Maryne Jay

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.

Background: Bacteria belonging to the genus Mycoplasma are small‐sized, have no cell walls and small genomes. They commonly cause respiratory disorders in their animal hosts. Three species have been found in the respiratory tract of horses worldwide, that is., Mycoplasma ( M. ) equirhinis , M. pulmonis and M. felis , but their role in clinical cases remains unclear. Objectives: The aim of this study was to i) develop and validate tools to detect, isolate and identify different Mycoplasma spp. strains in clinical equine respiratory‐tract specimens and ii) subsequently define the prevalence of the three species in France depending on sample types and horse characteristics (age, breed, sex). Study design: Validation of a workflow for mycoplasma diagnosis and subsequent prevalence study. Methods: Mycoplasma‐free tracheal wash samples spiked with numerated strains and DNA dilutions were used to validate the culture methods and real‐time PCR (rt‐PCR) assay. Isolated strains were identified by 16S rRNA gene sequencing. Prevalences were determined on a population of 616 horses with respiratory disorders, sampled in France in 2020. Results: In total, 104 horses (16.9%) were found to be positive for Mycoplasma spp. by at least one method. M. equirhinis was the predominant circulating species, accounting for 85% of the rt‐PCR‐positive samples and 98% of the 40 cultured strains. Main limitation: The proposed pre‐enrichment procedure improves the sensitivity of detection but hinders the quantification of the initial mycoplasma load in the clinical specimens. Conclusions: Prevalence of mycoplasma varied with age, breed, and type of sample.

Antimicrobial resistance (AMR) surveillance of mycoplasmas of veterinary importance has been held back for years due to lack of harmonized methods for antimicrobial susceptibility testing (AST) and interpretative criteria, resulting in a crucial shortage of data. To address AMR in ruminant mycoplasmas, we mobilized a long-established clinical surveillance network called “Vigimyc.” Here we describe our surveillance strategy and detail the results obtained during a 2-year monitoring period. We also assess how far our system complies with current guidelines on AMR surveillance and how it could serve to build epidemiological cut-off values (ECOFFs), as a first attainable criterion to help harmonize monitoring efforts and move forward to clinical breakpoints. Clinical surveillance through Vigimyc enables continuous collection, identification and preservation of Mycoplasma spp. isolates along with metadata. The most frequent pathogens, i.e., M. bovis and species belonging to M. mycoides group, show stable clinicoepidemiological trends and were included for annual AST. In the absence of interpretative criteria for ruminant mycoplasmas, we compared yearly minimum inhibitory concentration (MIC) results against reference datasets. We also ran a SWOT (Strengths, Weaknesses, Opportunities, Threats) analysis on the overall service provided by our AMR surveillance strategy. Results of the 2018–2019 surveillance campaign were consistent with the reference datasets, with M. bovis isolates showing high MIC values for all antimicrobial classes except fluoroquinolones, and species of the Mycoides group showing predominantly low MIC values. A few new AMR patterns were detected, such as M. bovis with lower spectinomycin MICs. Our reference dataset partially complied with European Committee on Antimicrobial Susceptibility Testing (EUCAST) requirements, and we were able to propose tentative epidemiological cut-off values (TECOFFs) for M. bovis with tilmicosin and spectinomycin and for M. mycoides group with tilmicosin and lincomycin. These TECOFFs were consistent with other published data and the clinical breakpoints of Pasteurellaceae, which are often used as surrogates for mycoplasmas. SWOT analysis highlighted the benefit of pairing clinical and antimicrobial resistance surveillance despite the AST method-related gaps that remain. The international community should now direct efforts toward AST method harmonization and clinical interpretation.

Contagious agalactia (CA) is a disease caused equally by four Mycoplasma species, in single or mixed infections. Clinical signs are multiple, including mastitis, arthritis, keratoconjunctivitis, pneumonia, and septicemia, non-specific, and expressed differently depending whether sheep or goats are affected, on causative mycoplasmas as well as type of husbandry. CA has been reported worldwide and its geographic distribution maps to that of small ruminant breeding areas. However, as current diagnostic tests are expensive and difficult to implement, it is certainly underdiagnosed and prevalence data are only available for a few countries. CA control relies on vaccines, chemotherapy and good herd management practices. It requires long-term commitment but is often unsuccessful, with frequent clinical relapses. The persistence of the etiological agents, despite their overall susceptibility to antimicrobials, comes from their genetic plasticity and capacity to escape the host immune response. The existence of asymptomatic carriers and the numerous sources of infections contribute to rapid spread of the disease and complicate the control and prevention efforts. Here we review all these aspects in order to highlight recent progress made and identify gaps in knowledge or tools needed for better disease management. Discussion also underlines the detrimental effect of contagious agalactia on small ruminant welfare.

Several Brucella isolates have been described in wild-caught and “exotic” amphibians from various continents and identified as B. inopinata-like strains. On the basis of epidemiological investigations conducted in June 2017 in France in a farm producing domestic frogs (Pelophylax ridibundus) for human consumption of frog's legs, potentially pathogenic bacteria were isolated from adults showing lesions (joint and subcutaneous abscesses). The bacteria were initially misidentified as Ochrobactrum anthropi using a commercial identification system, prior to being identified as Brucella spp. by MALDI-TOF assay. Classical phenotypic identification confirmed the Brucella genus, but did not make it possible to conclude unequivocally on species determination. Conventional and innovative bacteriological and molecular methods concluded that the investigated strain was very close to B. microti species, and not B. inopinata-like strains, as expected. The methods included growth kinetic, antimicrobial susceptibility testing, RT-PCR, Bruce-Ladder, Suis-Ladder, RFLP-PCR, AMOS-ERY, MLVA-16, the ectoine system, 16S rRNA and recA sequence analyses, the LPS pattern, in silico MLST-21, comparative whole-genome analyses (including average nucleotide identity ANI and whole-genome SNP analysis) and HRM-PCR assays. Minor polyphasic discrepancies, especially phage lysis and A-dominant agglutination patterns, as well as, small molecular divergences suggest the investigated strain should be considered a B. microti-like strain, raising concerns about its environmental persistence and unknown animal pathogenic and zoonotic potential as for other B. microti strains described to date.

Canine brucellosis is a major underestimated zoonosis that remains endemic in many areas of the world. A recent phylogeographic investigation including 53 Brucella canis field isolates revealed the existence of two major lineages worldwide.

Bovine brucellosis is a worldwide zoonotic disease that still burdens several countries in the Mediterranean, Asia, Africa and Latin America. Although the disease is present in Ecuador, the Galapagos Islands seem to be free from the disease based on a survey conducted in 1997 where all tested animals showed negative results. This study aimed at estimating the probability of freedom from brucellosis in this Ecuadorian province in 2014. A survey was implemented on the three main cattle-producing islands of the province: Santa Cruz, Isabela and San Cristóbal. Thirty-three cattle farms and 410 cattle were tested for brucellosis using the Rose Bengal test and indirect ELISA. All animals showed negative results for both tests. Probability of freedom was estimated at 98%, 91% and 88% for Santa Cruz, Isabela and San Cristóbal, respectively, considering a herd-level design seroprevalence of 20% and animal-level design seroprevalence of 15%, and assuming a perfect specificity of the survey. The negative results found in 1997 and present surveys suggest that the Galapagos Islands are free from bovine brucellosis.

Canine brucellosis, due to Brucella canis, is a worldwide zoonosis that remains endemic in South America, including Brazil. Implementation of powerful whole-genome sequencing approaches allowed exploring the Brucella genus considered as monomorphic, with, to date, more than 500 genomes available in public databases. Nevertheless, with under-representation of B. canis genomes −only twenty complete or draft genomes−, lack of knowledge about this species is still considerable. This report describes a comparative genomics-based phylogeographic investigation of 53 B. canis strains, including 28 isolates paired-end sequenced in this work. Obtained results allow identifying a SNP panel species-specific to B. canis of 1086 nucleotides. In addition, high-resolution analyses assess the epidemiological relationship between worldwide isolates. Our findings show worldwide strains are distributed among 2 distinct lineages. One of them seems to be specific to South American strains, including Brazil. B. canis South American strains may be identified by a SNP panel of 15 nucleotides, whereas a 22 SNP panel is sufficient to define contamination origin from Brazil. These results lead to the proposal of a possible spread route for dog brucellosis through South America. Additionally, whole-genome analyses highlight the remarkable genomic stability of B. canis strains over time and the sustainability of the infection in São Paulo over 12 year-period. Significant increase of B. canis genomes available in public databases provides new insights into B. canis infection in South America, including Brazil, as well as in the world, and also offers new perspectives for the Brucella genus largo sensu.

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.