Reduce, Replace, Refine: Determining A Posteriori Reference Intervals for Biochemistry in Hermann's Tortoise (Testudo hermanni).

Biochemical and hematological analyses are important for the assessment of animal health. However, for most wild species their use is hindered by the scarcity of reliable reference intervals. Indeed, collecting body fluids (e.g., blood, urine) in free-ranging animals is often technically challenging. Further, sampling many individuals would be essential to consider major sources of variations, such as species, populations, sex, age, and seasons. One alternative, according to the reduction, refinement, and replacement framework, is to establish reference intervals a posteriori using literature survey and unpublished data. We produced reference intervals for free-ranging Hermann's tortoises (Testudo hermanni), using analyses performed on blood samples collected in previous studies and conservation programs conducted in the field between 2010 and 2016 in southern France (n=195 individuals). Thirteen parameters were analyzed: packed-cell volume, blood concentrations of corticosterone, testosterone, glycemia, cholesterol, triglyceride, urea, uric acid, calcium, sodium, potassium, asparagine aminotransferase (AST), and alanine aminotransferases (ALT). Reference intervals for subgroups defined by sex and season were relevant for corticosterone, triglyceride, and calcium (sex) and cholesterol (season). Comparing our results with those obtained in captive individuals in Germany, except for urea and AST levels the intervals from both free-ranging versus captive tortoises were similar, suggesting that reference intervals established from captive individuals may be suitable for free-ranging populations in this species.

[Brucellosis in Alpine ibex: 10 years of research and expert assessments]. / La brucellose du bouquetin des Alpes - Un exemple de dix années de recherche et d'expertise.

Brucellosis due to Brucella melitensis affects domestic and wild ruminants, as well as other mammals, including humans. Despite France being officially free of bovine brucellosis since 2005, two human cases of Brucella melitensis infection in the French Alps in 2012 led to the discovery of one infected cattle herd and of one infected population of wild Alpine ibex (Capra ibex). In this review, we present the results of 10 years of research on the epidemiology of brucellosis in this population of Alpine ibex. We also discuss the insights brought by research and expert assessments on the efficacy of disease management strategies used to mitigate brucellosis in the French Alps.

Title: La brucellose du bouquetin des Alpes - Un exemple de dix années de recherche et d'expertise. Abstract: La brucellose à Brucella melitensis touche les ruminants domestiques et sauvages, ainsi que d'autres mammifères, dont les humains. Bien que la France soit officiellement indemne depuis 2005, deux cas humains reportés en Haute-Savoie en 2012 ont conduit à la découverte de l'infection dans un élevage bovin et chez les bouquetins des Alpes (Capra ibex) du massif du Bargy. Nous présentons dans cette synthèse les principales découvertes de ces dix dernières années sur le système brucellose-bouquetins. Nous discuterons également de l'apport de la recherche et de l'expertise sur l'évaluation de l'efficacité des mesures de gestion sanitaire mises en place dans le massif du Bargy pour lutter contre la brucellose.

Temporal dynamics of antibody level against Lyme disease bacteria in roe deer: Tale of a sentinel?

Changes in the risk of exposure to infectious disease agents can be tracked through variations in antibody prevalence in vertebrate host populations. However, information on the temporal dynamics of the immune status of individuals is critical. If antibody levels persist a long time after exposure to an infectious agent, they could enable the efficient detection of the past circulation of the agent; if they persist only a short time, they could provide snap shots of recent exposure of sampled hosts. Here, we explored the temporal dynamics of seropositivity against Lyme disease agent Borrelia burgdorferi sensu lato (Bbsl) in individuals of a widespread medium-sized mammal species, the roe deer (Capreolus capreolus), in France. Using a modified commercially available immunoassay we tested 1554 blood samples obtained in two wild deer populations monitored from 2010 to 2020. Using multi-event capture-mark-recapture models, we estimated yearly population-, age-, and sex-specific rates of seroconversion and seroreversion after accounting for imperfect detection. The yearly seroconversion rates indicated a higher level of exposure in early (2010-2013) than in late years (2014-2019) to infected tick bites in both populations, without any detectable influence of sex or age. The relatively high rates of seroreversion indicated a short-term persistence of antibody levels against Bbsl in roe deer. This was confirmed by the analysis of samples collected on a set of captive individuals that were resampled several times a few weeks apart. Our findings show the potential usefulness of deer as a sentinel for tracking the risk of exposure to Lyme disease Bbsl, although further investigation on the details of the antibody response to Bbsl in this incompetent host would be useful. Our study also highlights the value of combining long-term capture-mark-recapture sampling and short-time analyses of serological data for wildlife populations exposed to infectious agents of relevance to wildlife epidemiology and human health.

Instability of fecal glucocorticoid metabolites at 4°C: Time to freeze matters.

The use of fecal corticosteroid metabolites (FCMs) has proven to be well suited to evaluate adrenocortical activity, a major component of the stress response, particularly in wildlife. As with any tools, confounding factors and drawbacks must be carefully considered. Among them, sample preservation and storage are of particular importance, as they can affect stability of FCMs and lead to biased results and interpretations. Arguably, immediate freezing of fecal samples upon collection is the best practice to preserve FCM integrity, however, for logistical reasons, this condition is rarely feasible in the field. It is generally argued that temporary storage of samples at low above-zero temperature is an acceptable way of preserving samples in the field before freezing them for long-term storage. However, to our knowledge, there is no empirical study that demonstrates the stability of fecal metabolites in samples stored at +4°C. In this study, we collected a fresh fecal sample from 20 captive roe deer, each of which was homogenized and split into three subsamples (60 subsamples in total) to investigate the effects on FCMs levels of temporary storage at +4°C for 24 h and 48 h before freezing versus immediate freezing at -20°C after feces collection. Compared to immediate freezing, mean FCMs levels decreased by 25% every 24 h when feces were stored at +4°C before freezing. The variance of FCMs levels followed the same pattern, leading to a clear reduction in the ability to detect biological effects. Minimizing the storage time at +4°C before freezing should therefore be seriously considered when establishing sampling and storage protocols for feces in the field for adequate hormonal profiling.

COMPARING ANTIBIOTIC RESISTANCE IN FREE-RANGING VS. CAPTIVE AFRICAN WILD HERBIVORES.

Antimicrobial resistance (AMR) is a critical challenge of the 21st century for public and animal health. The role of host biodiversity and the environment in the evolution and transmission of resistant bacteria between populations and species, and specifically at the wildlife-livestock-human interface, needs to be further investigated. We evaluated the AMR of commensal Escherichia coli in three mammalian herbivore species-impala (Aepyceros melampus), greater kudu (Tragelaphus strepsiceros), and plains zebra (Equus quagga)-targeting populations living under two conditions: captivity (French zoos) and free ranging (natural and private parks in Zimbabwe). From 137 fecal samples from these three host species, 328 E. coli isolates were isolated. We measured the AMR of each isolate against eight antibiotics, and we assessed the presence of AMR genes and mobile genetic element class 1 integrons (int1). Isolates obtained from captive hosts had a higher probability of being resistant than those obtained from free-ranging hosts (odds ratio, 293.8; confidence interval, 10-94,000). This statistically higher proportion of AMR bacteria in zoos than in natural parks was especially observed for bacteria resistant to amoxicillin. The percentage of int1 detection was higher when isolates were obtained from captive hosts, particularly captive impalas. Ninety percent of bacterial isolates with genes involved in antibiotic resistance also had the int1 gene. The sul1, sul2, blaTEM, and stra genes were found in 14, 19, 0, and 31%, respectively, of E. coli with respective antibiotic resistance. Finally, plains zebra carried AMR significantly more often than the other species.

First Expert Elicitation of Knowledge on Possible Drivers of Observed Increasing Human Cases of Tick-Borne Encephalitis in Europe.

Tick-borne encephalitis (TBE) is a viral disease endemic in Eurasia. The virus is mainly transmitted to humans via ticks and occasionally via the consumption of unpasteurized milk products. The European Centre for Disease Prevention and Control reported an increase in TBE incidence over the past years in Europe as well as the emergence of the disease in new areas. To better understand this phenomenon, we investigated the drivers of TBE emergence and increase in incidence in humans through an expert knowledge elicitation. We listed 59 possible drivers grouped in eight domains and elicited forty European experts to: (i) allocate a score per driver, (ii) weight this score within each domain, and (iii) weight the different domains and attribute an uncertainty level per domain. An overall weighted score per driver was calculated, and drivers with comparable scores were grouped into three terminal nodes using a regression tree analysis. The drivers with the highest scores were: (i) changes in human behavior/activities; (ii) changes in eating habits or consumer demand; (iii) changes in the landscape; (iv) influence of humidity on the survival and transmission of the pathogen; (v) difficulty to control reservoir(s) and/or vector(s); (vi) influence of temperature on virus survival and transmission; (vii) number of wildlife compartments/groups acting as reservoirs or amplifying hosts; (viii) increase of autochthonous wild mammals; and (ix) number of tick species vectors and their distribution. Our results support researchers in prioritizing studies targeting the most relevant drivers of emergence and increasing TBE incidence.

Age and spatio-temporal variations in food resources modulate stress-immunity relationships in three populations of wild roe deer.

Living in variable and unpredictable environments, organisms face recurrent stressful situations. The endocrine stress response, which includes the secretion of glucocorticoids, helps organisms to cope with these perturbations. Although short-term elevations of glucocorticoid levels are often associated with immediate beneficial consequences for individuals, long-term glucocorticoid elevation can compromise key physiological functions such as immunity. While laboratory works highlighted the immunosuppressive effect of long-term elevated glucocorticoids, it remains largely unknown, especially in wild animals, whether this relationship is modulated by individual and environmental characteristics. In this study, we explored the co-variation between integrated cortisol levels, assessed non-invasively using faecal cortisol metabolites (FCMs), and 12 constitutive indices of innate, inflammatory, and adaptive immune functions, in wild roe deer living in three populations with previously known contrasting environmental conditions. Using longitudinal data on 564 individuals, we further investigated whether age and spatio-temporal variations in the quantity and quality of food resources modulate the relationship between FCMs and immunity. Negative covariation with glucocorticoids was evident only for innate and inflammatory markers of immunity, while adaptive immunity appeared to be positively or not linked to glucocorticoids. In addition, the negative covariations were generally stronger in individuals facing harsh environmental constraints and in old individuals. Therefore, our results highlight the importance of measuring multiple immune markers of immunity in individuals from contrasted environments to unravel the complex relationships between glucocorticoids and immunity in wild animals. Our results also help explain conflicting results found in the literature and could improve our understanding of the link between elevated glucocorticoid levels and disease spread, and its consequences on population dynamics.

Variations in immune parameters with age in a wild rodent population and links with survival.

Recent findings suggest that immune functions do not unidirectionally deteriorate with age but that a potentially adaptive remodeling, where functions of the immune system get downregulated while others get upregulated with age could also occur. Scarce in wild populations, longitudinal studies are yet necessary to properly understand the patterns and consequences of age variations of the immune system in the wild. Meanwhile, it is challenging to understand if the observed variations in immune parameters with age are due to changes at the within-individual level or to selective (dis)appearance of individuals with peculiar immune phenotypes. Thanks to a long-term and longitudinal monitoring of a wild Alpine marmot population, we aimed to understand within- and between-individual variation in the immune phenotype with age, in order to improve our knowledge about the occurrence and the evolutionary consequences of such age variations in the wild. To do so, we recorded the age-specific leukocyte concentration and leukocyte profile in repeatedly sampled dominant individuals. We then tested whether the potential changes with age were attributable to within-individual variations and/or selective (dis)appearance. Finally, we investigated if the leukocyte concentration and profiles were correlated to the probability of death at a given age. The leukocyte concentration was stable with age, but the relative number of lymphocytes decreased, while the relative number of neutrophils increased, over the course of an individual's life. Moreover, between individuals of the same age, individuals with fewer lymphocytes but more neutrophils were more likely to die. Therefore, selective disappearance seems to play a role in the age variations of the immune parameters in this population. Further investigations linking age variations in immune phenotype to individual fitness are needed to understand whether remodeling of the immune system with age could or could not be adaptive.

Covariation between glucocorticoids, behaviour and immunity supports the pace-of-life syndrome hypothesis: an experimental approach.

The biomedical literature has consistently highlighted that long-term elevation of glucocorticoids might impair immune functions. However, patterns are less clear in wild animals. Here, we re-explored the stress-immunity relationship considering the potential effects of behavioural profiles. Thirteen captive roe deer (Capreolus capreolus) were monitored over an eight-week period encompassing two capture events. We assessed how changes in baseline faecal cortisol metabolite (FCM) concentrations following a standardized capture protocol and an immune challenge using anti-rabies vaccination affected changes in 13 immune parameters of innate and adaptive immunity, and whether these changes in baseline FCM levels and immune parameters related to behavioural profiles. We found that individuals with increased baseline FCM levels also exhibited increased immunity and were characterized by more reactive behavioural profiles (low activity levels, docility to manipulation and neophilia). Our results suggest that the immunity of large mammals may be influenced by glucocorticoids, but also behavioural profiles, as it is predicted by the pace-of-life syndrome hypothesis. Our results highlight the need to consider covariations between behaviour, immunity and glucocorticoids in order to improve our understanding of the among-individual variability in the stress-immunity relationships observed in wildlife, as they may be underpinned by different life-history strategies.

Combining seroprevalence and capture-mark-recapture data to estimate the force of infection of brucellosis in a managed population of Alpine ibex.

In wildlife, epidemiological data are often collected using cross-sectional surveys and antibody tests, and seroprevalence is the most common measure used to monitor the transmission dynamics of infectious diseases. On the contrary, the force of infection, a measure of transmission intensity that can help understand epidemiological dynamics and monitor management interventions, remains rarely used. The force of infection can be derived from age-stratified cross-sectional serological data, or from longitudinal data (although less frequently available in wildlife populations). Here, we combined seroprevalence and capture-mark-recapture data to estimate the force of infection of brucellosis in an Alpine ibex (Capra ibex) population monitored from 2012 to 2018. Because the seroprevalence of brucellosis was 38% in this population in 2012, managers conducted two culling operations in 2013 and 2015, as well as captures every year since 2012, where seronegative individuals were marked and released, and seropositive individuals were removed. We obtained two estimates of the force of infection and its changes across time, by fitting (i) a catalytic model to age-seroprevalence data obtained from unmarked animals (cross-sectional), and (ii) a survival model to event time data obtained from recaptures of marked animals (longitudinal). Using both types of data allowed us to make robust inference about the temporal dynamics of the force of infection: indeed, there was evidence for a decrease in the force of infection between mid-2014 and late 2015 in both datasets. The force of infection was estimated to be reduced from 0.115 year-1 [0.074-0.160] to 0.016 year-1 [0.001-0.057]. These results confirm that transmission intensity decreased during the study period, probably due to management interventions and natural changes in infection dynamics. Estimating the force of infection could therefore be a valuable complement to classical seroprevalence analyses to monitor the dynamics of wildlife diseases, especially in the context of ongoing disease management interventions.

DNA methylation as a tool to explore ageing in wild roe deer populations.

DNA methylation-based biomarkers of ageing (epigenetic clocks) promise to lead to new insights into evolutionary biology of ageing. Relatively little is known about how the natural environment affects epigenetic ageing effects in wild species. In this study, we took advantage of a unique long-term (>40 years) longitudinal monitoring of individual roe deer (Capreolus capreolus) living in two wild populations (Chizé and Trois-Fontaines, France) facing different ecological contexts, to investigate the relationship between chronological age and levels of DNA methylation (DNAm). We generated novel DNA methylation data from n = 94 blood samples, from which we extracted leucocyte DNA, using a custom methylation array (HorvathMammalMethylChip40). We present three DNA methylation-based estimators of age (DNAm or epigenetic age), which were trained in males, females, and both sexes combined. We investigated how sex differences influenced the relationship between DNAm age and chronological age using sex-specific epigenetic clocks. Our results highlight that old females may display a lower degree of biological ageing than males. Further, we identify the main sites of epigenetic alteration that have distinct ageing patterns between the two sexes. These findings open the door to promising avenues of research at the crossroads of evolutionary biology and biogerontology.

Targeted strategies for the management of wildlife diseases: the case of brucellosis in Alpine ibex.

The management of infectious diseases in wildlife reservoirs is challenging and faces several limitations. However, detailed knowledge of host-pathogen systems often reveal heterogeneity among the hosts' contribution to transmission. Management strategies targeting specific classes of individuals and/or areas, having a particular role in transmission, could be more effective and more acceptable than population-wide interventions. In the wild population of Alpine ibex (Capra ibex-a protected species) of the Bargy massif (French Alps), females transmit brucellosis (Brucella melitensis) infection in ~90% of cases, and most transmissions occur in the central spatial units ("core area"). Therefore, we expanded an individual-based model, developed in a previous study, to test whether strategies targeting females or the core area, or both, would be more effective. We simulated the relative efficacy of realistic strategies for the studied population, combining test-and-remove (euthanasia of captured animals with seropositive test results) and partial culling of unmarked animals. Targeting females or the core area was more effective than untargeted management options, and strategies targeting both were even more effective. Interestingly, the number of ibex euthanized and culled in targeted strategies were lower than in untargeted ones, thus decreasing the conservation costs while increasing the sanitary benefits. Although there was no silver bullet for the management of brucellosis in the studied population, targeted strategies offered a wide range of promising refinements to classical sanitary measures. We therefore encourage to look for heterogeneity in other wildlife diseases and to evaluate potential strategies for improving management in terms of efficacy but also acceptability.

Interaction Patterns between Wildlife and Cattle Reveal Opportunities for Mycobacteria Transmission in Farms from North-Eastern Atlantic Iberian Peninsula.

Interactions taking place between sympatric wildlife and livestock may contribute to interspecies transmission of the Mycobacterium tuberculosis complex or non-tuberculous mycobacteria, leading to the spread of relevant mycobacterioses or to interferences with the diagnosis of tuberculosis. The aim of this study was to characterize the spatiotemporal patterns of interactions between wildlife and cattle in a low bovine tuberculosis prevalence Atlantic region. Camera traps were set during a one-year period in cattle farms with a history of tuberculosis and/or non-tuberculous mycobacterioses. The frequency and duration of wildlife visits, and the number of individuals per visit, were analysed through generalized linear mixed models. The seasons, type of place, type of point, and period of the day were the explanatory variables. A total of 1293 visits were recorded during 2741 days of camera observation. Only 23 visits showed direct contacts with cattle, suggesting that mycobacteria transmission at the wildlife-livestock interface would occur mainly through indirect interactions. Cattle pastures represented the most appropriate habitat for interspecies transmission of mycobacteria, and badgers' latrines appear to be a potential hotspot for mycobacteria circulation between badgers, wild boars, foxes, and cattle. According to both previous epidemiological information and the interaction patterns observed, wild boars, badgers, foxes, and small rodents are the species or group most often in contact with livestock, and thus may be the most involved in the epidemiology of mycobacterioses in the wildlife-livestock interface in this area.

Pathogen-mediated selection favours the maintenance of innate immunity gene polymorphism in a widespread wild ungulate.

Toll-like receptors (TLR) play a central role in recognition and host frontline defence against a wide range of pathogens. A number of recent studies have shown that TLR genes (Tlrs) often exhibit large polymorphism in natural populations. Yet, there is little knowledge on how this polymorphism is maintained and how it influences disease susceptibility in the wild. In previous work, we showed that some Tlrs exhibit similarly high levels of genetic diversity as genes of the Major Histocompatibility Complex (MHC), and signatures of contemporary balancing selection in roe deer (Capreolus capreolus), the most abundant cervid species in Europe. Here, we investigated the evolutionary mechanisms by which pathogen-mediated selection could shape this innate immunity genetic diversity by examining the relationships between Tlr (Tlr2, Tlr4 and Tlr5) genotypes (heterozygosity status and presence of specific alleles) and infections with Toxoplasma and Chlamydia, two widespread intracellular pathogens known to cause reproductive failure in ungulates. We showed that Toxoplasma and Chlamydia exposures vary significantly across years and landscape features with few co-infection events detected and that the two pathogens exert antagonistic selection on Tlr2 polymorphism. By contrast, we found limited support for Tlr heterozygote advantage. Our study confirmed the importance of looking beyond Mhc genes in wildlife immunogenetic studies. It also emphasized the necessity to consider multiple pathogen challenges and their spatiotemporal variation to improve our understanding of vertebrate defence evolution against pathogens.

Maternal effects shape offspring physiological condition but do not senesce in a wild mammal.

In vertebrates, offspring survival often decreases with increasing maternal age. While many studies have reported a decline in fitness-related traits of offspring with increasing maternal age, the study of senescence in maternal effect through age-specific changes in offspring physiological condition is still at its infancy. We assessed the influence of maternal age and body mass on offspring physiological condition in two populations of roe deer (Capreolus capreolus) subjected to markedly different environmental conditions. We measured seven markers to index body condition and characterize the immune profile in 86 fawns which became recently independent of their known-aged mothers. We did not find striking effects of maternal age on offspring physiological condition measured at 8 months of age. This absence of evidence for senescence in maternal effects is likely due to the strong viability selection observed in the very first months of life in this species. Offspring physiological condition was, on the other hand, positively influenced by maternal body mass. Between-population differences in environmental conditions experienced by fawns also influenced their average body condition and immune phenotype. Fawns facing food limitation displayed lower values in some markers of body condition (body mass and haemoglobin levels) than those living in good quality habitat. They also allocated preferentially to humoral immunity, contrary to those living in good conditions, which allocated more to cellular response. These results shed a new light on the eco-physiological pathways mediating the relationship between mother's mass and offspring condition.

Estimating disease prevalence and temporal dynamics using biased capture serological data in a wildlife reservoir: The example of brucellosis in Alpine ibex (Capra ibex).

The monitoring of the disease prevalence in a population is an essential component of its adaptive management. However, field data often lead to biased estimates. This is the case for brucellosis infection of ibex in the Bargy massif (France). A test-and-cull program is being carried out in this area to manage the infection: captured animals are euthanized when seropositive, and marked and released when seronegative. Because this mountainous species is difficult to capture, field workers tend to focus the capture effort on unmarked animals. Indeed, marked animals are less likely to be infected, as they were controlled and negative during previous years. As the proportion of marked animals in the population becomes large, captured animals can no longer be considered as an unbiased sample of the population. We designed an integrated Bayesian model to correct this bias, by estimating the seroprevalence in the population as the combination of the separate estimates of the seroprevalence among unmarked animals (estimated from the data) and marked animals (estimated with a catalytic infection model, to circumvent the scarcity of the data). As seroprevalence may not be the most responsive parameter to management actions, we also estimated the proportion of animals in the population with an active bacterial infection. The actual infection status of captured animals was thus inferred as a function of their age and their level of antibodies, using a model based on bacterial cultures carried out for a sample of animals. Focusing on the population of adult females in the core area of the massif, i.e. with the highest seroprevalence, this observational study shows that seroprevalence has been divided by two between 2013 (51%) and 2018 (21%). Moreover, the likely estimated proportion of actively infected females in the same population, though very imprecise, has decreased from a likely estimate of 34% to less than 15%, suggesting that the management actions have been effective in reducing infection prevalence.

Short-term telomere dynamics is associated with glucocorticoid levels in wild populations of roe deer.

While evidence that telomere length is associated with health and mortality in humans and birds is accumulating, a large body of research is currently seeking to identify factors that modulate telomere dynamics. We tested the hypothesis that high levels of glucocorticoids in individuals under environmental stress should accelerate telomere shortening in two wild populations of roe deer (Capreolus capreolus) living in different ecological contexts. From two consecutive annual sampling sessions, we found that individuals with faster rates of telomere shortening had higher concentrations of fecal glucocorticoid metabolites, suggesting a functional link between glucocorticoid levels and telomere attrition rate. This relationship was consistent for both sexes and populations. This finding paves the way for further studies of the fitness consequences of exposure to environmental stressors in wild vertebrates.

Under cover of the night: context-dependency of anthropogenic disturbance on stress levels of wild roe deer Capreolus capreolus.

Wildlife populations are increasingly exposed to human-induced modifications of their habitats. To cope with anthropogenic stressors, animals can adjust their behaviour-for example, by shifting their activity to more sheltered habitats, or becoming more nocturnal. However, whether use of spatial and temporal adjustments in behaviour may regulate the endocrine response is poorly documented. Here, we analyzed faecal cortisol metabolites (FCMs) of wild roe deer (Capreolus capreolus) living in a human-dominated agro-ecosystem. Using Global Positioning System monitoring of 116 individuals, we assessed their spatial behaviour and tested whether proximity to anthropogenic structures (linear distance to built-up areas) and the use of refuge habitats (woodland and hedges) influenced FCM levels. In accordance with our predictions, individuals ranging closer to anthropogenic structures during daytime had higher FCM levels, but this relationship was buffered as use of refuge habitat increased. In addition, this link between proximity to anthropogenic structures and FCM levels disappeared when we analyzed spatial behaviour at night. Finally, FCM levels were higher when the ambient temperature was lower, and during years of low resource availability. Our results demonstrate that the stress levels of large mammals may be strongly influenced by their proximity to anthropogenic activities, but that these effects may be buffered by behavioural adjustments in terms of space use and circadian rhythm. Whereas most studies have focused on the influence of environmental heterogeneity, our analysis highlights the need to also consider the fine-scale spatial response of individuals when studying the hormonal response of wild animals to human disturbance. We emphasize the potential to mitigate this hormonal stress response, and its potential negative consequences on population dynamics, through the preservation or restoration of patches of refuge habitat in close proximity to human infrastructure.

Genetic epidemiology of the Alpine ibex reservoir of persistent and virulent brucellosis outbreak.

While it is now broadly accepted that inter-individual variation in the outcomes of host-pathogen interactions is at least partially genetically controlled, host immunogenetic characteristics are rarely investigated in wildlife epidemiological studies. Furthermore, most immunogenetic studies in the wild focused solely on the major histocompatibility complex (MHC) diversity despite it accounts for only a fraction of the genetic variation in pathogen resistance. Here, we investigated immunogenetic diversity of the Alpine ibex (Capra ibex) population of the Bargy massif, reservoir of a virulent outbreak of brucellosis. We analysed the polymorphism and associations with disease resistance of the MHC Class II Drb gene and several non-MHC genes (Toll-like receptor genes, Slc11A1) involved in the innate immune response to Brucella in domestic ungulates. We found a very low neutral genetic diversity and a unique MHC Drb haplotype in this population founded few decades ago from a small number of individuals. By contrast, other immunity-related genes have maintained polymorphism and some showed significant associations with the brucellosis infection status hence suggesting a predominant role of pathogen-mediated selection in their recent evolutionary trajectory. Our results highlight the need to monitor immunogenetic variation in wildlife epidemiological studies and to look beyond the MHC.

Pattern of latrine use by domestic cats on dairy farms and the implications for Toxoplasma gondii transmission.

Toxoplasma gondii is the parasite responsible for toxoplasmosis, a highly prevalent zoonosis that affects humans and warm-blooded animals. Faeces of infected cats can contain millions of T. gondii oocysts, which remain infectious in the environment for months. Sites repeatedly used by cats for defecation ('latrines') are recognised as hotspots of T. gondii soil contamination, but this contamination varies from one latrine to another. To understand this spatial heterogeneity, camera traps were deployed in 39 cat latrines on three dairy farms with high-density cat populations and programmed to record visits during sixteen 10-day sessions, rotating between three farms over a period of a year. Generalized Linear Mixed Models were used to test the effects of cat sexual maturity, latrine location and season on the number of cat faeces deposited and on the number of cats defecating per latrine, as determined from the analysis of 41,282 video recordings. Sexually immature cats defecated 6.60-fold (95% CI = [2.87-15.25]) more often in latrines located close to a feeding site than in other latrines. This pattern was also observed for mature males (odds ratio [OR] = 9.42, 95% CI = [3.29-26.91]), especially during winter, but not for mature females (OR = 1.77, 95% CI = [0.80-3.94]). The number of defecating cats was also 2.67-fold (95% CI = [1.66-4.30], P < 0.001) higher in latrines located close to a feeding point than in those located far from it, regardless of cat category and season. Visits by intermediate T. gondii hosts (micromammals, birds and others) were also recorded. Out of the 39 latrines, 30 (76.92%) were visited by at least one intermediate host during the study period, and some latrines were highly frequented (up to 8.74 visits/day on average). These results provide evidence that the location of food resources in dairy farms influences the latrine use pattern by cats. Highly frequented latrines can be of high risk of T. gondii infection for definitive and intermediate hosts.