[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.

Evaluation of a Lateral Flow Immunochromatography Assay (LFIA) for Diagnosis and Surveillance of Brucellosis in French Alpine Ibex (Capra ibex).

France has been officially free of bovine brucellosis since 2005. Nevertheless, in 2012, as the source of two human cases, a bovine outbreak due to B. melitensis biovar 3 was confirmed in the French Alpine Bargy massif, due to a spillover from wild, protected Alpine ibex (Capra ibex). In order to reduce high Brucella prevalence in the local ibex population, successive management strategies have been implemented. Lateral flow immunochromatography assay (LFIA) was thus identified as a promising on-site screening test, allowing for a rapid diagnosis far from the laboratory. This study compared a commercial LFIA for brucellosis diagnosis with the WOAH-recommended tests for small ruminants (i.e., Rose Bengal test (RBT), Complement fixation test, (CFT) and Indirect ELISA, (iELISA)). LFIA showed the same analytical sensitivity as iELISA on successive dilutions of the International Standard anti-Brucella melitensis Serum (ISaBmS) and the EU Goat Brucella Standard Serum (EUGBSS). Selectivity was estimated at 100% when vaccinated ibex sera were analyzed. When used on samples from naturally infected ibex, LFIA showed high concordance, as well as relative sensitivity and specificity (>97.25%) in comparison with RBT and CFT. This work shows high reliability and ensures a better standardization of LFIA testing for wild ruminants.

"BACACIX", a spatial index combining proxies of bovine and badger space use associated with extended Mycobacterium bovis circulation in France.

To better prevent and control multi-host pathogen circulation over large areas, it is essential to identify patterns of disease persistence within host communities involved in pathogen circulation at a macroscale. The aim of this study was to design and calculate "BACACIX", a spatial index of indirect contacts between cattle and badgers, two species involved in the circulation of Mycobacterium bovis, one of the main causative agents of bovine tuberculosis (bTB), in some areas of France. The index combined spatial models of land use distribution (the probable distribution defining animal use of space) based on pasture location for cattle, and based on land cover for badgers, with proxies for animal density for both species. For badgers, we used two series of census data of badger setts in two regions of France to evaluate our model of badger space use distribution (also known as utilization distribution), and analyzed the relationship between BACACIX and the upsurge of bovine tuberculosis observed in several regions of France during the decade after the country obtained the officially bTB-free status in 2001. We observed high values of BACACIX from the southwest to the northeast of France and from Brittany to the Channel coast. Conversely, in two areas (north-central area and Mediterranean coast), index values were low, suggesting that indirect cattle-badger contacts were unlikely. In the two series of census data of badger setts that we analyzed, 96.5% and 87% of the global positioning system (GPS) locations of badger setts, respectively were located in the calculated badger space use distribution. A logistic regression model showed that after controlling bTB over the previous decade, the value of the index was positively associated with the risk of cattle outbreaks between 2001 and 2010 (OR = 1.57). In addition, the risk of bTB occurrence in cattle decreased when the pasture area outside the badger space use distribution increased. In the future, the spatial index of indirect cattle-badger contacts we propose could help to better target bTB surveillance and control in France.

Estimation of Bait Uptake by Badgers, Using Non-invasive Methods, in the Perspective of Oral Vaccination Against Bovine Tuberculosis in a French Infected Area.

Although France is officially declared free of bovine tuberculosis (TB), Mycobacterium bovis infection is still observed in several regions in cattle and wildlife, including badgers (Meles meles). In this context, vaccinating badgers should be considered as a promising strategy for the reduction in M. bovis transmission between badgers and other species, and cattle in particular. An oral vaccine consisting of live Bacille Calmette-Guérin (BCG) contained in bait is currently under assessment for badgers, for which testing bait deployment in the field and assessing bait uptake by badgers are required. This study aimed to evaluate the bait uptake by badgers and determine the main factors influencing uptake in a TB-infected area in Burgundy, north-eastern France. The baits were delivered at 15 different setts located in the vicinity of 13 pastures within a TB-infected area, which has been subject to intense badger culling over the last decade. Pre-baits followed by baits containing a biomarker (Rhodamine B; no BCG vaccine) were delivered down sett entrances in the spring (8 days of pre-baiting and 4 days of baiting) and summer (2 days of pre-baiting and 2 days of baiting) of 2018. The consumption of the marked baits was assessed by detecting fluorescence, produced by Rhodamine B, in hair collected in hair traps positioned at the setts and on the margins of the targeted pastures. Collected hairs were also genotyped to differentiate individuals using 24 microsatellites markers and one sex marker. Bait uptake was estimated as the proportion of badgers consuming baits marked by the biomarker over all the sampled animals (individual level), per badger social group, and per targeted pasture. We found a bait uptake of 52.4% (43 marked individuals of 82 genetically identified) at the individual level and a mean of 48.9 and 50.6% at the social group and pasture levels, respectively. The bait uptake was positively associated with the presence of cubs (social group level) and negatively influenced by the intensity of previous trapping (social group and pasture levels). This study is the first conducted in France on bait deployment in a badger population of intermediate density after several years of intensive culling. The results are expected to provide valuable information toward a realistic deployment of oral vaccine baits to control TB in badger populations.

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.

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.

How to Strengthen Wildlife Surveillance to Support Freedom From Disease: Example of ASF Surveillance in France, at the Border With an Infected Area.

Using a risk-based approach, the SAGIR network (dedicated to wildlife disease surveillance) had to strengthen surveillance activities after ASF was confirmed in Belgium in September 2018, very near the French border. Three new active dead wild boars search protocols supplemented opportunistic surveillance in Level III risk areas: patrols by volunteer hunters, professional systematic combing, and dog detection. Those protocols were targeted in terms of location and time and complemented each other. The main objectives of the designed surveillance system were (i) to assure early detection in case of introduction of the disease and (ii) to support the free status of the zone. Compiling the surveillance effort was thus a necessity to assure authorities and producer representatives that the sometimes low number of carcasses detected was not a consequence of no surveillance activities. The human involvement in implementing those activities was significant: more than 1000 8-h days just for the time spent in the field on active search activities. We calculated a specific indicator to enable a comparison of the surveillance results from different zones, including non-infected Belgian zones with strengthened surveillance activities. This was a first step in the evaluation of the efficacy of our surveillance activities in a WB population. Field experiments and modelling dead WB detection probability are planned to supplement this evaluation. Belgium regained its ASF-free status in November 2020, and ASF was not detected in France in either the WB or domestic pig populations.

Evaluation of Three Medetomidine-Based Anesthetic Protocols in Free-Ranging Wild Boars (Sus scrofa).

Three medetomidine-based drug protocols were compared by evaluating time courses, reliability and physiological effects in wild boars. A total of 21 cage-trapped wild boars (Sus scrofa) were immobilized using one of the following drug combinations; MTZ: medetomidine (0.2 mg/kg) + tiletamine-zolazepam (2.0 mg/kg), MK: medetomidine (0.15 mg/kg) + ketamine (5 mg/kg), and MKB: medetomidine (0.1 mg/kg) + ketamine (5.0 mg/kg) + butorphanol (0.2 mg/kg). Induction time, recovery time, and physiological variables were recorded and arterial blood gas analysis measured twice, before and after 15 min of oxygen supplementation (0.5-1.0 L/min). For reversal, 4 mg of atipamezole per mg of medetomidine was administered intramuscularly. The boars recovered in the cage and were released once ataxia resolved. The MK group had significantly longer recovery times (mean 164 min ± 79 SD) compared to the other groups. MKB elicited longer and incomplete induction compared to the other groups (mean induction time 20 min ± 10 SD), decreasing the efficiency of the capture and increasing the risk of hyperthermia. Both ketamine-based protocols required additional ketamine intramuscularly to prolong the anesthesia after 20-40 min from induction. Agreement between the pulse oximeter and the blood gas analyzer was low, with the pulse oximeter underestimating the real values of arterial oxyhemoglobin saturation, particularly at higher readings. Mild acute respiratory acidosis (PaCO2 45-60 mmHg) and mild to moderate hypoxemia (PaO2 69-80 mmHg) occurred in most boars, regardless of the treatment group but especially in the MKB group. The acid-base status improved and hypoxemia resolved in all boars during oxygen supplementation, with the PaO2 rising above the physiological reference range (81.6-107.7 mmHg) in many individuals. MK and MKB induced safe and reliable immobilization of wild boars for at least 20 min. Supplemental oxygen delivery is recommended in order to prevent hypoxemia in wild boars immobilized with the protocols used in the present study. Long and ataxic recoveries occurred in most animals, regardless of the protocol, but especially in the MKB group.

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.

Evaluation of a commercial ELISA for detection of epizootic haemorrhagic disease antibodies in domestic and wild ruminant sera.

Bluetongue (BT) and epizootic haemorrhagic disease (EHD) are vector-borne viral diseases affecting domestic and wild ruminants. Both are notifiable under OIE rules. BT and EHD viruses (BTV and EHDV) are closely related Orbiviruses with structural, antigenic and molecular similarities. Both viruses can produce analogous clinical signs in susceptible animals. Serological tests are commonly used for BT and EHD diagnosis and surveillance. Competitive ELISA (c-ELISA) is the most widely used serological test for the specific detection of BTV or EHDV viral protein 7 (VP7) antibodies (Abs). The specificity and sensitivity of the BTV c-ELISA kits available on the market are recognized for the detection of BTV Abs. Concerning EHD, a single commercial EHDV c-ELISA kit (ELISA A kit) commonly used for diagnosis in Europe and Africa was available between 2011 and 2018 but is now no longer on the market. In this study, we evaluated a new commercial c-ELISA to detect ruminant EHDV VP7 Abs in 2,199 serum samples from cattle, sheep, goats, wild deer and zoo animals. The results showed that this ELISA kit is specific and can detect the presence of IgG anti-EHDV VP7 with a very good diagnostic specificity and a satisfactory sensitivity in domestic ruminants, zoo animals and wild deer. Therefore, the evaluated c-ELISA can detect the introduction of EHDV into an area where BTV-seropositive domestic animals are present. The performance of this kit is similar to that of the c-ELISA A kit and can thus be used for diagnosis.