Combination of in silico and molecular techniques for discrimination and virulence characterization of marine Brucella ceti and Brucella pinnipedialis.

Introduction: Mammals are the main hosts for Brucella sp., agents of worldwide zoonosis. Marine cetaceans and pinnipeds can be infected by Brucella ceti and B. pinnipedialis, respectively. Besides classical bacteriological typing, molecular approaches such as MLVA, MLSA, and whole-genome sequencing (WGS) can differentiate these species but are cumbersome to perform. Methods: We compared the DNA and genome sequences of 12 strains isolated from nine marine mammals, with highly zoonotic B. melitensis, B. abortus, and B. suis, and the publicly available genomes of B. ceti and B. pinnipedialis. In silico pipelines were used to detect the antimicrobial resistance (AMR), plasmid, and virulence genes (VGs) by screening six open-source and one home-made library. Results and discussion: Our results show that easier-to-use HRM-PCR, Bruce-ladder, and Suis-ladder can separate marine Brucella sp., and the results are fully concordant with other molecular methods, such as WGS. However, the restriction fragment length polymorphism (RFLP) method cannot discriminate between B. pinnipedialis and B. ceti B1-94-like isolates. MLVA-16 results divided the investigated strains into three clades according to their preferred host, which was confirmed in WGS. In silico analysis did not find any AMR and plasmid genes, suggesting antimicrobial susceptibility of marine Brucella, while the presence of the VGs btpA gene was variable dependent on the clade. Conclusion: The HRM-PCR and Suis-ladder are quick, easy, and cost-effective methods to identify marine Brucella sp. Moreover, in silico genome analyses can give useful insights into the genetic virulence and pathogenicity potential of marine Brucella strains.

Epidemiology and pathogenicity of M. equirhinis in equine respiratory disorders.

Mycoplasmas are pathogens involved in respiratory disorders of various animal hosts. In horses, Mycoplasma (M.) equirhinis is the species most frequently detected in clinical respiratory specimens, with a prevalence of 12-16%, but its clinical implication in equine respiratory disorders remains unclear. Here we screened 1948 clinical specimens for the presence of M. equirhinis. The samples were both tracheal washes (TW) and bronchoalveolar lavages (BAL) collected by veterinarians in France in day-to-day work between 2020 and 2022. The samples were associated with a standardized form that served to collect key general and clinical information, such as horse age, breed, and living environment. M. equirhinis was detected using a combination of culture and post-enrichment PCR. Other diagnostic data included virology and bacteriology as well as neutrophil counts, when available. Prevalence of M. equirhinis was examined as a function of a clinical score based on four significant clinical signs (nasal discharge, cough, dyspnoea, and hyperthermia). Multivariate logistic regression analysis was run to identify risk factors for the presence of M. equirhinis, and comparative prevalence analysis was used to test for association with other bacteria and viruses. TW and BAL were analysed independently, as we found that TW samples were associated with a higher prevalence of M. equirhinis. As prevalence remained steady whatever the clinical score, M. equirhinis cannot be considered a primary pathogen. M. equirhinis was more frequently isolated in thoroughbreds and trotters and in horses living exclusively stabled compared to other horses or other living environments. M. equirhinis was never detected in BAL specimens with a 'normal' neutrophil count, i.e. 5%, suggesting it could be associated with an inflammatory response, similar to that observed in equine asthma. Prevalence of M. equirhinis was shown to increase in the presence of other bacteria such as Streptococcus equi subsp. zooepidemicus (S. zoo) or viruses, and S. zoo load was higher in M. equirhinis-positive samples, suggesting a potential increase of clinical signs in the event of co-infection.

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.

Detection of Mycoplasma spp. in horses with respiratory disorders.

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.

Seroprevalence and Risk Factors of Brucellosis in Ruminants in Dhofar Province in Southern Oman.

Objective: The aim of the present work was to raise awareness of Brucella infection and emphasize the use of serological tests for screening and confirmation of the presence of the infection in different localities in the Dhofar region in the Sultanate of Oman. Methods: A seroprevalence of Brucella infection in naturally infected livestock was undertaken in 50 farms (a total of 434 sera, 207 goats, 84 sheep, 54 cattle, and 89 camels) from different wilayat of the Dhofar region in the southern part of Oman. Rose Bengal (RBT), complement fixation (CFT), and indirect enzyme-linked immunosorbent assay (I-ELISA) tests were used to determine the presence of Brucella antibodies. Statistical analysis (Pearson chi-square, binary logistic regression, and univariate logistic regression) was used to investigate the significance between the prevalence and the categorical risk factors individually, with two or more levels (animal species, animal condition, and or location). Results: Our results show that the overall seroprevalence based on CFT, RBT, and I-ELISA was 3% (13/424, CI: 1.8-5.1%), 4.8% (21/434, CI: 3.1-7.3%), and 8% (35/434, CI: 5.8-10.9%), respectively. The highest seroprevalence was reported in goats (13% (27/207)) and animals from East Jabal (13% (21/161)), whereas the lowest was recorded in camels (3.4% (3/89)) and animals from deserts (1.4% (1/69)). Parameters such as the positive predictive value (PPV) and the negative predictive value (NPV) showed that the sensitivity of I-ELISA and CFT based on the RBT test was 61.9% and 57.1%, respectively, whereas the specificity of I-ELISA (94.6%) was less than that of CFT (97.33%). Conclusion: We concluded that three tests are confirmatory for the presence of Brucella infection.

Addressing the Antimicrobial Resistance of Ruminant Mycoplasmas Using a Clinical Surveillance Network.

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.

Population structure and antimicrobial susceptibility of Mycoplasma ovipneumoniae isolates in France.

Chronic non-progressive pneumonia in small ruminants caused by Mycoplasma (M.) ovipneumoniae is mainly controlled by chemotherapy. In France, during the last decade, a rise in M. ovipneumoniae cases was recorded in both sheep and goats, suggesting a possible emergence. Whether this rise is associated with antimicrobial resistance, as observed in other ruminant Mycoplasma species, has yet to be examined. The aim of the study was to characterize the diversity of M. ovipneumoniae strains circulating in France and assess their antimicrobial resistance, together with the underlying mechanisms, to help find an explanation for the increase in reported cases. The genetic diversity of 56 strains isolated between 2007 and 2018 from sheep and goats was assessed using different subtyping methods. Their susceptibility to six antimicrobial classes was profiled by estimating Minimum Inhibitory Concentrations (MICs) using an optimised agar dilution method. Resistance mechanisms were explored by sequence analysis of rRNA targets. A high genetic diversity of strains was evidenced, with consistent, marked animal-host clustering in the Hsp70 gene and whole genome sequence phylogeny. No clonal evolution could thus account for putative emergence. Apart from florfenicol, MICs were low except for a few isolates with increased values for tetracyclines, macrolides and lincosamides. Hotspot mutations in the target ribosomal gene could explain increased tetracycline MICs. Other mechanisms are suspected for macrolide-lincosamide and florfenicol resistance. The emergence of M. ovipneumoniae is thus not related to any increase in resistance or to a clonal spread. Explanations may lie in breeding practices.

Correction to: Investigation on Brucella infection in farm animals in Saham, Sultanate of Oman with reference to human brucellosis outbreak.

The original article [1] incorrectly presents final author, Eugene H. Johnson's name incorrectly whereby middle initial, 'H.' is mistakenly presented as a Family Name.

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.