Bruno Garin-Bastuji

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.

Human Brucellosis in Maghreb: Existence of a Lineage Related to Socio-Historical Connections with Europe

Despite control/eradication programs, brucellosis, major worldwide zoonosis due to the Brucella genus, is endemic in Northern Africa and remains a major public health problem in the Maghreb region (Algeria/Morocco/Tunisia). Brucella melitensis biovar 3 is mostly involved in human infections and infects mainly small ruminants. Human and animal brucellosis occurrence in the Maghreb seems still underestimated and its epidemiological situation remains hazy. This study summarizes official data, regarding Brucella melitensis infections in Algeria, from 1989 to 2012, with the purpose to provide appropriate insights concerning the epidemiological situation of human and small ruminant brucellosis in Maghreb. Algeria and Europe are closely linked for historical and economical reasons. These historical connections raise the question of their possible impact on the genetic variability of Brucella strains circulating in the Maghreb. Other purpose of this study was to assess the genetic diversity among Maghreb B. melitensis biovar 3 strains, and to investigate their possible epidemiological relationship with European strains, especially with French strains. A total of 90 B. melitensis biovar 3 Maghreb strains isolated over a 25 year-period (1989–2014), mainly from humans, were analysed by MLVA-16. The obtained results were compared with genotypes of European B. melitensis biovar 3 strains. Molecular assays showed that Algerian strains were mainly distributed into two distinct clusters, one Algerian cluster related to European sub-cluster. These results led to suggest the existence of a lineage resulting from socio-historical connections between Algeria and Europe that might have evolved distinctly from the Maghreb autochthonous group. This study provides insights regarding the epidemiological situation of human brucellosis in the Maghreb and is the first molecular investigation regarding B. melitensis biovar 3 strains circulating in the Maghreb.

Assessment of the diagnostic sensitivity and specificity of an indirect ELISA kit for the diagnosis of Brucella ovis infection in rams

BackgroundBrucella ovis causes an infectious disease responsible for infertility and subsequent economic losses in sheep production. The standard serological test to detect B. ovis infection in rams is the complement fixation test (CFT), which has imperfect sensitivity and specificity in addition to technical drawbacks. Other available tests include the indirect enzyme-linked immunosorbent assays (I-ELISA) but no I-ELISA kit has been fully evaluated.The study aimed to compare an I-ELISA kit and the standard CFT. Our study was carried out on serum samples from 4599 rams from the South of France where the disease is enzootic. A Bayesian approach was used to estimate tests characteristics (diagnostic sensitivity, Se and diagnostic specificity, Sp). The tests were then studied together in order to optimise testing strategies to detect B. ovis.ResultsAfter optimising the cut-off values in order to avoid doubtful results without deteriorating the concordance between the results of the two tests, the I-ELISA appeared to be slightly more sensitive than CFT (Se I-ELISAu2009=u20090.917 [0.822; 0.992], 95% Credibility Interval (CrI) compared to Se CFTu2009=u20090.860 [0.740; 0.967], 95% CrI). However, CFT was slightly more specific than I-ELISA (Sp CFTu2009=u20090.988 [0.947; 1.0], 95% CrI) compared to Sp I-ELISA =0.952 [0.901; 1.0], 95% CrI).The tests were then associated with two different interpretation schemes. The series association increased the specificity of screening and could be used for pre-movement testing in rams from uninfected flocks. The parallel association increased sequence sensitivity, thus appearing more suitable for eradicating the disease in infected flocks.ConclusionsThe high sensitivity and acceptable specificity of this I-ELISA kit support its potential interest to avoid the limitations of CFT. The two tests could also be used together or combined with other diagnostic methods such as semen culture to improve the testing strategy. The choice of test sequence and interpretation criteria depends on the epidemiological context, screening objectives and the financial and practical constraints.

Examination of taxonomic uncertainties surrounding Brucella abortus bv. 7 by phenotypic and molecular approaches.

ABSTRACT Brucella taxonomy is perpetually being reshuffled, at both the species and intraspecies levels. Biovar 7 of Brucella abortus was suspended from the Approved Lists of Bacterial Names Brucella classification in 1988, because of unpublished evidence that the reference strain 63/75 was a mixture of B. abortus biovars 3 and 5. To formally clarify the situation, all isolates previously identified as B. abortus bv. 7 in the AHVLA and ANSES strain collections were characterized by classical microbiological and multiple molecular approaches. Among the 14 investigated strains, including strain 63/75, only four strains, isolated in Kenya, Turkey, and Mongolia, were pure and showed a phenotypic profile in agreement with the former biovar 7, particularly agglutination with both anti-A/anti-M monospecific sera. These results were strengthened by molecular strategies. Indeed, genus- and species-specific methods allowed confirmation that the four pure strains belonged to the B. abortus species. The combination of most approaches excluded their affiliation with the recognized biovars (biovars 1 to 6 and 9), while some suggested that they were close to biovar 3.These assays were complemented by phylogenetic and/or epidemiological methods, such as multilocus sequence analysis (MLSA) and variable-number tandem repeat (VNTR) analysis. The results of this polyphasic investigation allow us to propose the reintroduction of biovar 7 into the Brucella classification, with at least three representative strains. Interestingly, the Kenyan strain, sharing the same biovar 7 phenotype, was genetically divergent from other three isolates. These discrepancies illustrate the complexity of Brucella taxonomy. This study suggests that worldwide collections could include strains misidentified as B. abortus bv. 7, and it highlights the need to verify their real taxonomic position.

Assessment of the sensitivity of the gamma-interferon test and the single intradermal comparative cervical test for the diagnosis of bovine tuberculosis under field conditions.

In some French départements, the eradication of bovine tuberculosis is incomplete and usual skin tests [single intradermal tuberculin test (SIT) and single intradermal comparative cervical test (SICCT)] have poor specificity due to cross-reactions with non-pathogenic mycobacteria, causing economic losses. In Côte dOr (Burgundy, France), an experimental serial testing scheme based on the combination of SICCT and gamma-interferon (IFN-γ) tests has been initiated in order to shorten the interval between suspicion and its invalidation in herds with false-positive results to skin tests. Our aim was to assess the schemes sensitivity and to compare it to the sensitivity of the screening scheme recommended by the European Commission. Our study included 1768 animals from Côte dOr. The sensitivities of both schemes were estimated using a Bayesian approach. The individual sensitivity of the IFN-γ test [88·1%, 95% credibility interval (CrI) 72·8-97·5] was not significantly different from individual SICCT sensitivity (80·3%, 95% CrI 61·6-98·0) and individual SIT sensitivity (84·2%, 95% CrI 59·0-98·2). The individual specificity of the IFN-γ test was 62·3% (95% CrI 60·2-64·5). No significant difference could be demonstrated between the sensitivities of the serial testing scheme used in Côte dOr (73·1%, 95% CrI 41·1-100) and the European Union serial testing scheme (70·1%, 95% CrI 31·5-100·0).

Evaluation of five serological tests for the diagnosis of porcine brucellosis in French Polynesia

Porcine brucellosis due to Brucella suis biovar 1 raises important issues for pig breeders in French Polynesia. In this region, the disease is enzootic, spreads silently and engenders economic losses in infected farms as well as sporadic human cases. While serological tests are essential in surveillance and control programmes of animal diseases, to date none of the available tests have been shown to be reliable enough to be used as a gold standard in routine individual diagnosis of porcine brucellosis. Few studies about the estimation of the sensitivity and the specificity of porcine brucellosis screening tests have been published, none of them dealing with French Polynesia. The studied population included 1,595 pigs from French Polynesia. Five tests were evaluated: Rose Bengal test, fluorescence polarisation assay, indirect ELISA, and two competitive ELISAs (C-ELISA). The sensitivity and the specificity of each test were estimated. C-ELISA2 was the most sensitive test (Se C-ELISA2u2009=u20090.954 [0.889; 0.992] 95xa0% credibility interval (CrI)) while both C-ELISA and Rose Bengal test (RBT) were the most specific ones (Sp C-ELISA1u2009=u20090.856 [0.806; 0.915] 95xa0% CrI; Sp C-ELISA2u2009=u20090.849 [0.817; 0.879] 95xa0% CrI; Sp RBTu2009=u20090.853 [0.812; 0.898] 95xa0% CrI).

La tuberculose à mycobacterium bovis chez le cerf et le sanglier en France : Émergence et risque pour l'élevage bovin

Depuis les annees 1960, la tuberculose due a Mycobacterium bovis a ete decrite dans la faune sauvage de plusieurs pays a travers le monde. Selon les cas, les differentes especes de mammiferes trouvees infectees par M. bovis peuvent etre considerees comme reservoirs primaires, reservoirs secondaires ou culs-de-sac epidemiologiques. En 2000, la France a ete declaree officiellement indemne de tuberculose bovine et, jusqua cette date, M. bovis navait jamais ete isole chez un animal sauvage en liberte. Cependant, depuis 2001, linfection a ete identifiee chez des ongules sauvages dans quatre regions distinctes : tout dabord en Normandie (2001), puis en Corse et en Bourgogne (2003), et dans les Pyrenees (2005). En 2001-2002, dans la foret normande de Brotonne, une enquete epidemiologique a revele des prevalences apparentes dinfection de 28 % chez le sanglier et 14 % chez le cerf. La plupart des animaux ne presentaient pas de lesions visibles a linspection par les chasseurs. Des mesures de lutte (accroissement de la pression de chasse, interdiction daffouragement, destruction des visceres des animaux tues...) ont ete immediatement prises pour tenter de contenir lepizootie. Malgre cela, le phenomene sest aggrave : en effet, en 2005-2006, la prevalence apparente a atteint plus de 30 % chez le sanglier et plus de 20 % chez le cerf, avec une augmentation du taux de lesions ouvertes dans les deux especes Par contre, dans les autres regions, les cas semblent demeurer sporadiques (1 % < p< 5%). Les memes genotypes de M. bovis ont ete retrouves chez les ongules sauvages et les bovins qui auraient pu etre a leur contact. Ceci laisse supposer quil existe un reel lien epidemiologique entre les animaux sauvages et domestiques Ces resultats posent la question de lemergence de la tuberculose dans la faune sauvage, de son origine, et du risque de transmission au betail.

Contagious epididymitis due to Brucella ovis: relationship between sexual function, serology and bacterial shedding in semen

BackgroundContagious Epididymitis (CE) due to Brucella ovis (B. ovis) is a contagious disease that impairs rams’ fertility due to epididymis, testicle and accessory sexual gland alterations. An increased incidence of CE has been observed in South Eastern France (“PACA” region) since the Rev.1 vaccination against B. melitensis has been stopped in 2008. The objective of this study was to evaluate the relationship between the infection by B. ovis and the sexual function of rams.Two-hundred eighteen sexually-mature rams, from 11 seropositive flocks, were submitted to a clinical examination of the genital tract, a semen collection by electro-ejaculation for spermogram and culture, and a serological examination for anti-B. ovis antibodies by complement fixation test (CFT) and indirect ELISA (I-ELISA). The relationships between clinical, seminal, bacteriological and serological parameters were studied using the Fisher exact test and a logistic regression model (binomial logit).ResultsB. ovis shedding in semen was significantly associated with seropositivity (CFT and I-ELISA; pu2009<u20090.001 and 0.01 respectively), genital tract alterations (pu2009<u20090.05) and poor semen quality (pu2009<u20090.001). Seropositive rams presented significantly more genital tract alterations (pu2009<u20090.001) and a poor seminal score (pu2009<u20090.001) than seronegative rams.ConclusionsSince semen culture is not routinely feasible in field conditions, a control plan of CE should be based, where Rev.1 vaccination is not possible, on both systematic clinical and serological examination of rams, followed by the culling of seropositive and/or genital tract alterations carrier rams.

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.