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.

Generalist nematodes dominate the nemabiome of roe deer in sympatry with sheep at a regional level.

The growth of livestock farming and the recent expansion of wild ungulate populations in Europe favor opportunities for direct and/or indirect cross-transmission of pathogens. Comparatively few studies have investigated the epidemiology of gastro-intestinal nematode parasites, an ubiquitous and important community of parasites of ungulates, at the wildlife/livestock interface. In this study, we aimed to assess the influence of livestock proximity on the gastrointestinal nematode community of roe deer in a rural landscape located in southern France. Using ITS-2 rDNA nemabiome metabarcoding on fecal larvae, we analysed the gastrointestinal nematode communities of roe deer and sheep. In addition, we investigated Haemonchus contortus nad4 mtDNA diversity to specifically test parasite circulation among domestic and wild host populations. The dominant gastrointestinal nematode species found in both the roe deer and sheep were generalist species commonly found in small ruminant livestock (e.g. H. contortus), whereas the more specialised wild cervid nematode species (e.g. Ostertagia leptospicularis) were only present at low frequencies. This is in marked contrast with previous studies that found the nemabiomes of wild cervid populations to be dominated by cervid specialist nematode species. In addition, the lack of genetic structure of the nad4 mtDNA of H. contortus populations between host species suggests circulation of gastrointestinal nematodes between roe deer and sheep. The risk of contact with livestock only has a small influence on the nemabiome of roe deer, suggesting the parasite population of roe deer has been displaced by generalist livestock parasites due to many decades of sheep farming, not only for deer grazing close to pastures, but also at a larger regional scale. We also observed some seasonal variation in the nemabiome composition of roe deer. Overall, our results demonstrate significant exchange of gastrointestinal nematodes between domestic and wild ungulates, with generalist species spilling over from domestic ungulates dominating wild cervid parasite communities.

Lack of efficacy of fenbendazole against Giardia duodenalis in a naturally infected population of dogs in France.

Giardiosis is a worldwide intestinal parasitosis, affecting both humans and animals. Treatment in dogs remains limited and the lack of efficacy of the few approved medications is a rising concern. In this study, 23 dogs raised by veterinary students and naturally infected with Giardia duodenalis were treated in home conditions with fenbendazole (50 mg/kg orally for 5 consecutive days). Fecal samples were collected immediately before treatment (FS1), 2-4 days after treatment (FS2) and 8-10 days after treatment (FS3). Giardia duodenalis cyst excretion was measured quantitatively by direct immunofluorescence assay (DFA) at FS1, FS2 and FS3. Molecular typing with a nested PCR targeting the SSU rDNA locus was also performed at FS1 and FS2. Fecal consistency improved in 16/21 dogs (76%) and mean cyst shedding was reduced by 84% after treatment. However, only 8/23 dogs (35%) achieved therapeutic success (≥90% reduction of cysts) and only 4/23 dogs (17%) had complete elimination of G. duodenalis. Molecular typing showed that dogs harbored only canine-specific assemblages, with a high prevalence of assemblage C in analyzed samples (30/39). We also detected different assemblages after treatment and nucleotide substitutions in assemblage C sequences that have not been described previously. Eight to ten days after treatment, high Giardia cyst excretion was measured, suggesting possible reinfection despite hygiene measures and/or multiplication. These data suggest that fenbendazole treatment may improve fecal consistency but has limited therapeutic efficacy against giardiosis in this population of dogs. Further research is still needed to assess the efficacy of fenbendazole against canine giardiosis.

Title: Absence d'efficacité du fenbendazole contre Giardia duodenalis dans une population de chiens naturellement infectés en France. Abstract: La giardiose est une parasitose intestinale mondiale, touchant à la fois l'homme et les animaux. Chez le chien, le traitement reste limité et le manque d'efficacité des quelques médicaments autorisés inquiète de plus en plus. Dans cette étude, 23 chiens d'étudiants vétérinaires et infectés naturellement par Giardia duodenalis ont été traités en conditions réelles avec du fenbendazole (50 mg/kg par voie orale pendant 5 jours consécutifs). Des échantillons de selles ont été collectés juste avant le traitement (FS1), 2­4 jours après traitement (FS2) et 8­10 jours après traitement (FS3). L'excrétion de kystes de G. duodenalis a été mesurée quantitativement par immunofluorescence directe (IFD) à FS1, FS2 et FS3. Un génotypage par PCR nichée ciblant le locus SSU ADNr a également été réalisé à FS1 et FS2. La consistance des selles a été améliorée chez 16/21 (76 %) chiens et la moyenne d'excrétion des kystes a été réduite de 84 % juste après le traitement. Seulement 8/23 (35 %) chiens ont atteint un succès thérapeutique (≥ 90 % de réduction d'excrétion de kystes) et 4/23 (17 %) chiens ont eu une élimination complète de G. duodenalis. L'analyse des séquences a montré que les chiens présentaient seulement des assemblages génotypiques spécifiques de l'espèce canine, avec une forte prévalence de l'assemblage C dans les échantillons analysés (30/39). Des changements d'assemblage après traitement et des substitutions nucléotidiques jamais décrites au sein de l'assemblage C ont également été observés. Huit à dix jours après traitement, une forte excrétion de kystes de G. duodenalis a été mesurée : malgré les mesures hygiéniques, une réinfection et/ou une multiplication semblent probables. Ces données suggèrent que le traitement au fenbendazole peut améliorer la consistance des selles mais a une efficacité thérapeutique limitée contre la giardiose dans cette population de chiens. Des recherches supplémentaires sont encore nécessaires pour évaluer l'efficacité du fenbendazole contre la giardiose canine.

Metabarcoding in two isolated populations of wild roe deer (Capreolus capreolus) reveals variation in gastrointestinal nematode community composition between regions and among age classes.

BACKGROUND: Gastrointestinal nematodes are ubiquitous for both domestic and wild ungulates and have varying consequences for health and fitness. They exist as complex communities of multiple co-infecting species, and we have a limited understanding of how these communities vary in different hosts, regions and circumstances or of how this affects their impacts. METHODS: We have undertaken ITS2 rDNA nemabiome metabarcoding with next-generation sequencing on populations of nematode larvae isolated from 149 fecal samples of roe deer of different sex and age classes in the two isolated populations of Chizé and Trois Fontaines in France not co-grazing with any domestic ungulate species. RESULTS: We identified 100 amplified sequence variants (ASVs) that were assigned to 14 gastrointestinal nematode taxa overall at either genus (29%) or species (71%) level. These taxa were dominated by parasites classically found in cervids-e.g. Ostertagia leptospicularis, Spiculopteragia spp. Higher parasite species diversity was present in the Trois Fontaines population than in the Chizé population including the presence of species more typically seen in domestic livestock (Haemonchus contortus, Bunostomum sp., Cooperia punctata, Teladorsagia circumcincta). No differences in parasite species diversity or community composition were seen in the samples collected from three zones of differing habitat quality within the Chizé study area. Young roe deer hosted the highest diversity of gastrointestinal nematodes, with more pronounced effects of age apparent in Trois Fontaines. The effect of host age differed between gastrointestinal nematode species, e.g. there was little effect on O. leptospicularis but a large effect on Trichostrongylus spp. No effect of host sex was detected in either site. CONCLUSIONS: The presence of some livestock parasite species in the Trois Fontaines roe deer population was unexpected given the isolation of this population away from grazing domestic livestock since decades. Overall, our results illustrate the influence of host traits and the local environment on roe deer nemabiome and demonstrate the power of the nemabiome metabarcoding approach to elucidate the composition of gastrointestinal nematode communities in wildlife.

Reproductive females and young mouflon (Ovis gmelini musimon × Ovis sp.) in poor body condition are the main spreaders of gastrointestinal parasites.

Several individual, environmental and parasitic factors can influence the impacts of parasites on host's fitness and on host's ability to transmit these parasites to new hosts. Identifying these factors and the individuals who play a greater role in parasite transmission is of main concern for the development of parasite control strategies. In the present study, we aimed to describe the diversity of gastrointestinal parasites and to identify the individual factors influencing the faecal spreading of parasites in a free-ranging population of Mediterranean mouflon. From the analysis of 433 faecal samples, we found Eimeria spp. and gastrointestinal strongyles (GIS) were the most common parasites (>94%). The faecal oocyst counts of Eimeria spp. were the highest during the first years of life. It was 1.6 times higher in females than in males and 2.5 times higher in individuals in poor than in good body condition. Similarly, the faecal egg count of GIS was higher in females and decreased with age, but only in males. Finally, reproductive females had GIS faecal egg count values 2.6 times higher than non-reproductive females. Management strategies of parasites should thus primarily focus on reproductive females and young individuals in poor body condition as they represent the main contamination source of the environment.

Does host socio-spatial behavior lead to a fine-scale spatial genetic structure in its associated parasites?

Gastro-intestinal nematodes, especially Haemonchus contortus, are widespread pathogenic parasites of small ruminants. Studying their spatial genetic structure is as important as studying host genetic structure to fully understand host-parasite interactions and transmission patterns. For parasites having a simple life cycle (e.g., monoxenous parasites), gene flow and spatial genetic structure are expected to strongly rely on the socio-spatial behavior of their hosts. Based on five microsatellite loci, we tested this hypothesis for H. contortus sampled in a wild Mediterranean mouflon population (Ovis gmelini musimon × Ovis sp.) in which species- and environment-related characteristics have been found to generate socio-spatial units. We nevertheless found that their parasites had no spatial genetic structure, suggesting that mouflon behavior was not enough to limit parasite dispersal in this study area and/or that other ecological and biological factors were involved in this process, for example other hosts, the parasite life cycle, or the study area history.

TITLE: Le comportement socio-spatial de l'hôte conduit-il à une structure génétique à fine échelle de ses parasites ? ABSTRACT: Les nématodes gastro-intestinaux, et plus particulièrement Haemonchus contortus, sont cosmopolites et pathogènes chez les petits ruminants. Étudier leur structure génétique spatiale est aussi important que d'étudier celle des hôtes pour pleinement comprendre les interactions hôtes-parasites et les processus de transmission. Pour les parasites ayant des cycles de vie simples (par exemple, les parasites monoxènes), on s'attend à ce que les flux de gènes et la structure génétique spatiale dépendent fortement du comportement socio-spatial de leurs hôtes. En utilisant cinq loci microsatellites, nous avons testé cette hypothèse pour des H. contortus échantillonnés dans une population sauvage de mouflons méditerranéens (Ovis gmelini musimon × Ovis sp.) dans laquelle les caractéristiques de l'espèce et de l'environnement génèrent des unités socio-spatiales. Nous avons néanmoins mis en évidence que leurs parasites ne présentent pas de structure génétique spatiale, ce qui suggère que le comportement des mouflons ne restreint pas la dispersion des parasites dans cette aire d'étude et/ou que d'autres facteurs biologiques et écologiques tels que d'autres hôtes, le cycle de vie du parasite, ou l'histoire de l'aire d'étude jouent un rôle dans ce processus.

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.

Both candidate gene and neutral genetic diversity correlate with parasite resistance in female Mediterranean mouflon.

BACKGROUND: Parasite infections can have substantial impacts on population dynamics and are accordingly a key challenge for wild population management. Here we studied genetic mechanisms driving parasite resistance in a large herbivore through a comprehensive approach combining measurements of neutral (16 microsatellites) and adaptive (MHC DRB1 exon 2) genetic diversity and two types of gastrointestinal parasites (nematodes and coccidia). RESULTS: While accounting for other extrinsic and intrinsic predictors known to impact parasite load, we show that both neutral genetic diversity and DRB1 are associated with resistance to gastrointestinal nematodes. Intermediate levels of multi-locus heterozygosity maximized nematodes resistance, suggesting that both in- and outbreeding depression might occur in the population. DRB1 heterozygosity and specific alleles effects were detected, suggesting the occurrence of heterozygote advantage, rare-allele effects and/or fluctuating selection. On the contrary, no association was detected between genetic diversity and resistance to coccidia, indicating that different parasite classes are impacted by different genetic drivers. CONCLUSIONS: This study provides important insights for large herbivores and wild sheep pathogen management, and in particular suggests that factors likely to impact genetic diversity and allelic frequencies, including global changes, are also expected to impact parasite resistance.

Prevalence and genetic diversity of the intestinal parasites Blastocystis sp. and Cryptosporidium spp. in household dogs in France and evaluation of zoonotic transmission risk.

Several parasites including the protozoa Blastocystis sp. and Cryptosporidium spp. may be causative agents of gastrointestinal symptoms in domestic dogs, and there may be a potential risk of transmission to owners. While France is one of the largest European countries in terms of its canine population, little data is available about the molecular epidemiology of these two parasites. The purpose of this study was to determine the prevalence of intestinal parasites in household dogs in France, and to evaluate the zoonotic risk of Blastocystis sp. and Cryptosporidium spp. by genotyping the corresponding isolates. To this end, 116 faecal samples were collected from household dogs regardless of breed, age or gender, living in the Lyons area, France. Various intestinal protozoa and helminths were identified by light microscopy. Screening for Blastocystis sp. and Cryptosporidium spp. were subsequently performed by PCR targeting the small subunit (SSU) rDNA coding region, followed by direct sequencing of the PCR products and analysis of the sequences obtained for genotyping. The overall prevalence of dogs infected with at least one gastrointestinal parasite was 42.2% (49/116). After light microscopy examination of faecal samples, the most common parasites found were the protozoa Giardia sp. (25.0%) and Cystoisospora sp. (19.8%). Using molecular methods, four dogs (3.4%) were shown to be infected by Blastocystis sp. and carried either subtype (ST) 2, commonly identified in various animal groups, or ST10, frequently found in bovids. Three dogs (2.6%) were positive for C. canis, infecting humans episodically. The low prevalence of both parasites, combined with the identification of C. canis and Blastocystis sp. ST2 and ST10 in the canine population, strongly suggests that dogs play a negligible role as zoonotic reservoirs for both parasites and do not seem to be natural hosts of Blastocystis sp.

The cryopreservation of ovarian tissue: uses and indications in veterinary medicine.

Animal experiments have shown that cryopreservation of the ovarian cortex, containing primordial follicles, could be used to preserve gametes thereby restoring fertility in humans and animals. During the last 100 years, many hundreds of species have been lost, and a third of the breeding animals are threatened with extinction. To preserve genetic diversity, notably for the conservation of endangered species, it is essential to conserve female and male gametes. Today, biotechnologies such as artificial insemination and embryo transfer are used in breeding programs and are well developed. However, even using these advanced techniques, there are problems due to the limited number of individuals used as the source of gametes, so that the risk of inbreeding is high, even in large populations. To preserve genetic diversity, it is necessary to create gene banks of male and female gametes and embryos, using a very large number of individual donors. Cryopreservation of ovarian tissue could present a means for enlarging the gene pool. Cryopreserved ovarian tissue could be used in auto- or xenografts, or for in vitro maturation (IVM) of primordial follicles. In this review, we describe the processes for cryopreservation of ovarian tissue and the various possibilities for using it.