysts on day 7 postinfection. The immunofluorescent assay was based on a FITC-labeled anti-25-kDa monoclonal antibody, while the direct microscopy was performed on midguts stained with 2% mercurochrome. Young oocysts were easily detected by their typical and bright green-fluorescing Pfs25 positive coat and their characteristic pattern of pigment granules under transmitted white light examination. The agreement between the results of the two methods was assessed using the Kappa coefficient on prevalences of positive infections and the interclass correlation coefficient on arithmetic mean oocyst load per infected midgut. The results indicated a low agreement between the two methods for the comparison of prevalences of infected mosquitoes. However, this agreement was near perfect for the comparison of mean oocyst intensities. Prevalences of positive infections and the overall number of parasites per positive gut were significantly correlated for both methods. Thus, the immunofluorescent test could be an appropriate tool for early determination of malaria infection in mosquitoes, particularly under laboratory conditions. The possible applications of this immuno-fluorescent technique are discussed.

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; Babesiosis is a tick-transmitted disease of mammalian hosts, caused by the intraerythrocytic protozoan parasites of the genus Babesia. Transmission of Babesia parasites from the vertebrate host to the tick is mediated by sexual stages, the gametocytes which are the only intraerythrocytic stages that survive and develop inside the vector. Very few data are available concerning these parasite stages and some markers are needed in order to refine our knowledge of Babesia life cycle inside the tick and to permit the monitoring of parasite transmission from vertebrate to vector. We previously identified some potential markers of the Babesia divergens gametocytes using an in silico post-genomic approach based on sequence identity between the available genomes of Plasmodium and Babesia spp. Here, one of the identified proteins, BdCCp2, was validated as a marker of sexual stages of B. divergens, in infected ticks challenged with antisera directed against recombinant BdCCp2 protein. The BdCCp2 protein was detected by Western blot in some infected ticks, as a discrete band of approximately 171 kDa, while no signal was detected in the laboratory-reared non-infected tick. BdCCp2 was also detected, by immunohistochemical analyses, in piriform or ovoid bodies, measuring 2.5-4.5 μm in diameter, in the gut of partially engorged ticks that were experimentally infected. This molecular marker can then be used in the future to characterize and analyze the biology of B. divergens gametocytes.

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.

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

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.

Bartonella spp. are facultative intracellular vector-borne bacteria associated with several emerging diseases in humans and animals all over the world. The potential for involvement of ticks in transmission of Bartonella spp. has been heartily debated for many years. However, most of the data supporting bartonellae transmission by ticks come from molecular and serological epidemiological surveys in humans and animals providing only indirect evidences without a direct proof of tick vector competence for transmission of bartonellae. We used a murine model to assess the vector competence of Ixodes ricinus for Bartonella birtlesii. Larval and nymphal I. ricinus were fed on a B. birtlesii-infected mouse. The nymphs successfully transmitted B. birtlesii to naïve mice as bacteria were recovered from both the mouse blood and liver at seven and 16 days after tick bites. The female adults successfully emitted the bacteria into uninfected blood after three or more days of tick attachment, when fed via membrane feeding system. Histochemical staining showed the presence of bacteria in salivary glands and muscle tissues of partially engorged adult ticks, which had molted from the infected nymphs. These results confirm the vector competence of I. ricinus for B. birtlesii and represent the first in vivo demonstration of a Bartonella sp. transmission by ticks. Consequently, bartonelloses should be now included in the differential diagnosis for patients exposed to tick bites., Author Summary Bartonella spp. are bacteria that infect the red blood cells and that are associated with several diseases in humans and animals all over the world. They are transmitted by arthropod vectors including fleas, lice and sand-flies, but new potential vectors are suspected and in particular ticks. Diseases transmitted by ticks, currently in emergence, have diverse etiology (viral, bacterial, parasitic) and are responsible for high morbidity and mortality rates around the world. The potential for involvement of ticks in transmission of Bartonella spp. has been heartily debated for many years because of the numerous but indirect proofs of its existence. In this study, the authors used a murine model to assess the ability of the tick Ixodes ricinus to transmit Bartonella bacteria to mice. Results of the study confirm the vector competence of I. ricinus and represent the first in vivo demonstration of a Bartonella sp. transmission by ticks. Consequently, bartonelloses should be now included in the differential diagnosis for patients exposed to tick bites.

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.

Article Number 21; International audience; Babesia sp. (EU1), first characterized in 2003, has been implicated in human cases of babesiosis in Italy, Austria and Germany. It has been identified in roe deer and in its suspected tick vector, Ixodes ricinus, in several European countries. The aim of the present study was to validate the competence of I. ricinus as a vector of Babesia sp. (EU1) via experimental infections. For this purpose, a parasite strain isolated from roe deer was cloned in sheep erythrocytes. After experimental infections, parasite DNA was successfully amplified by PCR in both eggs and larvae originating from infected I. ricinus females and in the salivary glands of females exposed to Babesia sp. (EU1) as nymphs. We also demonstrate that infected females were able to transmit parasite DNA during a new blood meal. Together with previous epidemiological studies, these results validate I. ricinus as a competent vector for Babesia sp. (EU1).

International audience; Babesia, the causal agent of babesiosis, are tick-borne apicomplexan protozoa. True babesiae (Babesia genus sensu stricto) are biologically characterized by direct development in erythrocytes and by transovarial transmission in the tick. A large number of true Babesia species have been described in various vertebrate and tick hosts. This review presents the genus then discusses specific adaptations of Babesia spp. to their hosts to achieve efficient transmission. The main adaptations lead to long-lasting interactions which result in the induction of two reservoirs: in the vertebrate host during low long-term parasitemia and throughout the life cycle of the tick host as a result of transovarial and transstadial transmission. The molecular bases of these adaptations in vertebrate hosts are partially known but few of the tick-host interaction mechanisms have been elucidated.

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.

Contains fulltext : 57970.pdf (Publisher’s version ) (Closed access) Summary Quantitatively assessing the impact of naturally occurring transmission-blocking (TB) immunity on malaria parasite sporogonic development may provide a useful interpretation of the underlying mechanisms. Here, we compare the effects of plasma derived from 23 naturally infected gametocyte carriers (OWN) with plasma from donors without previous malaria exposure (AB) on the early sporogonic development of Plasmodium falciparum in Anopheles gambiae. Reduced parasite development efficiency was associated with mosquitoes taking a blood meal mixed with the gametocyte carriers' own plasma, whereas replacing autologous plasma with non-immune resulted in the highest level of parasite survival. Seven days after an infective blood meal, 39.1% of the gametocyte carriers' plasma tested showed TB activity as only a few macrogametocytes ingested along with immune plasma ended up as ookinetes but subsequent development was blocked in the presence of immune plasma. In other experiments (60.9%), the effective number of parasites declined dramatically from one developmental stage to the next, and resulted in an infection rate that was two-fold lower in OWN than in AB infection group. These findings are in agreement with those in other reports and go further by quantitatively examining at which transition stages TB immunity exerts its action. The transitions from macrogametocytes to gamete/zygote and from gamete/zygote to ookinete were identified as main targets. However, the net contribution of host plasma factors to these interstage parasite reductions was low (5-20%), suggesting that irrespective of the host plasma factors, mosquito factors might also lower the survival level of parasites during the early sporogonic development.

International audience; Malaria transmission from humans to mosquitoes was assessed in two neighbouring villages in a rural area near Yaoundé, Cameroon during high and low transmission seasons during 1998-2000, using several indices previously evaluated in different areas endemic for malaria but never directly compared. These indices were estimated from human parasitological data and mosquito infection rates and, for each individual, thick blood films were prepared at the same time as experimental infection of laboratory-bred mosquitoes. Among the 685 volunteers examined, the prevalence of Plasmodium falciparum gametocyte carriers was 16%, and 8% of individuals were able to infect mosquitoes. The percentage of mosquitoes that became infected by feeding on the infectious individuals was 21%. Children aged < 10 years contributed to about 75% of the infectious reservoir, although they constituted only 35% of the total population. Differences were found between the transmission seasons and the villages, and varied according to the index examined. Although there were more infectious individuals in one of the two villages, they were less infectious than those in the other village during the high transmission season. Comparative analysis of the transmission indices suggests the existence of functioning transmission-blocking immunity in one of the villages, which until now has been only hypothetically considered to play a role in malaria transmission in a natural setting. The epidemiological value of all the indices used and their accuracy in estimating the human infectious reservoir and its natural or induced variations are discussed.

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.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., Ixodes ricinus is the most widespread and abundant tick in Europe, frequently bites humans, and is the vector of several pathogens including those responsible for Lyme disease, Tick-Borne Encephalitis, anaplasmosis, babesiosis and bartonellosis. These tick-borne pathogens are transmitted to vertebrate hosts via tick saliva during blood feeding, and tick salivary gland (SG) factors are likely implicated in transmission. In order to identify such tick factors, we characterized the transcriptome of female I. ricinus SGs using next generation sequencing techniques, and compared transcriptomes between Bartonella henselae-infected and non-infected ticks. High-throughput sequencing of I. ricinus SG transcriptomes led to the generation of 24,539 isotigs. Among them, 829 and 517 transcripts were either significantly up- or down-regulated respectively, in response to bacterial infection. Searches based on sequence identity showed that among the differentially expressed transcripts, 161 transcripts corresponded to nine groups of previously annotated tick SG gene families, while the others corresponded to genes of unknown function. Expression patterns of five selected genes belonging to the BPTI/Kunitz family of serine protease inhibitors, the tick salivary peptide group 1 protein, the salp15 super-family, and the arthropod defensin family, were validated by qRT-PCR. IrSPI, a member of the BPTI/Kunitz family of serine protease inhibitors, showed the highest up-regulation in SGs in response to Bartonella infection. IrSPI silencing impaired tick feeding, as well as resulted in reduced bacterial load in tick SGs. This study provides a comprehensive analysis of I. ricinus SG transcriptome and contributes significant genomic information about this important disease vector. This in-depth knowledge will enable a better understanding of the molecular interactions between ticks and tick-borne pathogens, and identifies IrSPI, a candidate to study now in detail to estimate its potentialities as vaccine against the ticks and the pathogens they transmit., XYL was supported by funds from the China Scholarship Council (CSC). This work was funded by INRA and EU grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext037 (http://www.edenext.eu). This work was also partially supported by the Spanish Secretaría de Estado de Investigación, Desarrollo e Innovación, Ministerio de Economía y Competitividad project BFU2011-23896.

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.

Human infectiousness to mosquitoes can be estimated by 2 tests : direct feeding on the skin and membrane feeding on venous blood. To validate the membrane feeding assay, the infectiousness of #Plasmodium falciparum$ gametocyte carriers to #Anopheles gambiae$ was estimated by these 2 methods in the same individuals in a rural area of Cameroon. Results from 37 experiments showed that direct feeding gave significantly higher infection rates than membrane feeding. We observed an average of 19.4% infected mosquitoes by direct feeding compared with 12.1% by membrane feeding, and a mean oocyst load of 5.63 by direct feeding compared with 2.65 by membrane feeding. However, there was a very good concordance between the 2 tests : 84.3% with the Kappa test on percentages of infected mosquitoes and 98.7% with the interclass correlation coefficient on oocyst loads. In addition, we found a good linear correlation between the 2 methods. (Résumé d'auteur)

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.

http://web.oie.int/boutique/index.php?page=ficprod&id_prec=1407&id_produit=1516&lang=fr&fichrech=1; International audience; Vector-borne diseases are principally zoonotic diseases transmitted to humans by animals. Pathogens such as bacteria, parasites and viruses are primarily maintained within an enzootic cycle between populations of non-human primates or other mammals and largely non-anthropophilic vectors. This 'wild' cycle sometimes spills over in the form of occasional infections of humans and domestic animals. Lifestyle changes, incursions by humans into natural habitats and changes in agropastoral practices create opportunities that make the borders between wildlife and humans more permeable. Some vector-borne diseases have dispensed with the need for amplification in wild or domestic animals and they can now be directly transmitted to humans. This applies to some viruses (dengue and chikungunya) that have caused major epidemics. Bacteria of the genus Bartonella have reduced their transmission cycle to the minimum, with humans acting as reservoir, amplifier and disseminator. The design of control strategies for vector-borne diseases should be guided by research into emergence mechanisms in order to understand how a wild cycle can produce a pathogen that goes on to cause devastating urban epidemics.