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.

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.

Comparative assessment of two commonly used commercial ELISA tests for the serological diagnosis of contagious agalactia of small ruminants caused by Mycoplasma agalactiae.

BACKGROUND: Contagious agalactia (CA) of sheep and goats caused by Mycoplasma agalactiae is a widely occurring economically important disease that is difficult to control. The ELISA is commonly used for the serological detection of CA but it has some limitations and the performance of the available tests have not been properly evaluated.Two commercial ELISA kits are widely used, one involving a fusion protein as target antigen and the other a total antigen. The objectives were to compare these tests by evaluating:i. Their diagnostic sensitivity and specificity, the relevance of the recommended cut-off points, the correlation between the two tests, and, the correlation between serology data and the milk shedding of M. agalatiae;ii. The influence of extrinsic factors such as the targeted animal species, geographical origin of the samples, intra-specific variability of M. agalactiae and concurrent mycoplasma infections.A sample of 5900 animals from 211 farms with continuous CA monitoring for 20 years and no prior vaccination history was used. The infection status was known from prior bacteriological, epidemiological and serological monitoring with a complementary immunoblotting test. RESULTS: The average diagnostic sensitivity was 56% [51.8-59.8] for the fusion protein ELISA and 84% [81.3-87.2] for the total antigen ELISA, with noteworthy flock-related variations. The average diagnostic specificity for the fusion protein ELISA was 100% [99.9-100], and for the total antigen ELISA differed significantly between goats and sheep: 99.3% [97.4-99.9] and 95.7% [93.8-97.2] respectively.Experimental inoculations with different M. agalactiae strains revealed that the ELISA kits poorly detected the antibody response to certain strains. Furthermore, test performances varied according to the host species or geographical origin of the samples.Finally, the correlation between milk shedding of M. agalactiae and the presence of detectable antibodies in the blood was poor. CONCLUSIONS: These serological tests are not interchangeable. The choice of a test will depend on the objectives (early detection of infection or disease control program), on the prevalence of infection and the control protocol used. Given the variety of factors that may influence performance, a preliminary assessment of the test in a given situation is recommended prior to widespread use.

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.

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.

High Shedding Potential and Significant Individual Heterogeneity in Naturally-Infected Alpine ibex (Capra ibex) With Brucella melitensis.

Wildlife reservoirs of infectious diseases raise major management issues. In Europe, brucellosis has been eradicated in domestic ruminants from most countries and wild ruminants have not been considered important reservoirs so far. However, a high prevalence of Brucella melitensis infection has been recently identified in a French population of Alpine ibex (Capra ibex), after the emergence of brucellosis was confirmed in a dairy cattle farm and two human cases. This situation raised the need to identify the factors driving the persistence of Brucella infection at high prevalence levels in this ibex population. In the present paper, we studied the shedding pattern of B. melitensis in ibex from Bargy Massif, French Alps. Bacteriological examinations (1-15 tissues/samples per individual) were performed on 88 seropositive, supposedly infected and euthanized individuals. Among them, 51 (58%) showed at least one positive culture, including 45 ibex with at least one Brucella isolation from a urogenital sample or a lymph node in the pelvic area (active infection in organs in the pelvic area). Among these 45 ibex, 26 (30% of the total number of necropsied animals) showed at least one positive culture for a urogenital organ and were considered as being at risk of shedding the bacteria at the time of capture. We observed significant heterogeneity between sex-and-age classes: seropositive females were most at risk to excrete Brucella before the age of 5 years, possibly corresponding to abortion during the first pregnancy following infection such as reported in the domestic ruminants. The high shedding potential observed in young females may have contributed to the self-sustained maintenance of infection in this population, whereas males are supposed to play a role of transmission between spatial units through venereal transmission during mating. This heterogeneity in the shedding potential of seropositive individuals should be considered in the future to better evaluate management scenarios in this system as well as in others.

Identification and Characterization of Mycoplasma feriruminatoris sp. nov. Strains Isolated from Alpine Ibex: A 4th Species in the Mycoplasma mycoides Cluster Hosted by Non-domesticated Ruminants?

The genus Mycoplasma, a group of free-living, wall-less prokaryotes includes more than 100 species of which dozens are primary pathogens of humans and domesticated animals. Mycoplasma species isolated from wildlife are rarely investigated but could provide a fuller picture of the evolutionary history and diversity of this genus. In 2013 several isolates from wild Caprinae were tentatively assigned to a new species, Mycoplasma (M.) feriruminatoris sp. nov., characterized by an unusually rapid growth in vitro and close genetic proximity to ruminant pathogenic species. We suspected that atypical isolates recently collected from Alpine ibex in France belonged to this new species. The present study was undertaken to verify this hypothesis and to further characterize the French ibex isolates. Phylogenetic analyses were performed to identify the isolates and position them in trees containing several other mycoplasma species pathogenic to domesticated ruminants. Population diversity was characterized by genomic macrorestriction and by examining the capacity of different strains to produce capsular polysaccharides, a feature now known to vary amongst mycoplasma species pathogenic to ruminants. This is the first report of M. feriruminatoris isolation from Alpine ibex in France. Phylogenetic analyses further suggested that M. feriruminatoris might constitute a 4th species in a genetic cluster that so far contains only important ruminant pathogens, the so-called Mycoplasma mycoides cluster. A PCR assay for specific identification is proposed. These French isolates were not clonal, despite being collected in a restricted region of the Alps, which signifies a considerable diversity of the new species. Strains were able to concomitantly produce two types of capsular polysaccharides, ß-(1→6)-galactan and ß-(1→6)-glucan, with variation in their respective ratio, a feature never before described in mycoplasmas.

Sociospatial structure explains marked variation in brucellosis seroprevalence in an Alpine ibex population.

In a context of (re)emerging infectious diseases with wildlife reservoirs, understanding how animal ecology shapes epidemiology is a key issue, particularly in wild ungulates that share pathogens with domestic herbivores and have similar food requirements. For the first time in Europe, brucellosis (Brucella melitensis), a virulent zoonosis, persisted in an Alpine ibex (Capra ibex) population and was transmitted to cattle and humans. To better understand disease dynamics, we investigated the relationships between the spatial ecology of ibex and the epidemiology of brucellosis. Combining home range overlap between 37 GPS-collared individuals and visual observations of 148 visually-marked individuals monitored during the 2013-2016 period, we showed that females were spatially segregated in at least 4 units all year round, whereas males were more prone to move between female units, in particular during the rutting period. In addition to ibex age, the spatial structure in females largely contributed to variation in seroprevalence in the whole population. These results suggest that non-sexual routes are the most likely pathways of intraspecific transmission, crucial information for management. Accounting for wildlife spatial ecology was hence decisive in improving our ability to better understand this health challenge involving a wildlife reservoir.

Brucella melitensis in France: persistence in wildlife and probable spillover from Alpine ibex to domestic animals.

Bovine brucellosis is a major zoonosis, mainly caused by Brucella abortus, more rarely by Brucella melitensis. France has been bovine brucellosis officially-free since 2005 with no cases reported in domestic/wild ruminants since 2003. In 2012, bovine and autochthonous human cases due to B. melitensis biovar 3 (Bmel3) occurred in the French Alps. Epidemiological investigations implemented in wild and domestic ruminants evidenced a high seroprevalence (>45%) in Alpine ibex (Capra ibex); no cases were disclosed in other domestic or wild ruminants, except for one isolated case in a chamois (Rupicapra rupicapra). These results raised the question of a possible persistence/emergence of Brucella in wildlife. The purpose of this study was to assess genetic relationships among the Bmel3 strains historically isolated in humans, domestic and wild ruminants in Southeastern France, over two decades, by the MLVA-panel2B assay, and to propose a possible explanation for the origin of the recent bovine and human infections. Indeed, this genotyping strategy proved to be efficient for this microepidemiological investigation using an interpretation cut-off established for a fine-scale setting. The isolates, from the 2012 domestic/human outbreak harbored an identical genotype, confirming a recent and direct contamination from cattle to human. Interestingly, they clustered not only with isolates from wildlife in 2012, but also with local historical domestic isolates, in particular with the 1999 last bovine case in the same massif. Altogether, our results suggest that the recent bovine outbreak could have originated from the Alpine ibex population. This is the first report of a B. melitensis spillover from wildlife to domestic ruminants and the sustainability of the infection in Alpine ibex. However, this wild population, reintroduced in the 1970s in an almost closed massif, might be considered as a semi-domestic free-ranging herd. Anthropogenic factors could therefore account with the high observed intra-species prevalence.

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.