[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.

Brucella spp. are facultative anaerobic bacteria under denitrifying conditions.

IMPORTANCE: Respiration is a fundamental and complex process that bacteria use to produce energy. Despite aerobic respiration being the most common, some bacteria make use of a mode of respiration in the absence of oxygen, called anaerobic respiration, which can yield advantages in adaptation to various environmental conditions. Denitrification is part of this respiratory process ensuring higher respiratory flexibility under oxygen depletion. Here, we report for the first time the evidence of anaerobic growth of Brucella spp. under denitrifying conditions, which implies that this genus should be reconsidered as facultative anaerobic. Our study further describes that efficient denitrification is not equally found within the Brucella genus, with atypical species showing a greater ability to denitrify, correlated with higher expression of the genes involved, as compared to classical species.

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.

The emergence of Brucella canis as a public health threat in Europe: what we know and what we need to learn.

The zoonotic bacteria, Brucella canis, is becoming the leading cause of canine brucellosis in Europe. In dogs, it causes reproductive problems as well as non-specific lameness or discospondilitis. In humans, B. canis can be origin of chronic debilitating conditions characteristic to its genus such as undulant fever, splenomegaly, and lymphadenopathy. Although B. canis shows some pathogenic characteristics similar to B. abortus and B. melitensis, it lacks surface O-polysaccharide, like nonzoonotic B. ovis. This review shows that host-B. canis interactions are still poorly understood, with many knowledge and capability gaps, causing relatively poor sensitivity and specificity of existing diagnostic tools. Currently, there is no vaccine for this rough Brucella species. Besides, antimicrobial therapy does not guarantee bacterial elimination, and infection relapses are frequently reported, increasing the risks of antibiotic resistance development. B. canis has been detected in dogs in almost all European countries which increased human exposure, but currently there is no systematic surveillance. Moreover, B. canis caused brucellosis is not included in Animal Health Law, and therefore there is no legal framework to tackle this emerging infectious disease. To map out the diagnostic strategies, identify risks for human infections and propose management scheme for infected pet and kennel dogs, we present current understanding of canine B. canis caused brucellosis, outline major knowledge gaps and propose future steps. To address and highlight challenges veterinary and public health services encounter in Europe, we developed two B. canis infection scenarios: of a single household pet and of a kennel dog in larger group.

Molecular Investigations of Two First Brucella suis Biovar 2 Infections Cases in French Dogs.

Despite Brucella suis biovar 2's (BSB2) active circulation in wildlife, no canine infections have been reported. The present paper is the first to describe two cases of BSB2 infections in French dogs. The first case occurred in 2020 and concerned a 13-year-old male neutered Border Collie with clinical signs of prostatitis. The urine culture revealed the excretion of significant levels of Brucella in the sample. The second case concerned a German Shepherd with bilateral orchitis, in which it was possible to detect Brucella colonies following neutering. HRM-PCR and classical biotyping methods classified both isolated strains as BSB2, in contrast to expected B. canis, which is usually the etiological agent of canine brucellosis in Europe. The wgSNP and MLVA analyses highlighted the genetic proximity of two isolates to BSB2 strains originating from wildlife. No pig farms were present in the proximity of either dog's residence, ruling out potential spill over from infected pigs. Nevertheless, the dogs used to take walks in the surrounding forests, where contact with wildlife (i.e., wild boars or hares, or their excrements) was possible. These cases highlight the importance of adopting a One Health approach to control the presence of zoonotic bacteria in wild animals and avoid spillovers into domestic animals and, potentially, humans.

Comparative Genome-Wide Transcriptome Analysis of Brucella suis and Brucella microti Under Acid Stress at pH 4.5: Cold Shock Protein CspA and Dps Are Associated With Acid Resistance of B. microti.

Brucellae are facultative intracellular coccobacilli causing brucellosis, one of the most widespread bacterial zoonosis affecting wildlife animals, livestock and humans. The genus Brucella comprises classical and atypical species, such as Brucella suis and Brucella microti, respectively. The latter is characterized by increased metabolic activity, fast growth rates, and extreme acid resistance at pH 2.5, suggesting an advantage for environmental survival. In addition, B. microti is more acid-tolerant than B. suis at the intermediate pH of 4.5. This acid-resistant phenotype of B. microti may have major implications for fitness in soil, food products and macrophages. Our study focused on the identification and characterization of acid resistance determinants of B. suis and B. microti in Gerhardt's minimal medium at pH 4.5 and 7.0 for 20 min and 2 h by comparative RNA-Seq-based transcriptome analysis, validated by RT-qPCR. Results yielded a common core response in both species with a total of 150 differentially expressed genes, and acidic pH-dependent genes regulated specifically in each species. The identified core response mechanisms comprise proton neutralization or extrusion from the cytosol, participating in maintaining physiological intracellular pH values. Differential expression of 441 genes revealed species-specific mechanisms in B. microti with rapid physiological adaptation to acid stress, anticipating potential damage to cellular components and critical energy conditions. Acid stress-induced genes encoding cold shock protein CspA, pseudogene in B. suis, and stress protein Dps were associated with survival of B. microti at pH 4.5. B. suis response with 284 specifically regulated genes suggested increased acid stress-mediated protein misfolding or damaging, triggering the set-up of repair strategies countering the consequences rather than the origin of acid stress and leading to subsequent loss of viability. In conclusion, our work supports the hypothesis that increased acid stress resistance of B. microti is based on selective pressure for the maintenance of functionality of critical genes, and on specific differential gene expression, resulting in rapid adaptation.

The Use of Flocked Swabs with a Protective Medium Increases the Recovery of Live Brucella spp. and DNA Detection.

Brucellosis is a worldwide zoonosis caused by bacteria from the genus Brucella. Once established, it is very hard to eradicate this disease, since it contaminates animals, the environment, and humans, causing problems for veterinary and public health as well as wildlife protection programs. Swabs are used for sampling in bacteriological and/or molecular diagnostics, from seropositive animals with disease symptoms, from genitalia or tissue lesions, as well as from contaminated environments. The aim of this study was to compare main of the commercially used swab types for sampling and diagnostics of Brucella spp. and determine the optimal storage conditions and time frame for testing. To achieve this, we tested bacterial and molecular methods for detection of Brucella abortus, Brucella melitensis, and Brucella suis using nine swab types, all with different tip materials, treated immediately after spiking, after 72 h at +4°C, and after 72 h at -20°C. Flocked swabs showed the highest capacity to preserve bacterial viability and DNA quality, regardless the storage conditions. Flocked swabs immersed in a protective medium provided the best conditions for Brucella survival in all three storage conditions. At the same time, the efficacy of quantitative PCR (qPCR) detection for all swabs, including the positive control, was above 50%, irrespective of the storage conditions, while bacterial survival was significantly lowered when swabs were kept at +4°C or -20°C for 72 h (48.2% and 27.5%, respectively). Compared to the positive control and other types, the flocked swabs maintained higher reproducibility regarding their capacity to preserve live bacteria in all three storage conditions. IMPORTANCE In order to protect public and veterinary health from highly zoonotic bacteria such as members of the genus Brucella and prevent their dissemination into the environment, direct diagnostics are of utmost importance. However, in addition to the highly specific diagnostic tests, the sampling methods, time necessary for specimens to reach the laboratories, and transport conditions are important factors to consider in order to increase the sensitivity of performed tests, especially bacterial culturing and qPCR. This paper shows how different swab types and storage conditions influence classical bacteriological diagnostics of the most prevalent Brucella species - B. melitensis, B. abortus, and B. suis - but have little impact on molecular methods. The presented results highlight (i) the choice of swab regarding the storage and transport conditions, (ii) the importance of immediate swab treatment upon sampling, and (iii) that molecular methods do not depend on storage conditions, unlike classical bacteriological isolation.

New Brucella variant isolated from Croatian cattle.

BACKGROUND: A novel Brucella strain closely related to Brucella (B.) melitensis biovar (bv) 3 was found in Croatian cattle during testing within a brucellosis eradication programme. CASE PRESENTATION: Standardised serological, brucellin skin test, bacteriological and molecular diagnostic screening for Brucella infection led to positive detection in one dairy cattle herd. Three isolates from that herd were identified to species level using the Bruce ladder method. Initially, two strains were typed as B. melitensis and one as B. abortus, but multiplex PCR based on IS711 and the Suis ladder showed that all of them to belong to B. melitensis, and the combination of whole-genome and multi-locus sequencing as well as Multi-Locus Variable numbers of tandem repeats Analysis (MLVA) highlighted a strong proximity within the phylogenetic branch of B. melitensis strains previously isolated from Croatia, Albania, Kosovo and Bosnia and Herzegovina. Two isolates were determined to be B. melitensis bv. 3, while the third showed a unique phylogenetic profile, growth profile on dyes and bacteriophage typing results. This isolate contained the 609-bp omp31 sequence, but not the 723-bp omp31 sequence present in the two isolates of B. melitensis bv. 3. CONCLUSIONS: Identification of a novel Brucella variant in this geographic region is predictable given the historic endemicity of brucellosis. The emergence of a new variant may reflect a combination of high prevalence among domestic ruminants and humans as well as weak eradication strategies. The zoonotic potential, reservoirs and transmission pathways of this and other Brucella variants should be explored.

Brucella microti-like prevalence in French farms producing frogs.

In the last 10 years, many atypical novel members of Brucella species have been reported, including several Brucella inopinata-like strains in wild-caught and "exotic" amphibians from various continents. In 2017, a strain of Brucella was isolated for the first time in animals from a French farm producing frogs-Pelophylax ridibundus-for human consumption and identified as B. microti-like. Following this first isolation, investigations were performed in this farm as well as in the farm of the research unit that provided the domestic frog strain to estimate the prevalence of B. microti-like infection and its presence in the surrounding environment. Farming practices were investigated and samples including frogs at different development stages, surface tank swabs, water, feed and soil were analysed by real-time PCR and bacteriological methods. High B. microti-like prevalence values (higher than 90%) were obtained in frog samples in the commercial farm, and its presence was highlighted in the environmental samples except feed. In the research unit farm, B. microti-like species was also isolated and detected in frog and environmental samples. These results show that B. microti-like organisms are able to colonize amphibians and persist in their environment. Its presence could constitute a possible risk for consumers and workers proving the importance of assessing the zoonotic and pathogenic potentials of these new and atypical Brucella species.

Brucella melitensis Rev.1 vaccination generates a higher shedding risk of the vaccine strain in Alpine ibex (Capra ibex) compared to the domestic goat (Capra hircus).

Epidemiological investigations implemented in wild and domestic ruminants evidenced a reservoir for Brucella in Capra ibex in the French Alps. Vaccination was considered as a possible way to control Brucella infection in this wildlife population. Twelve ibexes and twelve goats were allocated into four groups housed separately, each including six males or six non-pregnant females. Four to five animals were vaccinated and one or two animals were contact animals. Half of the animals were necropsied 45 days post-vaccination (pv), and the remaining ones at 90 days pv. Additional samples were collected 20 and 68 days pv to explore bacterial distribution in organs and humoral immunity. Neither clinical signs nor Brucella-specific lesions were observed and all vaccinated animals seroconverted. Brucella distribution and antibody profiles were highly contrasted between both species. Proportion of infected samples was significantly higher in ibex compared to goats and decreased between 45 and 90 days pv. Two male ibex presented urogenital excretion at 20 or 45 days pv. The bacterial load was higher 45 days in ibexes compared to goats, whereas it remained moderate to low 90 days pv in both species with large variability between animals. In this experiment, differences between species remained the main source of variation, with low impact of other individual factors. To conclude, multiplicative and shedding capacity of Rev.1 was much higher in ibex compared to goats within 90 days. These results provide initial information on the potential use in natura of a commercial vaccine.

Lethality of Brucella microti in a murine model of infection depends on the wbkE gene involved in O-polysaccharide synthesis.

Brucella microti was isolated a decade ago from wildlife and soil in Europe. Compared to the classical Brucella species, it exhibits atypical virulence properties such as increased growth in human and murine macrophages and lethality in experimentally infected mice. A spontaneous rough (R) mutant strain, derived from the smooth reference strain CCM4915T, showed increased macrophage colonization and was non-lethal in murine infections. Whole-genome sequencing and construction of an isogenic mutant of B. microti and Brucella suis 1330 revealed that the R-phenotype was due to a deletion in a single gene, namely wbkE (BMI_I539), encoding a putative glycosyltransferase involved in lipopolysaccharide (LPS) O-polysaccharide biosynthesis. Complementation of the R-strains with the wbkE gene restored the smooth phenotype and the ability of B. microti to kill infected mice. LPS with an intact O-polysaccharide is therefore essential for lethal B. microti infections in the murine model, demonstrating its importance in pathogenesis.

The Glutaminase-Dependent Acid Resistance System: Qualitative and Quantitative Assays and Analysis of Its Distribution in Enteric Bacteria.

Neutralophilic bacteria have developed several strategies to overcome the deleterious effects of acid stress. In particular, the amino acid-dependent systems are widespread, with their activities overlapping, covering a rather large pH range, from 6 to <2. Recent reports showed that an acid resistance (AR) system relying on the amino acid glutamine (AR2_Q), the most readily available amino acid in the free form, is operative in Escherichia coli, Lactobacillus reuteri, and some Brucella species. This system requires a glutaminase active at acidic pH and the antiporter GadC to import L-glutamine and export either glutamate (the glutamine deamination product) or GABA. The latter occurs when the deamination of glutamine to glutamate, via acid-glutaminase (YbaS/GlsA), is coupled to the decarboxylation of glutamate to GABA, via glutamate decarboxylase (GadB), a structural component of the glutamate-dependent AR (AR2) system, together with GadC. Taking into account that AR2_Q could be widespread in bacteria and that until now assays based on ammonium ion detection were typically employed, this work was undertaken with the aim to develop assays that allow a straightforward identification of the acid-glutaminase activity in permeabilized bacterial cells (qualitative assay) as well as a sensitive method (quantitative assay) to monitor in the pH range 2.5-4.0 the transport of the relevant amino acids in vivo. The qualitative assay is colorimetric, rapid and reliable and provides several additional information, such as co-occurrence of AR2 and AR2_Q in the same bacterial species and assessment of the growth conditions that support maximal expression of glutaminase at acidic pH. The quantitative assay is HPLC-based and allows to concomitantly measure the uptake of glutamine and the export of glutamate and/or GABA via GadC in vivo and depending on the external pH. Finally, an extensive bioinformatic genome analysis shows that the gene encoding the glutaminase involved in AR2_Q is often nearby or in operon arrangement with the genes coding for GadC and GadB. Overall, our results indicate that AR2_Q is likely to be of prominent importance in the AR of enteric bacteria and that it modulates the enzymatic as well as antiport activities depending on the imposed acidic stress.

The Glutaminase-Dependent System Confers Extreme Acid Resistance to New Species and Atypical Strains of Brucella.

Neutralophilic bacteria have developed specific mechanisms to cope with the acid stress encountered in environments such as soil, fermented foods, and host compartments. In Escherichia coli, the glutamate decarboxylase (Gad)-dependent system is extremely efficient: it requires the concerted action of glutamate decarboxylase (GadA/GadB) and of the glutamate (Glu)/γ-aminobutyrate antiporter, GadC. Notably, this system is operative also in new strains/species of Brucella, among which Brucella microti, but not in the "classical" species, with the exception of marine mammals strains. Recently, the glutaminase-dependent system (named AR2_Q), relying on the deamination of glutamine (Gln) into Glu and on GadC activity, was described in E. coli. In Brucella genomes, a putative glutaminase (glsA)-coding gene is located downstream of the gadBC genes. We found that in B. microti these genes are expressed as a polycistronic transcript. Moreover, using a panel of Brucella genus-representative strains, we show that the AR2_Q system protects from extreme acid stress (pH ≤2.5), in the sole presence of Gln, only the Brucella species/strains predicted to have functional glsA and gadC. Indeed, mutagenesis approaches confirmed the involvement of glsA and gadC of B. microti in AR2_Q and that the acid-sensitive phenotype of B. abortus can be ascribed to a Ser248Leu substitution in GlsA, leading to loss of glutaminase activity. Furthermore, we found that the gene BMI_II339, of unknown function and downstream of the gadBC-glsA operon, positively affects Gad- and GlsA-dependent AR. Thus, we identified novel determinants that allow newly discovered and marine mammals Brucella strains to be better adapted to face hostile acidic environments. As for significance, this work may contribute to the understanding of the host preferences of Brucella species and opens the way to alternative diagnostic targets in epidemiological surveillance of brucellosis.

Gene and protein expression in response to different growth temperatures and oxygen availability in Burkholderia thailandensis.

Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors.