Different distribution of antimicrobial resistance genes and virulence profiles of Staphylococcus aureus strains isolated from clinical mastitis in six countries.

Staphylococcus aureus is recognized worldwide as one of the main contagious mastitis agents in cattle and can express a set of antimicrobial resistance genes and virulence-associated genes that explain the wide range of outcomes of intramammary infections. Staphylococcus aureus strains are heterogeneous: their different resistance and virulence patterns, associated with host-level factors and treatment factors, are related to the severity of infection. The aim of this study was to determine phenotypic antibiotic susceptibility, occurrence of selected antimicrobial resistance genes and other virulence genes in 93 S. aureus strains isolated from clinical mastitis in 6 countries: Argentina, Brazil, Germany, Italy, the United States (New York State), and South Africa. These isolates were tested against a total of 16 drugs (amoxicillin-clavulanate, ampicillin, cefazolin, cefoperazone, cefquinome, enrofloxacin, erythromycin, gentamicin, kanamycin, lincomycin, oxacillin, penicillin, rifampin, spiramycin, sulfamethoxazole/trimethoprim, tylosin) by minimum inhibitory concentration (MIC) assay, and examined for the presence of 6 antibiotic-resistance genes (blaZ, mecA, mecC, ermA, ermB, ermC) and 6 virulence-associated genes (scn, chp, sak, hla, hlb, sea) via PCR analysis. The phenotypic results of this study revealed the presence of 19.4% penicillin-resistant strains, whereas 22.6% of the strains were classified as having resistance (5.4%) or intermediate resistance (17.2%) to erythromycin. Most (96.8%) of the isolates were inhibited by cephalosporins, and all were susceptible to amoxicillin-clavulanate. Two strains (1 from Germany, 1 from Italy) were resistant to oxacillin and were positive for mecA. Among the other antimicrobial resistance genes, the most frequently detected was blaZ (46.2%), and 32.3% of the isolates were positive for erm genes: ermC (21.5%) and ermB (10.8%). The most prevalent virulence gene was hla (100%), followed by hlb (84.9%) and sea (65.6%). These results show a low prevalence of antibiotic multidrug resistance in S. aureus isolates, even if the detection of selected antimicrobial resistance genes did not always correspond with the occurrence of phenotypic antibiotic resistance; the immune evasion cluster gene prevalence was quite low in the samples analyzed.

Evaluation of the systemic innate immune response and metabolic alterations of nonlactating cows with diet-induced subacute ruminal acidosis.

Subacute ruminal acidosis (SARA) increases lipopolysaccharide endotoxin in the rumen, which might translocate into the systemic circulation, triggering a cascade of clinical and immunological alterations. The objective of this study was to characterize the clinical immune and metabolic responses to ruminal-derived lipopolysaccharide in nonlactating cows induced with SARA using 2 challenges, a grain-based SARA challenge (GBSC) or an alfalfa-pellet SARA challenge (APSC). Six dry, nonlactating Holstein cows were used in a 3 × 3 Latin square arrangement of treatments with 4-wk experimental cycles. All cows received the control diet containing 70% forage and 30% mixed concentrates (dry matter basis) for 3 wk. In wk 4, cows received a control diet, GBSC (38% wheat-barley pellets, 32% other mixed concentrate, and 30% forages), or APSC (45% mixed concentrate, 32% alfalfa pellets, and 23% other forages). Total plasma proteins and immunology-related proteins, acute phase proteins, blood cells, serum chemistry, mRNA gene expression of peripheral blood cell surface markers, and selected proinflammatory cytokines were evaluated. Ruminal pH was lower in both groups with induced SARA compared with a control group. Ruminal endotoxins were higher in GBSC; however, plasma endotoxin was not detected in any study group. No significant differences in feed intake, rectal temperature, white blood cell counts, or differentials were found between control and SARA challenge groups; changes in glucose, urea, Ca, and Mg were observed in SARA groups. Total plasma proteins were lower in both SARA groups, and acute phase proteins were higher in GBSC. The expression of CD14, MD2, and TLR4 mRNA in peripheral blood leukocytes was not affected by SARA induction. The induction of SARA as a result of GBSC or APSC challenge was successful; however, LPS was not detected in plasma. Changes in clinical, metabolic, and inflammatory responses were not observed in the SARA-challenged cows, suggesting that, in this study, SARA was not associated with a systemic response to inflammation.

Noninferiority trial on the efficacy of premilking teat disinfectant against naturally occurring new intramammary infections using a novel 2-step diagnostic process.

The objective of the study was to evaluate premilking teat disinfectant efficacy with the use of a novel diagnostic protocol against a positive control. The evaluation of efficacy was based on establishing noninferiority of a new premilking teat disinfectant compared with an existing premilking teat disinfectant. Approximately 200 cows were randomly allocated to 1 of 2 groups. Study personnel were blinded as to cow allocation and identification of the premilking disinfectants. Quarters were disinfected using a dip cup before milking with either the control or experimental product. The disinfectant was left on the teat for approximately 30s before being wiped off in preparation for milking. Quarter-level bacteriological infection status was established at the beginning of the study and quarter milk samples were collected biweekly thereafter. Bacteriological analyses were performed only when somatic cell counts in milk samples crossed a parity-specific threshold. Poisson regression models were used to analyze data. The difference in the rate of new intramammary infections was small and the upper bound of the 95% confidence interval of the rate difference was smaller than a predefined noninferiority limit. Based on the observed difference in new intramammary infections rate between the experimental and control product, it was concluded that the experimental disinfectant was not inferior compared with the positive control. The protocol used in this study proposes a valid and economically attractive methodology to evaluate the efficacy of teat disinfectants relative to a positive control.

Milk selenium concentration and its association with udder health in Atlantic Canadian dairy herds.

Soils and plants in Atlantic Canadian provinces are known to contain low concentrations of selenium (Se). Earlier studies have indicated that dairy producers in Atlantic Canada are providing insufficient supplementary Se in the ration to meet the Se requirements of dairy cattle, as measured by herd-level milk Se concentration. The objective of this study was to evaluate the association between milk Se concentration and somatic cell count (SCC) and the risk of new intramammary infection (IMI) in the dry period, in Atlantic Canadian dairy cows. Eighteen dairy farms participating in the Canadian Bovine Mastitis Research Network cohort study were selected as a convenience sample. On each farm 15 cows to be dried off were selected. Quarter milk samples were collected at 4 and 2 wk before drying-off, within 24 h after calving, and at 7 d after calving to evaluate IMI status. Composite milk samples were analyzed for SCC and Se concentration. Mean milk Se concentration was marginal in 14% of the cows that were on pasture during the grazing season. Milk Se concentration was not associated with the overall odds of new IMI in the dry period; however, the odds of having a new Streptococcus spp. and other gram-positive pathogen IMI in the dry period increased with increasing milk Se concentration. Somatic cell count increased with milk Se concentration, even after adjusting for IMI status. The dairy population in our study had higher ranges for milk Se concentration, whereas ranges for prevalence of IMI, and SCC were lower, compared with those in studies where a negative relationship between Se status and udder health was first noted. Therefore, under the current management conditions, milk Se concentration did not appear to be a principal determinant of udder health.

The effect of selenium supplementation before calving on early-lactation udder health in pastured dairy heifers.

Selenium (Se) deficiency has been associated with lowered resistance to mastitis in dairy cattle. However, little published data exists on the effect of Se supplementation before calving on udder health of pastured dairy heifers. Further, the relative efficacy of injectable barium selenate and oral organic Se for improving udder health in cows has not previously been tested. The objectives of this study were to determine the effects of precalving Se supplementation and type of supplementation on the blood activity of glutathione peroxidase and measures of udder health immediately after calving and during the first month of lactation in pastured dairy heifers. One hundred forty pregnant Chilean Holstein-Friesian heifers were fed a basal diet containing, on average, 0.15 mg of Se/kg of dry matter. One month before predicted calving, heifers were allocated to 1 of 3 groups. Group 1 (n=49) received no supplementary Se, group 2 (n=46) received a single subcutaneous injection of Se (1 mg/kg of live weight, as barium selenate), and group 3 (n=45) was fed Se yeast (3 mg/heifer/d until calving). Heifers supplemented with barium selenate had a higher glutathione peroxidase activity from 14 d in milk onwards. Selenium supplementation, irrespective of source, tended to reduce the prevalence of intramammary infection (IMI) and decrease the prevalence of quarters with high somatic cell count (SCC) at calving. Overall, Se supplementation did not result in a reduction of the incidence of new IMI or clinical mastitis or in decreased SCC during the balance of the first month of lactation. However, in pasture-based heifers injected with barium selenate before calving, and fed diets with 1.3 and 2.5 mg of Se/d precalving and during lactation, respectively, no cases of clinical mastitis were observed in the first month of lactation.

Lack of evidence for Mycoplasma spp. in bulk tank milk of herds located in mid-western Colombia / Falta de evidencia para Mycoplasma spp. en leche de tanques de enfriamiento de hatos del centro-occidente colombiano

ABSTRACT Mycoplasma spp. is reported as a highly contagious mastitis-causing bacteria in dairy cattle, without successful or low response to most common antibiotic treatments due to the lack of cell wall. In Colombia it has been reported in the Central Andean region during 2014. The aim was to estimate the prevalence of Mycoplasma spp. in bulk tank milk using microbiological and molecular diagnosis. A random longitudinal study enrolling 220 commercial dairy farms located in four provinces of the mid-western region of Colombia from four pasteurizer companies was performed. Bulk tank milk samples were collected once monthly for three months period for determining somatic cell count (SCC) and microbiological and molecular diagnosis of Mycoplasma spp. cultures were done without pre-enrichment procedures directly in mycoplasma agar with cefoperazone to inhibit growth of opportunistic microorganisms, plates were incubated under 37° C and atmosphere of 10% CO2 and inspected during a 10d period. Molecular analysis was done by a multiplex PCR using specific primers targeting the 16S-23S rARN gene of Mycoplasma spp. and from non-pathogenic bacteria occasionally found in milk. LnSCC average of included dairy farms was 6.19 x103 cells/mL, Mycoplasma spp. was not isolated during microbiological cultures, and no DNA belonging to the species was detected by PCR in the 220 bulk tanks milk, with an estimated prevalence lower than 2.3%. This finding shows that there is not microbiological or molecular evidence that demonstrates the presence of the pathogen in the milk from the mid-western region of Colombia at herd level.

RESUMEN Mycoplasma spp. está descrito como una bacteria causante de mastitis altamente contagiosa en ganado lechero, sin o con baja respuesta a tratamientos antibióticos convencionales debido a que carece de pared celular. En Colombia ha sido reportado en la región Andina Central durante 2014. El objetivo fue estimar la prevalencia de Mycoplasma spp. en leche de tanques de enfriamiento empleando diagnósticos microbiológicos y moleculares. Se realizó un estudio aleatorio longitudinal que incluyó 220 lecherías comerciales en cuatro departamentos del centro-occidente colombiano acopiadas por cuatro compañías pasteurizadoras. Se recolectaron muestras de leche del tanque de enfriamiento mensualmente durante tres meses para determinar el recuento de células somáticas (SCC) y el diagnóstico microbiològico y molecular de Mycoplasma spp. Los cultivos se realizaron sin procedimientos de preenriquecimiento directamente en agar micoplasma con cefoperazona para inhibir crecimiento de microorganismos oportunistas, los agares se incubaron a 37° C con una atmosfera del 10% CO2 e inspeccionados durante 10d. Los análisis moleculares se realizaron por PCR multiplex usando cebadores específicos para los genes 16S-23S rRNA del Mycoplasma spp. y de algunas bacterias oportunistas ocasionales en la leche. El promedio del LnSCC fue de 6.19 x103 células/ mL, Mycoplasma spp. no fue aislado de los cultivos microbiológicos y no se encontró ADN de a esta especie mediante PCR en los 220 tanques de leche. Lo anterior indica una prevalencia estimada menor a 2,3%. Se concluye que no existe evidencia micro-biológica ni molecular para demostrar la presencia del patógeno en la leche de la región centro-occidente colombiana a nivel de hato.

The association between subclinical mastitis around calving and reproductive performance in grazing dairy cows.

The objective of this study was to evaluate the effect of subclinical mastitis (SCM) on calving-to-first-service interval (CFS), calving-to-conception interval (CC), and on the number of services per conception (S/C) in grazing Holstein and Normande cows. Primiparous (n=43) and multiparous (n=165) cows were selected from five dairy herds. Two composite milk samples were aseptically collected from each cow at drying-off, and then every week during the first postpartum month. One sample was used for somatic cell count (SCC), and the other one for bacteriological analysis. Cows were followed up to 300 d after calving. Non-parametric and parametric survival models, and negative binomial regression were used to assess the association between SCM, evaluated by SCC and milk culture, and reproductive indices. Staphylococcus aureus, CNS, and Streptococcus uberis were the most frequent isolated pathogens. Subclinical mastitis in the first month of lactation was not associated with CFS; however, the CC interval was longer in cows with SCM compared to healthy cows, the former also had a higher number of S/C.