Prevalence of tick-borne pathogens in ticks collected from the wild mountain ungulates mouflon and chamois in 4 regions of France.

Ticks are major vectors of various pathogens of health importance, such as bacteria, viruses and parasites. The problems associated with ticks and vector-borne pathogens are increasing in mountain areas, particularly in connection with global climate change. We collected ticks (n = 2,081) from chamois and mouflon in 4 mountainous areas of France. We identified 6 tick species: Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata and Dermacentor marginatus. We observed a strong variation in tick species composition among the study sites, linked in particular to the climate of the sites. We then analysed 791 ticks for DNA of vector-borne pathogens: Babesia/Theileria spp., Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, and Rickettsia of the spotted fever group (SFG). Theileria ovis was detected only in Corsica in Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) and Anaplasma phagocytophilum (3 sites) were detected in I. ricinus. Anaplasma ovis was detected at one site in I. ricinus and Rh. sanguineus s.l. SFG Rickettsia were detected at all the study sites: R. monacensis and R. helvetica in I. ricinus at the 3 sites where this tick is present; R. massiliae in Rh. sanguineus s.l. (1 site); and R. hoogstraalii and Candidatus R. barbariae in Rh. bursa in Corsica. These results show that there is a risk of tick-borne diseases for humans and domestic and wild animals frequenting these mountain areas.

Title: Prévalence d'agents pathogènes vectorisés chez des tiques collectées chez des ongulés sauvages (mouflons, chamois) dans 4 zones montagneuses en France. Abstract: Les tiques sont des vecteurs majeurs de différents agents pathogènes d'importance sanitaire, tels que des bactéries, des virus et des parasites. Les problématiques liées aux tiques et aux pathogènes vectorisés augmentent en zones de montagne, en lien notamment avec le réchauffement climatique. Nous avons collecté des tiques (n = 2 081) sur des chamois et des mouflons dans 4 zones montagneuses en France. Six espèces ont été identifiées : Ixodes ricinus, Rhipicephalus bursa, Rh. sanguineus s.l., Haemaphysalis sulcata, H. punctata et Dermacentor marginatus. Nous avons observé une forte variation de la composition en espèces de tiques entre les sites d'étude, en lien notamment avec le climat des sites. Nous avons ensuite recherché les ADN d'agents pathogènes vectorisés sur 791 tiques : Babesia/Theileria spp, Borrelia burgdorferi s.l., Anaplasma phagocytophilum, A. marginale, A. ovis, et de Rickettsia du groupe des fièvres boutonneuses (SFG). Theileria ovis a été détecté uniquement en Corse chez Rh. bursa. Babesia venatorum (2 sites), Borrelia burgdorferi s.l. (B. afzelii and B. garinii; 2 sites) et Anaplasma phagocytophilum (3 sites) ont été détectés chez I. ricinus. Anaplasma ovis a été détecté dans un site chez I. ricinus et Rh. sanguineus s.l.. Les Rickettsia SFG ont été détectées dans tous les sites d'étude : Rickettsia monacensis et R. helvetica chez I. ricinus dans les 3 sites où cette tique est présente; R. massiliae chez Rh. sanguineus s.l. (1 site); et R. hoogstraalii et Candidatus R. barbariae chez Rh. bursa en Corse. Ces résultats montrent un risque de transmission de maladies par les tiques pour les personnes et les animaux domestiques et sauvages fréquentant ces zones de montagne.

Red deer (Cervus elaphus) Did Not Play the Role of Maintenance Host for Bluetongue Virus in France: The Burden of Proof by Long-Term Wildlife Monitoring and Culicoides Snapshots.

Bluetongue virus (BTV) is a Culicoides-borne pathogen infecting both domestic and wild ruminants. In Europe, the Red Deer (Cervus elaphus) (RD) is considered a potential BTV reservoir, but persistent sylvatic cycle has not yet been demonstrated. In this paper, we explored the dynamics of BTV1 and BTV8 serotypes in the RD in France, and the potential role of that species in the re-emergence of BTV8 in livestock by 2015 (i.e., 5 years after the former last domestic cases). We performed 8 years of longitudinal monitoring (2008-2015) among 15 RD populations and 3065 individuals. We compared Culicoides communities and feeding habits within domestic and wild animal environments (51,380 samples). Culicoides diversity (>30 species) varied between them, but bridge-species able to feed on both wild and domestic hosts were abundant in both situations. Despite the presence of competent vectors in natural environments, BTV1 and BTV8 strains never spread in RD along the green corridors out of the domestic outbreak range. Decreasing antibody trends with no PCR results two years after the last domestic outbreak suggests that seropositive young RD were not recently infected but carried maternal antibodies. We conclude that RD did not play a role in spreading or maintaining BTV in France.

Detection of tick-borne pathogens in questing Ixodes ricinus in the French Pyrenees and first identification of Rickettsia monacensis in France.

Ticks are important vectors of several human and animal pathogens. In this study, we estimated the prevalence of important tick-borne infections in questing ticks from an area in Southwestern France (Hautes-Pyrénées) inhabited by Pyrenean chamois (Rupicapra pyrenaica pyrenaica) experiencing high tick burden. We examined adult and nymph ticks collected by the flag dragging method from 8 to 15 sites in the Pic de Bazès during the years 2009, 2011, 2013 and 2015. PCR assays were conducted on selected ticks for the detection of Borrelia burgdorferi s.l., Babesia spp., Rickettsia spp., spotted fever group (SFG) Rickettsia and Anaplasma phagocytophilum. Randomly selected positive samples were submitted for sequence analysis. A total of 1971 questing ticks were collected including 95 males, 101 females and 1775 nymphs. All collected ticks were identified as Ixodes ricinus. Among them, 696 ticks were selected for pathogen detection and overall prevalence was 8.4% for B. burgdorferi s.l.; 0.4% for Babesia spp.; 6.1% for A. phagocytophilum; 17.6% for Rickettsia spp.; and 8.1% for SFG Rickettsia. Among the sequenced pathogens, we detected in this population of ticks the presence of Babesia sp. EU1 and Rickettsia helvetica, as well as Rickettsia monacensis for the first time in France. The detection of these pathogens in the Pic de Bazès highlights the potential infection risks for visitors to this area and the Pyrenean chamois population.

TITLE: Détection d'agents pathogènes présents chez les tiques errantes, Ixodes ricinus, dans les Pyrénées françaises et première identification de Rickettsia monacensis en France. ABSTRACT: Les tiques sont des vecteurs importants de plusieurs maladies animales et humaines. Dans cette étude, nous avons estimé la prévalence de maladies vectorisées chez les tiques errantes dans une région du sud-ouest de la France (Hautes-Pyrénées) occupée par des chamois des Pyrénées (Rupicapra pyrenaica pyrenaica) fortement infestés par les tiques. Nous avons utilisé des tiques adultes et des larves collectées par la technique du drapeau sur 8­15 sites du Pic de Bazès au cours des années 2009, 2011, 2013 et 2015. Des analyses PCR ont été menées sur des tiques sélectionnées pour la détection de Borrelia burgdorferi s.l., Babesia spp., Rickettsia spp., Rickettsia du groupe de la fièvre pourprée (GFP) et Anaplasma phagocytophilum. Des échantillons positifs sélectionnés au hasard ont été soumis à une analyse de séquences. Un total de 1971 tiques a été collecté dont 95 mâles, 101 femelles et 1775 larves. Toutes les tiques recueillies ont été identifiées comme étant Ixodes ricinus. Parmi celles-ci, 696 tiques ont été sélectionnées pour la détection des agents pathogènes et la prévalence globale était de 8,4 % pour B. burgdorferi s.l., 0,4 % pour Babesia spp., 6,1 % pour A. phagocytophilum, 17,6 % pour Rickettsia spp. et 8,1 % pour Rickettsia GFP. Parmi les agents pathogènes séquencés, nous avons détecté dans cette population de tiques la présence de Babesia sp. EU1, Rickettsia helvetica et, pour la première fois en France, Rickettsia monacensis. La détection de ces agents pathogènes dans le Pic de Bazès souligne les risques potentiels d'infection pour les visiteurs de cette région et pour la population de chamois des Pyrénées.

Assessing a multiplex-targeted proteomics approach for the clinical diagnosis of periodontitis using saliva samples.

AIM: The present study focused on the research of new biomarkers based on the liquid chromatography-multiple-reaction monitoring (MRM) proteomic profile in whole saliva of patients with periodontitis compared with periodontal healthy patients. METHODS: A 30-min multiplexed liquid chromatography-MRM method was used for absolute quantification of 35 plasma biomarkers in saliva from control patients and patients with periodontitis. RESULTS: Three proteins namely hemopexin, plasminogen and α-fibrinogen were shown to be clearly related to the presence of periodontitis compared with healthy patients. Apolipoprotein H was found to discriminate for the first time chronic and aggressive periodontitis. CONCLUSION: Our results indicate that this innovative MRM method could be used to screen for periodontitis in clinical environment. Furthermore, apolipoprotein H was found to be a discriminant biomarker of aggressive periodontitis.

Self-clearance of Pestivirus in a Pyrenean Chamois ( Rupicapra pyrenaica) Population.

Understanding the dynamics of host-pathogen interaction is key to the management of epidemics. A pestivirus belonging to the border disease virus group 4 emerged around 2001 in Pyrenean chamois ( Rupicapra pyrenaica) in Spain and France. The virus had significant demographic impact in some populations, but it was less harmful and more endemic in other places. The determinants of these local variations are still unclear. Here, we documented empirical evidence of self-clearance of the virus in a chamois population in France. This population has regularly been counted, and chamois were trapped and harvested each year, providing unique demographic and epidemiologic surveys of the population since 1984 and 1994, respectively. The virus was detected using direct (PCR) and indirect (antibody) testing. We showed that virus transmission declined in 2011-12 and likely ceased in 2013, leading to a decline in antibody prevalence since 2014. Self-clearance may be due to limited exchanges with other populations, decrease in population size after an epizootic, and herd immunity. The age structure of captured animals shifted to younger age classes after virus self-clearance, suggesting a return to a colonizing population structure. The possible consequences of virus re-entry are discussed. This observation suggests that pestivirus dynamics occurs at the scale of the metapopulation of Pyrenean chamois. Local self-clearance and re-emergence may help explain the variation of virus dynamics at the local scale.

Pseudomonas aeruginosa and Achromobacter sp. clonal selection leads to successive waves of contamination of water in dental care units.

Dental care unit waterlines (DCUWs) consist of complex networks of thin tubes that facilitate the formation of microbial biofilms. Due to the predilection toward a wet environment, strong adhesion, biofilm formation, and resistance to biocides, Pseudomonas aeruginosa, a major human opportunistic pathogen, is adapted to DCUW colonization. Other nonfermentative Gram-negative bacilli, such as members of the genus Achromobacter, are emerging pathogens found in water networks. We reported the 6.5-year dynamics of bacterial contamination of waterlines in a dental health care center with 61 dental care units (DCUs) connected to the same water supply system. The conditions allowed the selection and the emergence of clones of Achromobacter sp. and P. aeruginosa characterized by multilocus sequence typing, multiplex repetitive elements-based PCR, and restriction fragment length polymorphism in pulsed-field gel electrophoresis, biofilm formation, and antimicrobial susceptibility. One clone of P. aeruginosa and 2 clones of Achromobacter sp. colonized successively all of the DCUWs: the last colonization by P. aeruginosa ST309 led to the closing of the dental care center. Successive dominance of species and clones was linked to biocide treatments. Achromobacter strains were weak biofilm producers compared to P. aeruginosa ST309, but the coculture of P. aeruginosa and Achromobacter enhanced P. aeruginosa ST309 biofilm formation. Intraclonal genomic microevolution was observed in the isolates of P. aeruginosa ST309 collected chronologically and in Achromobacter sp. clone A. The contamination control was achieved by a complete reorganization of the dental health care center by removing the connecting tubes between DCUs.

Toxoplasmosis in natural populations of ungulates in France: prevalence and spatiotemporal variations.

Toxoplasmosis is characterized by a complex epidemiology. The risk of infection for humans depends on their contact with infective oocysts in a contaminated environment and on the amount of tissue cysts located within consumed meat. Unfortunately, the prevalence of tissue cysts is largely unknown for game species. Although herbivorous game species are a source of infection for humans, the level of infection found in wildlife can also be used to estimate environmental contamination. The aim of this study was to estimate the prevalence of Toxoplasma gondii infection and analyze its temporal dynamics in one population of chamois (Rupicapra rupicapra), one of mouflon (Ovis gmelini musimon), and two of roe deer (Capreolus capreolus) in France, surveyed during a period of 6 to 28 years. Taking into account individual risk factors, we specifically analyzed the relationship between T. gondii prevalence and meteorological conditions that may influence oocyst survival. Serum samples from 101 chamois, 143 mouflons, and 1155 roe deer were tested for antibodies against T. gondii using the modified agglutination test (MAT), an enzyme-linked immunosorbent assay (ELISA) assay, or both. Using MAT with a threshold of 1:6, seroprevalence was 14.7% in mouflon, 16.8% in chamois, and 43.7% in roe deer. In mouflon and roe deer, seroprevalence was positively correlated with age and/or body mass, in accordance with the hypothesis that antibodies have long-term persistence. In roe deer, seropositivity differed between the two populations and changed linearly over time between 1983 and 2010, increasing by a factor 1.75 every 10 years. Moreover, in this species, the highest prevalences were found during dry and cold years or during warm and moist years, depending on the population. Our results suggest that the risk for people to acquire infection through game meat increases over time, but with high variability according to the population of origin and meteorological conditions of the year.

Experimental infection of pregnant Pyrenean chamois (Rupicapra pyrenaica) with border disease virus subtype 4.

Border disease virus (BDV) causes high mortality in Pyrenean chamois (Rupicapra pyrenaica) on the French and Spanish sides of the Pyrenees Mountains. We investigated the pathology induced by BDV in pregnant chamois via experimental infection. Three females were inoculated during the second third of pregnancy with a BDV-4 subgroup strain isolated from a wild Pyrenean chamois during an acute epizootic. A fourth pregnant chamois and one nonpregnant ewe were kept as negative controls. Animals were monitored to assess clinical signs, hematology, viremia, and serology. Postmortem examinations included necropsy, histopathology, and quantification of viral RNA in organs. Pregnancy was unsuccessful in all inoculated animals. One died 24 days postinoculation (dpi) without showing any precursory clinical signs. The second animal had profuse diarrhea from 13 dpi to its death at 51 dpi. The third aborted at 46 dpi and was euthanized at 51 dpi. All animals were viremic from 4 dpi until death. Neutralizing antibodies against BDV-4 were detected from 12 dpi. Necropsies showed generalized lymphadenomegaly, associated in one case with disseminated petechial hemorrhages in the digestive tract. Seventy-eight of 79 organs from inoculated adults and their fetuses had detectable viral RNA. The main histologic lesions in adults were mild lymphohistiocytic encephalitis associated with moderate or moderately severe lymphoid depletion. Control animals remained negative for virus (in blood and organs), antibody, and lesions upon postmortem examination. BDV infection during pregnancy in Pyrenean chamois causes severe disease leading to abortion, then death. Despite 100% fetal death following inoculation, viral RNA was recovered from all organs of infected fetuses, suggesting that persistently infected offspring could be born. Our results may help explain the reported decrease in chamois populations in several areas and suggest that great care must be taken when interpreting infection status for wildlife.

Detection of Rickettsia helvetica in Ixodes ricinus ticks collected from Pyrenean chamois in France.

Seventy-one Ixodes ricinus ticks collected from Pyrenean chamois (Rupicapra pyrenaica) in the French Pyrenees were tested by real-time polymerase chain reaction to detect the presence of Rickettsia and Bartonella. Four ticks (6%) were positive for R. helvetica. The chamois carries infected ticks, and this enables the dissemination throughout the environment with this bacterium, a potential human pathogen.

Immune phenotype and body condition in roe deer: individuals with high body condition have different, not stronger immunity.

An efficient immunity is necessary for host survival, but entails energetic costs. When energy is limited, immunocompetence and body condition should co-vary positively among individuals and, depending on body condition, individuals should allocate more either in innate immunity or in adaptive response. We tested whether immune phenotype depends on body condition in large mammals, using data from two contrasted populations of roe deer Capreolus capreolus in France. Roe deer living at Chizé, a forest with poor habitat quality, were expected to show lower values for body condition and immune parameters than roe deer at Trois Fontaines, a forest with high habitat quality. From 285 blood samples collected between December 2009 and March 2011, we measured seven metabolic parameters and ten immunological parameters. A Principal Component Analysis showed that all indicators of body condition co-varied positively and were lowest at Chizé. Several immunological indicators correlated to body condition and differed between Trois Fontaines and Chizé. However, high body condition was not associated to a high average level of immunocompetence, but instead to high levels of indicators of acute inflammatory innate response, while low body condition was associated to high levels of monocytes and lymphocytes, possibly reflecting adaptive immunity. Limited data suggest that the difference between populations was not related to the presence of specific parasite species, however parasite exposure and stress have to be investigated to gain a more complete understanding of the determinants of immunity.

Emergence of atypical Mycoplasma agalactiae strains harboring a new prophage and associated with an alpine wild ungulate mortality episode.

The bacterium Mycoplasma agalactiae is responsible for contagious agalactia (CA) in small domestic ruminants, a syndrome listed by the World Organization for Animal Health and responsible for severe damage to the dairy industry. Recently, we frequently isolated this pathogen from lung lesions of ibexes during a mortality episode in the French Alps. This situation was unusual in terms of host specificity and tissue tropism, raising the question of M. agalactiae emergence in wildlife. To address this issue, the ibex isolates were characterized using a combination of approaches that included antigenic profiles, molecular typing, optical mapping, and whole-genome sequencing. Genome analyses showed the presence of a new, large prophage containing 35 coding sequences (CDS) that was detected in most but not all ibex strains and has a homolog in Mycoplasma conjunctivae, a species causing keratoconjunctivitis in wild ungulates. This and the presence in all strains of large integrated conjugative elements suggested highly dynamic genomes. Nevertheless, M. agalactiae strains circulating in the ibex population were shown to be highly related, most likely originating from a single parental clone that has also spread to another wild ungulate species of the same geographical area, the chamois. These strains clearly differ from strains described in Europe so far, including those found nearby, before CA eradication a few years ago. While M. agalactiae pathogenicity in ibexes remains unclear, our data showed the emergence of atypical strains in Alpine wild ungulates, raising the question of a role for the wild fauna as a potential reservoir of pathogenic mycoplasmas.

Factors associated with the use of emergency dental care facilities in a French public hospital.

This study was undertaken to qualitatively analyze patients' profiles and to identify the sociodemographic and oral health factors associated with emergency visits to the public dental service in Montpellier, France. A cross-sectional survey was conducted in the dental care service at Montpellier Hospital. Socioeconomic and clinical variables were compared between the patients using the emergency dental care service and those utilizing the general dental services, which were by appointment. An evaluation of the results indicated that younger patients and people from lower socioeconomic groups used the emergency dental service more frequently. Unemployed people (OR = 1.60) and manual workers (OR = 1.86) were also more likely to use this service. The need for treatment of caries was significantly higher in the group that used the emergency service. It appeared that the two groups of patients had different attendance behavior and showed significantly different socioeconomic and oral health status.

Diseases and reproductive success in a wild mammal: example in the alpine chamois.

Density-dependent and climatic factors affect reproduction and dynamics of wild ungulates. Parasites can also decrease reproductive success through either a direct abortive effect or a negative impact on host growth and body condition. However, few studies have investigated the effect of parasitism on fecundity of ungulates in natural conditions. We studied three bacterial infections caused by Salmonella enterica serovar Abortusovis, Chlamydophila abortus and Coxiella burnetii. These bacteria are leading causes of reproductive failure in sheep, goat and cattle, which raises the question of their influence on population dynamics of wild ungulates. A long-term study of demography and epidemiology of an alpine chamois (Rupicapra rupicapra, L.) population (Les Bauges Reserve, France) and a generalized linear modeling approach were used to analyze the reproductive success of chamois according to population density, weather conditions and the prevalence of antibodies against the three bacteria in females. This approach enabled us to identify the confounding effect of weather and parasitism on fecundity in a natural population. After accounting for density, the prevalence of antibodies against the three bacteria explained 36% of the annual variation in reproductive success, and weather conditions explained an additional 31%. This study was, to our knowledge, the first to compare the decrease in fecundity due to bacterial infections and weather conditions in a population of wild mountain ungulates.

Transmission of a pestivirus infection in a population of Pyrenean chamois.

Outbreaks of a previously unrecorded disease have recently affected Pyrenean chamois (Rupicapra pyrenaica pyrenaica) populations across the mountain range. A pestivirus was hypothesized to be the cause of this emerging disease and this type of virus can cross the species barrier and be transmitted to or from wildlife. Using an epidemiological survey conducted from 1995 to 2004 at Orlu, France, we characterized the virus and analyzed its transmission. A phylogenetic analysis of viral sequences and virus neutralization tests showed that the virus belonged to the newly described border disease virus-4 group. The increase of seroprevalence with age indicated that infection can occur at any age and resulted in lifelong immunity. Overall, 70.3% of 323 samples were positive for anti-p80 antibodies and 10.2% of 167 samples showed viremia, as demonstrated by either positive ELISA antigen test or RT-PCR. Infection has thus been widespread in this population since 1995, whereas no mass mortality or clinical signs have been observed. Incidence and seroprevalence varied seasonally and according to number of individuals aged less than 2 years old in the population, so viral transmission was dependent on host population age structure. We propose that the virus is now endemic in this population and is likely detrimental for reproduction and juveniles. Further investigation is needed to estimate the impact of pestivirus on host population dynamics and the risk of cross-transmission to farm animals.

Detection of a newly described pestivirus of Pyrenean chamois (Rupicapra pyrenaica pyrenaica) in France.

A pestivirus was detected and characterized in chamois (Rupicapra pyrenaica pyrenaica) originating from the French part of the Pyrenees. Phylogenetic analysis of the pestivirus was done on the basis of a fragment from the 5' noncoding region including 22 published nucleotide sequences of different pestivirus strains. Our strain was grouped within the clade of border disease viruses (BDV). However, it had an intermediate position between clade BDV and classical swine fever viruses representing a basal position to BDV strains of domestic sheep. Our strain was grouped as a sister unit to a novel pestivirus (Chamois-1) recently described from chamois in Spain. Therefore, we postulate that this virus occurs in the entire population of Pyrenean chamois. On the basis of the phylogenetic grouping of this isolate, a postulated cross-species transmission of pestivirus from domestic sheep to chamois via shared pastures seems to be unlikely.