Non-aureus Staphylococci and Bovine Udder Health: Current Understanding and Knowledge Gaps.

Despite considerable efforts to control bovine mastitis and explain its causes, it remains the most costly and common disease of dairy cattle worldwide. The role and impact of non-aureus staphylococci (NAS) in udder health are not entirely understood. These Gram-positive bacteria have become the most frequently isolated group of bacteria in milk samples of dairy cows and are associated with (mild) clinical and subclinical mastitis. Different species and strains of NAS differ in their epidemiology, pathogenicity, virulence, ecology and host adaptation, and antimicrobial resistance profiles. They have distinct relationships with the microbiome composition of the udder and may also have protective effects against other mastitis pathogens. Some appear to persist on the skin and in the teat canal and udder, while others seem to be transient residents of the udder from the environment. Analyzing genotypic and phenotypic differences in individual species may also hold clues to why some appear more successful than others in colonizing the udder. Understanding species-level interactions within the microbiome and its interactions with host genetics will clarify the role of NAS in bovine mastitis and udder health.

Ownership structure and financial status of Canadian dairy operations.

The ownership structure and financial status of Canadian dairy farms is described using Statistics Canada 2016 Census of Agriculture data. As herd size increased, family corporation became the most common ownership model after accounting for herd location by region. Regardless of location, gross farm receipts, operating expenses and profit margin increased significantly with herd size. Western Canadian dairies occupied significantly larger land bases and had significantly higher profit margins compared to all other regions. New trade agreements could affect the financial stability of Canada's dairy industry; these data provide a baseline for future comparison.

Structure de propriété et statut financier des opérations laitières canadiennes. La structure de propriété et le statut financier des fermes laitières canadiennes sont décrits en utilisant les données de Statistiques Canada du Recensement en agriculture de 2016. À mesure que la taille du troupeau augmentait, la corporation familiale devenait le modèle de propriété le plus fréquent après avoir pris en compte la localisation par région du troupeau. Sans égard à la localisation, le revenu brut par ferme, les dépenses d'opération et la marge de profit augmentèrent de manière significative avec la taille du troupeau. Les fermes laitières de l'ouest canadien occupaient des fonds de terre significativement plus grands et avaient des marges de profit significativement plus élevées comparativement à toutes les autres régions. Les nouvelles ententes d'échanges commerciaux pourraient affecter la stabilité financière de l'industrie laitière canadienne; ces données fournissent une mesure de base pour des comparaisons futures.(Traduit par Dr Serge Messier).

Update on demographics of the Canadian Dairy Industry for the period 2011 to 2016.

Between 2011 and 2016, the number of dairy operations in Canada decreased by 13.3%. Mean herd size increased from 65 to 73 animals per farm. The ratio of older (> 60 years old) to younger (< 31 years old) producers also increased. The age structure of the industry suggests that consolidation will continue for the next decade, which has implications for the provision of veterinary services to dairy operations.

Actualisation des données démographiques des exploitations laitières pendant la période de 2011 à 2016. De 2011 à 2016, le nombre d'exploitations laitières au Canada a diminué de 13,3 %. La taille moyenne des exploitations a augmenté de 65 à 73 animaux par ferme. Le ratio d'éleveurs plus âges (> 60 ans) de ceux plus jeunes (< 31 ans) a augmenté aussi. La structure par âge suggère que cette consolidation doive se poursuivra pendant la prochaine décennie et devrait toucher l'offre des services vétérinaires aux exploitations laitières.(Traduit par les auteurs).

Comprehensive Virulence Gene Profiling of Bovine Non-aureus Staphylococci Based on Whole-Genome Sequencing Data.

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.

Experimental and Theoretical Validation of System Variables That Control the Position of Particles at the Interface of Immiscible Liquids.

We construct a mathematical model describing the equilibrium flotation height of a spherical particle at the interface of immiscible liquids. The behavior of such a system depends on several experimentally measurable parameters, which include surface tensions, densities of all phases, and system scale. These parameters can be absorbed into three quantities that entirely determine the equilibrium position of the particle: the contact angle between the interface and particle, the Bond number, and the ratio of particle buoyant density to liquid phase densities-a new, dimensionless number that we introduce here. This experimentally convenient treatment allows us to make predictions that apply generally to the large parameter space of interesting systems. We find the model is in good agreement with experiments for particle size and interfacial tension spanning 3 orders of magnitude. We also consider the low interfacial tension case of aqueous two-phase systems (ATPSs) theoretically and experimentally. Such systems are more sensitive to changes in density than higher-tension aqueous/organic two-phase systems; we experimentally demonstrate that a millimeter-sized bead in an ATPS can be controllably positioned with between 5.9 and 95.1% of its surface area exposed to the bottom phase, whereas the same bead in an aqueous/organic system is limited to a range of 18.2-61.6%. Finally, we discuss the potential for wettability-based control for micron length-scale particles, which are not sensitive to changes in density. Our results can be used to simply define the experimentally controllable parameters that affect the equilibrium position and the length scales of a particle over which such parameters can be effectively tuned. A complete understanding of these properties is important for a number of applications including colloidal self-assembly and chemical patterning (e.g., formation of desymmetrized or Janus particles). By considering ATPSs, we broaden the potential uses to biological applications such as cell separation and interfacial tissue assembly.

Prevalence and Genetic Basis of Antimicrobial Resistance in Non-aureus Staphylococci Isolated from Canadian Dairy Herds.

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 ß-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.

Enrichment and Recovery of Mammalian Cells from Contaminated Cultures Using Aqueous Two-Phase Systems.

This Article describes a density-based method for removing contaminants, including microorganisms and nonviable cells, from mammalian cell cultures using an aqueous two-phase system (ATPS). The properties of a 7% w/w polyethylene glycol (PEG)-11% w/w Ficoll ATPS can be tuned to prepare a biocompatible system that removes contaminants with little to no adverse effects on the viability or growth of the cultured cells after treatment. This system can be used to enrich cell culture populations for viable cells and to reduce the number of microorganism contaminants in a culture, which increases the chances of subsequent antibiotic treatments being successful. We test the effectiveness of our method in model contaminated cultures of both adherent (HeLa) and suspension (HL-60 II) mammalian cells contaminated with bacteria (E. coli) and yeast (S. cerevisiae). An average of 70.2 ± 4.6% of HeLa cells added to the system are subsequently recovered, and 55.9 ± 2.1% of HL-60 II cells are recovered. After sedimenting to the interface of the ATPS, these cells have an average viability of 98.0 ± 0.2% and 95.3 ± 2.2%, respectively. By removing unwanted cells, desired cell populations can be recovered, and cultures that would otherwise need to be discarded can continue to be used.

Density separation of quiescent yeast using iodixanol.

As yeast are starved of nutrients, they enter G0, a quiescent state. Quiescent yeast (Q) cells retain viability for extended periods of time and resume growth following supplementation of missing nutrients. As such, Q cells have become a valuable model for studying longevity and self-renewal of chronologically aged cells. Traditional isolation of Q cells involves a relatively long centrifugation time through a continuous density gradient. Here, we describe a rapid and cost-effective Q-cell isolation technique that uses a single-density, one-step gradient prepared from media containing iodixanol.

Efficacy of recombinant bovine epidermal growth factor in the treatment of experimental subclinical Staphylococcus aureus mastitis in a ewe model.

Staphylococcus aureus is the most common contagious mastitis pathogen of dairy cattle. Antimicrobial treatment of infected cattle results in variable cure rates. Epidermal growth factor (EGF) plays an important role in the modulation of host innate immune responses and the regulation of mammary epithelial regeneration, indicating that EGF may be useful as a treatment for mastitis. A pilot study was conducted to evaluate the efficacy of recombinant bovine EGF (rbEGF) for the treatment of S aureus intramammary infection (IMI) using an ovine model. Each ewe was experimentally infected with S aureus in both udder halves. One udder half of each ewe received one of two treatments: EGF (n=13) or pirlimycin (n=13). The contralateral udder half of each ewe received sterile saline as a control. The bacteriological cure rate following rbEGF was significantly lower (15 per cent) than that attained with pirlimycin hydrochloride (61 per cent) and did not differ from that following treatment with sterile saline. Cure rates following treatment with rbEGF were not significantly different to those following sterile saline. Given that EGF is associated with modulation of host immunity and wound healing, future studies into EGF should not focus on whether EGF increases cure rates of S aureus IMI.

Quality of Adhesions After Sutured Paramedian vs. Laparoscopic Toggle Abomasopexy in an Ovine Model.

OBJECTIVE: To evaluate adhesions created by abomasopexies using either chromic gut or polydioxanone suture through a right paramedian approach and determine whether a laparoscopic toggle technique is an acceptable alternative to open abomasopexy using an ovine model. STUDY DESIGN: Experimental study. ANIMALS: Mature ewes (n=30). METHODS: Ewes received 1 of 3 abomasopexy techniques (n=10): right paramedian approach using chromic gut or polydioxanone, or a laparoscopic toggle technique. After euthanasia 8 weeks postoperatively, adhesions were removed en bloc and adhesion cross-sectional area (width × length) and depth (distance from abdominal wall to abomasum) were measured and given a grade of 0-3 based on the quality of adhesion. Surgical time was recorded and compared for each technique. Significance was set at P≤.05. RESULTS: Abomasopexies performed with either suture material resulted in a significantly larger mean cross-sectional area and higher adhesion grades compared to those performed using the toggle. Width and length of adhesions formed using chromic gut or polydioxanone were not significantly different; however, both were significantly wider and longer than those formed using the toggle. The laparoscopic toggle technique required significantly less surgical time than the sutured techniques. CONCLUSION: Polydioxanone is as effective as chromic gut suture material in inducing abomasal adhesion formation in our sheep model. The clinical significance of the size and grade of adhesions formed is unknown and requires further investigation before the laparoscopic toggle technique can be recommended as a replacement for paramedian abomasopexy in cattle for the treatment of displaced abomasum.

Comprehensive Phylogenetic Analysis of Bovine Non-aureus Staphylococci Species Based on Whole-Genome Sequencing.

Non-aureus staphylococci (NAS), a heterogeneous group of a large number of species and subspecies, are the most frequently isolated pathogens from intramammary infections in dairy cattle. Phylogenetic relationships among bovine NAS species are controversial and have mostly been determined based on single-gene trees. Herein, we analyzed phylogeny of bovine NAS species using whole-genome sequencing (WGS) of 441 distinct isolates. In addition, evolutionary relationships among bovine NAS were estimated from multilocus data of 16S rRNA, hsp60, rpoB, sodA, and tuf genes and sequences from these and numerous other single genes/proteins. All phylogenies were created with FastTree, Maximum-Likelihood, Maximum-Parsimony, and Neighbor-Joining methods. Regardless of methodology, WGS-trees clearly separated bovine NAS species into five monophyletic coherent clades. Furthermore, there were consistent interspecies relationships within clades in all WGS phylogenetic reconstructions. Except for the Maximum-Parsimony tree, multilocus data analysis similarly produced five clades. There were large variations in determining clades and interspecies relationships in single gene/protein trees, under different methods of tree constructions, highlighting limitations of using single genes for determining bovine NAS phylogeny. However, based on WGS data, we established a robust phylogeny of bovine NAS species, unaffected by method or model of evolutionary reconstructions. Therefore, it is now possible to determine associations between phylogeny and many biological traits, such as virulence, antimicrobial resistance, environmental niche, geographical distribution, and host specificity.

Skills required of dairy veterinarians in western Canada: a survey of practicing veterinarians.

This study determined skills required of entry-level veterinarians for dairy practice in western Canada and compared mixed and dairy practitioners in the skills that they perform. We surveyed western Canadian veterinarians involved in dairy practice, focusing primarily on clinical activity of respondents. Response rate was 39.4% (281/714). Respondents were classified as either mixed practitioners (< 10% time in dairy practice) or dairy practitioners (> 75% time in dairy practice). For both groups, individual animal medicine and surgery skills were performed more commonly than herd health skills. The most important skills identified were those required for basic theriogenology, physical examination, treatment of common disorders, and general surgery. These results underscore the continued importance of individual animal skills in food animal practice in western Canada.

Compétences requises par les vétérinaires laitiers de l'Ouest canadien: sondage auprès des vétérinaires praticiens. Cette étude a déterminé les compétences requises par les vétérinaires de niveau d'entrée pour la pratique laitière dans l'Ouest canadien et a comparé les praticiens mixtes et laitiers dans les compétences qu'ils mettent en pratique. Nous avons effectué une enquête auprès des vétérinaires de l'Ouest canadien œuvrant dans la pratique laitière, en nous concentrant principalement sur l'activité clinique des répondants. Le taux de réponse a été de 39,4 % (281/714). Les répondants ont été classés soient comme des praticiens mixtes (< 10 % du temps en pratique laitière) ou des praticiens laitiers (> 75 % du temps en pratique laitière). Pour les deux groupes, la médecine des animaux individuels et les compétences chirurgicales étaient mises en pratique plus couramment que les compétences de santé du troupeau. Les compétences les plus importantes identifiées étaient celles requises pour la thériogénélogie de base, l'examen physique, le traitement des affections courantes et la chirurgie générale. Ces résultats soulignent l'importance continue des compétences portant sur les animaux individuels dans la pratique des animaux destinés à l'alimentation de l'Ouest canadien.(Traduit par Isabelle Vallières).

Efficacy of vaccination against staphylococcal mastitis: a review and new data.

Infection of the heifer mammary gland with common mastitis pathogens, particularly staphylococci, prior to calving is well documented. Efforts to eliminate pre-partum intramammary infections (IMI) in heifers have focused primarily on intramammary antibiotic therapy shortly before or at the time of calving. Few studies have evaluated vaccination of heifers against staphylococcal mastitis. The objectives of the present study were to evaluate the efficacy of a commercially available Staphylococcus aureus bacterin in protecting against staphylococcal IMI (S. aureus and coagulase-negative staphylococci (CNS)), to study the effect of vaccination on milk SCC, and to evaluate the milk antibody isotype response to vaccination using a lactating cow model. Ninety Holstein-Friesian lactating dairy cows of various parities were systematically assigned to a vaccinated (n=44) or control (n=46) group. Vaccinates received two 5 ml doses of the bacterin 14 days apart starting on day 0. Quarter milk samples for bacterial culture were collected prior to each vaccination and approximately monthly thereafter for 6 months. Composite milk samples were collected on days 0, 14, 28, 49 and 70 for IgA, IgG(1), IgG(2), and IgM determinations and somatic cell count. No animals in either group developed a new S. aureus IMI after vaccination. The numbers of mammary quarters that developed a new CNS IMI, time to new CNS IMI, milk somatic cell count, and milk antibody isotype sample-to-positive ratio did not significantly differ between groups (P>0.05). In a herd with a 3% prevalence of S. aureus IMI and a 30% prevalence of CNS IMI, the vaccine did not reduce the new staphylococcal IMI rate. There may be insufficient vaccine-induced opsonizing antibody in milk to facilitate phagocytosis and clearance of staphylococci from the mammary gland.

Characterization of the antibody isotype response in serum and milk of heifers vaccinated with a Staphylococcus aureus bacterin (Lysigin).

The objective was to characterize the antibody isotype responses to vaccination with a commercial Staphylococcus aureus bacterin in dairy heifers. Twenty-five Holstein-Friesian dairy heifers were assigned at random to one of two groups, vaccinates (n=14) or controls (n=11). Vaccinates received two 5-ml doses of Lysigin 28 d apart in late gestation. Both groups were challenged with a heterologous serotype 5 strain of Staph. aureus by aseptic intramammary infusion on days 6, 7 and 8 of lactation. Samples for serum antibody isotype (IgG, IgG1, IgG2 and IgM) determinations were taken before each vaccination, immediately prior to challenge (6 d post-calving) and at the end of the study (28 d post-calving). Samples for milk antibody isotype determinations were taken immediately prior to challenge and at the end of the study. Antibody isotype sample-to-positive ratios (S:P ratio=(mean test sample OD-mean within-plate negative control OD)/(mean within-plate positive control OD-mean within-plate negative control OD)) were determined in milk and serum using a series of ELISAs coated with different strains of Staph. aureus belonging to capsular polysaccharide (CP) serotype 5 or 8 or surface polysaccharide (SP) serotype 336. Vaccinates had higher mean serum IgG1 and IgG2 S:P ratios than controls against all three strains of Staph. aureus (P < or = 0.023). Vaccinates had higher mean milk IgG S:P ratios than controls against CP8 and SP336 strains of Staph. aureus (P< or = 0.030). Hence, a humoural immune response to the vaccine was detected in serum and milk, but responses varied according to strain and antibody isotype tested.

Efficacy of different Lysigin formulations in the prevention of Staphylococcus aureus intramammary infection in dairy heifers.

The objective was to compare the efficacy of two experimental Staphylococcus aureus mastitis bacterins and a currently marketed five-isolate-based Staph. aureus bacterin (Lysigin, Boehringer Ingelheim Vetmedica, Inc.) with unvaccinated controls. Forty-seven Holstein-Friesian heifers were randomly assigned to one of four groups such that Group 1 (n=11) received a three-isolate experimental bacterin, Group 2 (n=11) received a five-isolate experimental bacterin, Group 3 (n=14) received Lysigin, and Group 4 (n=11) served as unvaccinated controls. Vaccinations were administered twice 28 d apart in late gestation. All groups were challenged with a heterologous strain of Staph. aureus (ATCC 29740) on days 6, 7, and 8 of lactation. Mastitis score, somatic cell count (SCC), milk culture yield, and total daily milk yield data were collected before and after challenge. All 47 cattle developed a Staph. aureus IMI post-challenge with three animals in Group 1 and one animal in Group 3 clearing their Staph. aureus IMI by the end of the study. However, there was no evidence of a difference between vaccinates and control with regard to Staph. aureus clearance rates post-challenge (P> or =0.214). Cattle vaccinated with Lysigin had a lower mean duration of clinical mastitis and lower total mastitis score post-challenge than controls (P=0.045 and P=0.046, respectively). Overall, there was no evidence that any of the vaccinated groups had a lower mean SCC than control (P> or =0.148) for the tested study days. Likewise there was no evidence that vaccinates had greater milk yield than controls post-challenge (P=0.617). Hence, there was no evidence that the vaccines reliably prevented Staph. aureus IMI, but Lysigin showed benefit in reducing the clinical severity and duration of clinical disease post-challenge. Neither of the experimental bacterins appeared to perform better than Lysigin.

Effect of prepartum intramammary treatment with pirlimycin hydrochloride on prevalence of early first-lactation mastitis in dairy heifers.

OBJECTIVE: To determine whether prepartum intramammary treatment of dairy heifers with pirlimycin hydrochloride would reduce the prevalence of intramammary infection (IMI) and lower the somatic cell count (SCC) during early lactation or improve 305-day mature equivalent milk production. DESIGN: Prospective clinical trial. ANIMALS: 183 Holstein-Friesian heifers (663 quarters) from 2 dairy farms. PROCEDURE: Heifers were assigned to treatment and control groups. Treated heifers received a single 50-mg dose of pirlimycin in each mammary quarter approximately 10 to 14 days prior to parturition. Prepartum mammary gland secretions and postpartum milk samples were collected for bacterial culture. Postpartum milk samples were also collected for determination of SCC or California mastitis testing and were tested for pirlimycin residues. Mature equivalent 305-day milk production data were recorded. RESULTS: Treated heifers in herd A had a higher overall cure rate, higher cure rates for IMI caused by coagulase-negative staphylococci (CNS) and Staphylococcus aureus, lower SCC, and lower prevalence of chronic IMI, compared with control heifers. Treated heifers in herd B had a higher overall cure rate and cure rate for IMI caused by CNS, compared with control heifers, but postpartum California mastitis test scores and prevalence of chronic IMI did not differ between groups. Mature equivalent 305-day milk production did not differ between herds or treatment groups. No pirlimycin residues were detected in postpartum milk samples. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that prepartum treatment of dairy heifers with pirlimycin may reduce the prevalence of early lactation IMI, particularly IMI caused by CNS, without causing pirlimycin residues in milk.

Surveillance of Staphylococcus aureus in veterinary teaching hospitals.

Staphylococcus aureus isolates (n = 70) from 65 patients (36 canine, 18 equine, 7 bovine, 2 avian, and 2 feline) at seven veterinary teaching hospitals in the United States were studied. The majority of patients (83%) with an S. aureus infection were canine and equine, but this may have reflected a sample bias based on clinic case loads and diagnostic lab submissions at the participating institutions. Fourteen percent of patients with an S. aureus infection were infected with a methicillin-resistant S. aureus (MRSA) isolate. Six of seven institutions had at least one MRSA infection during the study. Pulsed-field gel electrophoresis on 63 of the 70 isolates yielded 58 unique strains of S. aureus. None of the strain types of the MRSA isolates matched each other or the type of any other S. aureus isolate. The proportions of patients infected with an MRSA isolate were not significantly different between institutions or animal species (P > or = 0.222). Methicillin-resistant S. aureus isolates in this study seemed to be community acquired rather than hospital acquired.