Non-aureus staphylococci (NAS) are the most frequently isolated pathogens from intramammary infection (IMI) in dairy cattle. Virulence factors (VFs) and mechanisms by which NAS cause IMI are not fully known. Herein, we analyzed the distribution of 191 VFs in 441 genomes of 25 NAS species, after classifying VFs into functional categories: adherence (n = 28), exoenzymes (n = 21), immune evasion (n = 20), iron metabolism (n = 29), and toxins (n = 93). In addition to establishing VF gene profiles, associations of VF genes between and among functional categories were computed, revealing distinctive patterns of association among VFs for various NAS species. Associations were also computed for low, medium, and high somatic cell count (SCC) and clinical mastitis (CM) isolates, demonstrating distinctive patterns of associations for low SCC and CM isolates, but no differences between high SCC and CM isolates. To determine whether VF distributions had any association with SCC or CM, various clustering approaches, including complete linkages, Ward clustering, and t-distributed stochastic neighbor embedding, were applied. However, no clustering of isolates representing low SCC, medium SCC, or high SCC or CM was identified. Regression analysis to test for associations with individual VF functional categories demonstrated that each additional toxin and host immune evasion gene increased the odds of having high SCC or CM, although an overall increase in the number of VFs was not associated with increased SCC or occurrence of CM. In conclusion, we established comprehensive VF gene profiling, determined VF gene distributions and associations, calculated pathogenic potentials of all NAS species, and detected no clear link between VF genes and mastitis. IMPORTANCE Non-aureus staphylococci (NAS) are the most frequently isolated pathogens from milk in dairy cattle worldwide. The virulence factors (VFs) and mechanisms by which these bacteria cause udder infection are not fully known. We determined the distribution and associations of 191 VFs in 25 NAS species and investigated the relationship between VFs and disease. Although the overall number of VFs was not associated with disease severity, increasing numbers of toxin and host immune evasion genes specifically were associated with more severe disease outcomes. These findings suggest that the development of disease and the interactions of VFs with the host are complex and determined by the interplay of genes rather than just the presence of virulence genes. Together, our results provide foundational genetic knowledge to other researchers to design and conduct further experiments, focusing on understanding the synergy between VFs and roles of individual NAS species in IMI and characterizing species-specific effects on udder health.

Emergence and spread of antimicrobial resistance is a major concern for the dairy industry worldwide. Objectives were to determine: (1) phenotypic and genotypic prevalence of drug-specific resistance for 25 species of non-aureus staphylococci, and (2) associations between presence of resistance determinants and antimicrobial resistance. Broth micro-dilution was used to determine resistance profiles for 1,702 isolates from 89 dairy herds. Additionally, 405 isolates were sequenced to screen for resistance determinants. Antimicrobial resistance was clearly species-dependent. Resistance to quinupristin/dalfopristin was common in Staphylococcus gallinarum (prevalence of 98%), whereas S. cohnii and S. arlettae were frequently resistant to erythromycin (prevalence of 63 and 100%, respectively). Prevalence of resistance was 10% against β-lactams and tetracyclines. In contrast, resistance to antimicrobials critically important for human medicine, namely vancomycin, fluoroquinolones, linezolid and daptomycin, was uncommon (< 1%). Genes encoding multidrug-resistance efflux pumps and resistance-associated residues in deducted amino acid sequences of the folP gene were the most frequent mechanisms of resistance, regardless of species. The estimated prevalence of the mecA gene was 17% for S. epidermidis. Several genes, including blaZ, mecA, fexA, erm, mphC, msrA, and tet were associated with drug-specific resistance, whereas other elements were not. There were specific residues in gyrB for all isolates of species intrinsically resistant to novobiocin. This study provided consensus protein sequences of key elements previously associated with resistance for 25 species of non-aureus staphylococci from dairy cattle. These results will be important for evaluating effects of interventions in antimicrobial use in Canadian dairy herds.

ABSTRACT Staphylococcus aureus causes persistent clinical and subclinical bovine intramammary infections (IMI) worldwide. However, there is a lack of comprehensive information regarding genetic diversity, the presence of antimicrobial resistance (AMR), and virulence genes for S. aureus in bovine milk in Canada. Here, we performed whole-genome sequencing (WGS) of 119 Canadian bovine milk S. aureus isolates and determined they belonged to 8 sequence types (ST151, ST352, ST351, ST2187, ST2270, ST126, ST133, and ST8), 5 clonal complexes (CC151, CC97, CC126, CC133, and CC8), and 18 distinct Spa types. Pan-, core, and accessory genomes were composed of 6,340, 1,279, and 2,431 genes, respectively. Based on phenotypic screening for AMR, resistance was common against beta-lactams (19% of isolates) and sulfonamides (7% of isolates), whereas resistance against pirlimycin, tetracycline, ceftiofur, and erythromycin and to the combination of penicillin and novobiocin was uncommon (3, 3, 3, 2, and 2% of all isolates, respectively). We also determined distributions of 191 virulence factors (VFs) in 119 S. aureus isolates after classifying them into 5 functional categories (adherence [n = 28], exoenzymes [n = 21], immune evasion [n = 20], iron metabolism [n = 29], and toxins [n = 93]). Additionally, we calculated the pathogenic potential of distinct CCs and STs and determined that CC151 (ST151 and ST351) had the highest pathogenic potential (calculated by subtracting core-VFs from total VFs), followed by CC97 (ST352 and ST2187) and CC126 (ST126 and ST2270), potentially linked to their higher prevalence in bovine IMI worldwide. However, there was no statistically significant link between the presence of VF genes and mastitis. IMPORTANCE Staphylococcus aureus is a major cause of bovine intramammary infections, leading to significant economic losses to dairy industry in Canada and worldwide. There is a lack of knowledge regarding genetic diversity, the presence of antimicrobial resistance (AMR), and virulence genes for S. aureus isolated from bovine milk in Canada. Based on whole-genome sequencing and genomic analysis, we have determined the phylogeny and diversity of S. aureus in bovine milk and concluded that it had a large accessory genome, limited distribution of AMR genes, variable VF gene profiles and sequence types (ST), and clonal complex (CC)-specific pathogenic potentials. Comprehensive information on the population structure, as well as the virulence and resistance characteristics of S. aureus from bovine milk, will allow for source attribution, risk assessment, and improved therapeutic approaches in cattle.

Digital dermatitis (DD) presents as painful, ulcerative or proliferative lesions that lead to bovine lameness affecting economic efficiency and animal welfare. Although DD etiological agent(s) have not been established, it is widely accepted that DD is a polymicrobial disease significantly associated with species of Treponema and the non-linear disease progression may be attributed to interactions among infecting bacteria. We postulated the morphological changes associated with DD lesion grades are related to interactions among infecting species of Treponema. We developed a novel species-specific qPCR that can identify the absolute abundance of the four of the most common species of Treponema in DD, T. phagedenis, T. medium, T. pedis and T. denticola, in a single reaction. We found species abundance and the number of distinct Treponema species present is higher in active, ulcerative lesions than in healing lesions, chronic lesions, and DD-free skin. Treponema spp. were present in both DD-free skin and M3 lesions following treatment with oxytetracycline. We have also found positive correlations among T. phagedenis, T. medium and T. pedis indicating they are significantly more likely to be found together than apart and their absolute quantities tend to increase together, a relationship which is not present with T. denticola. Further, we found Treponema, particularly viable T. denticola, in lesions 5 days post treatment with oxytetracycline (M3). Our findings suggest that pathogenicity may be closely associated with Treponema abundance, particularly T. phagedenis, T. medium and T. pedis, and interactions among them, independent of T. denticola. Our results provide a novel, consistent method to identify species of Treponema within DD lesions and associate Treponema spp. and abundance with morphological changes related to host pathogenicity.Electronic supplementary materialThe online version of this article (10.1186/s13567-018-0605-z) contains supplementary material, which is available to authorized users.

Face coverings are a key component of preventive health measure strategies to mitigate the spread of respiratory illnesses. In this study five groups of masks were investigated that are of particular relevance to the SARS-CoV-2 pandemic: re-usable, fabric two-layer and multi-layer masks, disposable procedure/surgical masks, KN95 and N95 filtering facepiece respirators. Experimental work focussed on the particle penetration through mask materials as a function of particle diameter, and the total inward leakage protection performance of the mask system. Geometric mean fabric protection factors varied from 1.78 to 144.5 for the fabric two-layer and KN95 materials, corresponding to overall filtration efficiencies of 43.8% and 99.3% using a flow rate of 17 L/min, equivalent to a breathing expiration rate for a person in a sedentary or standing position conversing with another individual. Geometric mean total inward leakage protection factors for the 2-layer, multi-layer and procedure masks were <2.3, while 6.2 was achieved for the KN95 masks. The highest values were measured for the N95 group at 165.7. Mask performance is dominated by face seal leakage. Despite the additional filtering layers added to cloth masks, and the higher filtration efficiency of the materials used in disposable procedure and KN95 masks, the total inward leakage protection factor was only marginally improved. N95 FFRs were the only mask group investigated that provided not only high filtration efficiency but high total inward leakage protection, and remain the best option to protect individuals from exposure to aerosol in high risk settings. The Mask Quality Factor and total inward leakage performance are very useful to determine the best options for masking. However, it is highly recommended that testing is undertaken on prospective products, or guidance is sought from impartial authorities, to confirm they meet any implied standards.

Mastitis is the most prevalent and costly disease in dairy cattle worldwide, with implications for animal health and welfare as well as production and economics. Nonlactating heifers are an often-neglected group of animals concerning mastitis management, as they are assumed to be free of mastitis. An observational field study was conducted between 2007 and 2008 on 91 dairy herds across Canada, representative of provincial averages of bulk milk somatic cell count (BMSCC) and barn type. The aims of that study were to (1) estimate in early-lactating heifers overall and pathogen-specific incidence rate of clinical mastitis (IRCM), prevalence of intramammary infection (IMI), and prevalence of subclinical mastitis (SCM; defined as SCC ≥200,000 cells/mL); (2) compare these udder health parameters between heifers and multiparous cows; and (3) determine regional patterns and variations in these udder health parameters across BMSCC categories. During the first day of lactation, IRCM was higher in heifers than in multiparous cows (99 vs. 48 cases per 10,000 quarter-days at risk, respectively). Clinical mastitis affected 4% of heifers (0.73 cases per 100 quarters) in the first 30 d after calving, with the most common pathogens isolated being Staphylococcus aureus and Escherichia coli, whereas S. aureus and non-aureus staphylococci were the most commonly isolated pathogens in multiparous cows. The IRCM in heifers was highest in Ontario heifers, but overall IRCM did not vary by BMSCC category and it was only higher in multiparous cows than heifers in high-BMSCC Ontario herds. Intramammary infections were present in 33% of heifer quarters, with non-aureus staphylococci the most commonly isolated group of bacteria in both heifers (26% of quarters) and multiparous cows (18% of quarters). Pathogen-specific prevalence of IMI did not differ between heifers and multiparous cows, but we noted regional differences and differences across BMSCC categories in pathogen-specific prevalence of IMI. Prevalence of SCM in heifers was 13.6% and was lowest in Alberta herds. In all regions, SCM prevalence was higher in multiparous cows than in heifers. In conclusion, udder health of Canadian dairy heifers was similar to that of other countries, demonstrating the importance of the issue. Differences between heifers and multiparous cows early in lactation highlighted the need for management practices to target the precalving period in heifers, when exposure to risk factors differs from that in lactating cows.

Assessment of antimicrobial use (AMU) is vital for interpreting the origin of changes in antimicrobial resistance (AMR). The objectives of the present study were to estimate the association between AMU determined using on-farm treatment records (TR) and inventory of empty drug containers (INV). Herds were selected to represent Canadian dairy farms. Producers were asked to record animal health events and treatments on a standard General Health Event form. For inventory data, 40-L receptacles were placed at various locations considered convenient to deposit all empty drug containers. Antimicrobial defined-daily dosages (ADD) were calculated for 51 Canadian herds using the 2 methods. Estimation of AMU was 31,840 ADD using the INV and 14,487 ADD using the TR, indicating that for every TR entry, 2.20 times more treatments were observed using the INV. Mastitis, reproductive conditions, and dry cow therapy were the most frequent reasons for antimicrobial therapy when assessing TR. For all antimicrobials evaluated, mean ADD was higher using the INV versus TR. Regardless, a strong positive correlation (0.80) was observed between the 2 methods, indicating that herds with increased number of ADD recorded using the INV also had increased number of ADD recorded using TR. Furthermore, a positive association was observed for the 6 most commonly used antimicrobials. In comparison to methods used in surveillance programs on AMU in livestock that assume a constant use in all herds (i.e., sales data), INV provided a herd-level specific quantity of AMU positively correlated with AMU recorded at the animal level in general. The INV was easy to implement and provided a measure of total AMU in the herd. Availability of such information would be valuable for interpreting changes in AMR at the herd level and enabling evaluation of interventions for decreasing AMR.

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