Efficacy of a lactoferrin-penicillin combination to treat {beta}-lactam-resistant Staphylococcus aureus mastitis

The efficacy of intramammary (IM) treatments containing penicillin G (PG) alone or a combination of PG and bovine lactoferrin (bLF) was evaluated using a model of experimentally induced chronic bovine mastitis caused by a clinical isolate of Staphylococcus aureus highly resistant to β-lactam antibiotics. First, we confirmed that this strain could cause mastitis and infection could not be cured with PG alone. In a second trial, chronic mastitis was induced in 19 late-lactating cows by injecting a low dose of Staph. aureus through the teat canal of all quarters. After 15 d, cows with stable infections in their 4 quarters had their mammary quarters randomly assigned, within cow, to 1 of 4 IM treatments as follows: 1) citrate buffer, 2) 100,000 IU of PG, (3) 1 g of bLF, or 4) 1 g of bLF + 100,000 IU of PG. Treatments were repeated twice a day for 5 d. A third trial was undertaken to investigate the effect of an extended therapy on chronic mastitis acquired in a previous lactation. One month before dry-off, 20 gravid cows regrouped by dates of calving were infected in their 4 quarters. Once infections were established, cows were dried off abruptly. After calving, aseptic milk samples were collected separately from all quarters for 4 wk to monitor infection. Mammary quarters from enrolled cows were then randomly assigned, within cow, to 1 of 2 treatments as follows: 1) 100,000 IU of PG or 2) 250 mg of bLF + 100,000 IU of PG. Treatments were administered IM twice a day for 7 d. For all trials, milk samples were taken to monitor bacterial concentration and somatic cell count. Bacteriological cure rate was determined using milk samples taken 3 and 4 wk after initiation of treatments. For the second trial, cure rate was null for control quarters, 11.1% for bLF, 9.1% for PG, and 45.5% for the bLF + PG combination. For cows infected in their previous lactation, cure rate was higher for the bLF + PG combination (33.3%) compared with PG alone (12.5%). In conclusion, bLF added to PG is an effective combination (i.e., 3- to 5-times higher cure rate) for the treatment of stable Staph. aureu infections highly resistant to β-lactam antibiotics.     

Host adaptation of bovine Staphylococcus aureus seems associated with bacteriological cure after lactational antimicrobial treatment

Staphylococcus aureus causes a wide range of diseases in multiple species. Some sequence types (ST) are observed in a variety of hosts, whereas other strains are mainly associated with bovine mastitis, suggesting host adaptation. We propose that host adaptation of Staph. aureus may influence bacteriological cure of bovine subclinical mastitis after antimicrobial treatment. To test this hypothesis, multilocus sequence typing was performed on Staph. aureus isolates from 60 treated and 79 untreated control quarters that were obtained from well-defined cohorts of dairy cows from a recently conducted randomized field trial on early treatment of subclinical mastitis. Bovine-associated ST were distinguished from non-bovine-associated ST based on the literature and public databases. The association between host adaptation and bacteriological cure was investigated using population-averaged logistic regression models. Thirteen ST were identified, with approximately 80% of isolates belonging to bovine-associated ST. The odds for cure were around 2.5 times as high for non-bovine-associated ST as for bovine ST in treated quarters, whereas no difference in spontaneous cure was observed in untreated control quarters. In addition, host adaptation was related to known predictors of cure, such as penicillin susceptibility and somatic cell count. All isolates belonging to non-bovine-associated ST were resistant to penicillin, whereas the majority of isolates belonging to bovine-associated ST were penicillin susceptible. Penicillin-resistant bovine-associated strains were associated with high somatic cell counts compared with other strains. The correlation between penicillin resistance, cell counts, and host adaptation may affect the association between host adaptation and cure. For diagnostic purposes, a simple and fast alternative to multilocus sequence typing of Staph. aureus to determine host adaptation may be valuable.     

Strain diversity and infection durations of Staphylococcus spp. and Streptococcus spp. causing intramammary infections in dairy cows

To effectively prevent and control bovine mastitis, farmers and their advisors need to take infection pathways and durations into account. Still, studies exploring both aspects through molecular epidemiology with sampling of entire dairy cow herds over longer periods are scarce. Therefore, quarter foremilk samples were collected at 14-d intervals from all lactating dairy cows (n = 263) over 18 wk in one commercial dairy herd. Quarters were considered infected with Staphylococcus aureus, Streptococcus uberis, or Streptococcus dysgalactiae when ≥100 cfu/mL of the respective pathogen was detected, or with Staphylococcus epidermidis or Staphylococcus haemolyticus when ≥500 cfu/mL of the respective pathogen was detected. All isolates of the mentioned species underwent randomly amplified polymorphic DNA (RAPD)-PCR to explore strain diversity and to distinguish ongoing from new infections. Survival analysis was used to estimate infection durations. Five different strains of Staph. aureus were isolated, and the most prevalent strain caused more than 80% of all Staph. aureus infections (n = 46). In contrast, 46 Staph. epidermidis and 69 Staph. haemolyticus strains were isolated, and none of these caused infections in more than 2 different quarters. The 3 most dominant strains of Strep. dysgalactiae (7 strains) and Strep. uberis (18 strains) caused 81% of 33 and 49% of 37 infections in total, respectively. The estimated median infection duration for Staph. aureus was 80 d, and that for Staph. epidermidis and Staph. haemolyticus was 28 and 22 d, respectively. The probability of remaining infected with Strep. dysgalactiae or Strep. uberis for more than 84 and 70 d was 58.7 and 53.5%, respectively. Staphylococcus epidermidis and Staph. haemolyticus were not transmitted contagiously and the average infection durations were short, which brings into question whether antimicrobial treatment of intramammary infections with these organisms is justified. In contrast, infections with the other 3 pathogens lasted longer and largely originated from contagious transmission.     

Efficacy of extended cefquinome treatment of clinical Staphylococcus aureus mastitis

Clinical Staphylococcus aureus mastitis is difficult to cure. Extended antimicrobial treatment is often advocated as a practical approach to improve cure rates; however, scientific evidence of this hypothesis is lacking. A multi-centered, nonblinded, randomized, positive-controlled clinical trial was conducted in 5 European countries—France, Hungary, Italy, the Netherlands, and the United Kingdom—to study the efficacy of an extended intramammary cefquinome treatment (5 d) compared with a standard intramammary cefquinome treatment (1.5 d) of Staph. aureus clinical mastitis. Least squares means estimates of bacteriological cure during lactation were 34% [standard error (SE) = 9.9%] for the standard treatment group and 27% (SE = 8.4%) for the extended treatment group. In the final model, extended therapy was not significantly better. The only factor predicting bacteriological cure was pretreatment cow somatic cell count (SCC). Cows with >250,000 cells/mL in milk before treatment were less likely to cure. Least squares means of clinical cure during lactation was 60% (SE = 19%) for the standard treatment group and 82% (SE = 12%) for the extended treatment group. In the final model, clinical cure after extended treatment was significantly better. Pretreatment cow udder firmness predicted clinical cure. Firm udders were less likely to cure clinically. Irrespective of treatment regimen, new infection rates with pathogens other than Staph. aureus were higher (42%) after bacteriological cure than after nonbacteriological cure (22%) and cured cows had a significantly lower SCC. In conclusion, independent of the treatment protocol, cows with an SCC <250,000 cells/mL before treatment showed a higher probability of bacteriological cure. It appears that successful treatment of clinical Staph. aureus mastitis with cefquinome is associated with an increased number of new infections with coagulase-negative staphylococci. Extended treatment improved clinical, but not bacteriological, cure rates compared with the standard treatment. These results indicate that extending treatment of clinical Staph. aureus mastitis with cefquinome should not be recommended.     

Therapeutic effects of antimicrobial treatment during lactation of recently acquired bovine subclinical mastitis: Two linked randomized field trials

Two linked randomized field trials were performed on 39 herds in the Netherlands to 1) determine therapeutic effects of antimicrobial treatment of recently acquired subclinical mastitis (RASCM) during lactation, 2) evaluate the effect of duration of subclinical mastitis on therapeutic outcome, and 3) identify factors related to the therapeutic success of RASCM. Cows with a first elevated composite somatic cell count (CSCC) after 2 consecutive low CSCC measurements were eligible for enrollment in trial 1 (treatment at the first elevated CSCC). Quarter milk samples were collected to determine bacteriological status for major pathogens and coagulase-negative staphylococci. Cows with one or more culture-positive quarters with a quarter somatic cell count (QSCC) ≥100,000 cells/mL were defined to have RASCM and were randomly assigned treatment or control (no treatment). Untreated cows from trial 1 that had a second elevated CSCC at the next milk recording were eligible for enrollment in trial 2 (treatment at the second elevated CSCC). In trial 2, staphylococci-positive cows (Staphylococcus aureus and coagulase-negative staphylococci) were randomly assigned to treatment or control. Farmers used their own treatment protocols to treat quarters in both trials. Bacteriological cure was defined as absence of the pathogen identified pre-intervention in 2 samples post-intervention; QSCC, CSCC, and milk yield were also analyzed. Hierarchical logistic and linear models were used to determine therapeutic effects and to identify factors related to therapy outcome. Treated quarters had a higher bacteriological cure rate than control quarters for all pathogens in both trials. Treatment resulted in lower QSCC and CSCC, whereas milk yield was not affected by treatment. Bacteriological cure of RASCM was better in quarters with a low QSCC pre-intervention and in coagulase-negative staphylococci-positive quarters. Control quarters with a single culture-positive sample pre-intervention also had a higher bacteriological cure than control quarters with ≥2 culture-positive samples. Time of antimicrobial treatment affected bacteriological cure for penicillin-sensitive Staph. aureus. Bacteriological cure tended to be higher for Staph. aureus after treatment at the first elevated CSCC compared with treatment at the second elevated CSCC. Thus, early treatment of Staph. aureus might be more effective than later treatment.     

Short communication: antimicrobial drug susceptibility of Staphylococcus aureus from subclinical bovine mastitis in Italy

The antimicrobial susceptibility of 68 Staphylococcus aureus isolates collected during 2004 from milk of cows affected by subclinical mastitis was examined. The antimicrobial agents tested were the β-lactams, penicillin G, amoxicillin, ampicillin, cloxacillin, amoxicillin + clavulanate, cephalonium, and cefoperazone; and other drugs including lincomycin, oxytetracycline, doxycycline, and kanamycin. Minimum inhibitory concentrations recorded show that only certain β-lactamase-resistant penicillins (specifically cloxacillin) or penicillin combinations (amoxicillin + clavulanate) were consistently effective against Staph. aureus, whereas the other β-lactam derivatives and drugs from other pharmacological groups were either moderately effective or ineffective. Thus, β-lactamase-resistant penicillins are to be considered the antimicrobial agents of choice for treatment of bovine mastitis resulting from infection by Staph. aureus.     

Relationships between milk culture results and treatment for clinical mastitis or culling in Norwegian dairy cattle

In quarter milk samples from 2,492 randomly sampled cows that were selected without regard to their current or previous udder health status, the relationships between the following outcome variables were studied: treatment of clinical mastitis; the joint event of either treatment or culling for mastitis; culling for all reasons; culling specifically for mastitis; and the covariates of positive milk culture for Staphylococcus aureus, Streptococcus spp., and coagulase-negative Staphylococcus spp., or other pathogens, or of negative culture for mastitis pathogens. Microbiological diagnoses were assigned at the cow level, and altogether 3,075 diagnoses were related to the outcome variables. The relation between the absence of pathogens and rich (>1,500 cfu/mL of milk) or sparse (≤1,500 cfu/mL of milk) growth of Staph. aureus were also assessed separately for each outcome variable. The hazard of treatment of clinical mastitis was greater for cows diagnosed with Staph. aureus compared with cows with no pathogens in all analyses. Cows with sparse growth of Staph. aureus upon microbiological analysis were more likely to be treated for clinical mastitis, and cows with rich growth of the bacteria experienced a higher overall risk of culling when the models adjusted for cow composite milk somatic cell count. No difference between rich and sparse growth of Staph. aureus was found when mastitis was defined as the joint event of either culling for mastitis or treatment of clinical mastitis, and when the relationship with culling specifically for mastitis was assessed. The combined outcome of treatment and culling for mastitis was related to a positive diagnosis of Strep. spp. after cow composite milk somatic cell count was omitted from the model. Presence of Streptococcus spp. was also related to culling specifically for mastitis, whereas culling for all reasons and treatment of clinical mastitis was not related to a positive culture of Strep. spp. Presence of coagulase-negative Staph. spp. or other pathogens was not associated with either of the outcome variables.     

Effect of a biological response modifier on expression of growth factors and cellular proliferation at drying off

Agents that increase natural protective mechanisms have been proposed for the prevention and treatment of intramammary infections. Staphylococcus aureus is a major pathogen causing primarily subclinical chronic mastitis that responds poorly to antibiotic therapy. The objectives of this study were to describe the effects of a single intramammary infusion of a lipopolysaccharide-based biological response modifier (BRM) on mammary epithelial cellular proliferation and expression of insulin-like growth factor-I (IGF-I) and vascular endothelial growth factor (VEGF) in uninfected and Staph. aureus-infected bovine mammary glands during involution. Three groups of 12 cows, 6 Staph. aureus-infected and 6 uninfected, were infused with BRM or placebo in 2 mammary quarters and killed at 7, 14, and 21 d of involution. The proportion of infected quarters, mammary cell proliferation, and IGF-I and VEGF expression were evaluated. Biological response modifier treatment decreased the proportion of Staph. aureus-infected mammary quarters at 7 d of involution, but a similar number of isolations were observed at 14 and 21 d of involution in either treated or control quarters. The percentage of proliferating mammary epithelial cells was higher in infected than uninfected quarters at every observation period, irrespective of the treatment administered, whereas uninfected BRM-treated quarters showed increased cell proliferation at 7 d of involution. Insulin-like growth factor-I expression in uninfected quarters was not affected by treatment and showed a decrease at 21 d of involution. Expression of IGF-I was greater in infected than uninfected quarters at every observation period, irrespective of the treatment received. Expression of VEGF was greater in BRM-treated uninfected quarters at 7 d of involution compared with controls. In infected quarters, VEGF expression was lowest in BRM-treated quarters at 7 d of involution and increased throughout the observation period. Conversely, untreated infected quarters showed the highest VEGF expression at 7 d and decreased at 21 d of involution. Mammary cell proliferation and expression of IGF-I and VEGF were increased in Staph. aureus-infected quarters. Increased mammary cell proliferation and VEGF expression were observed in BRM-treated quarters during the first week of involution.     

An improved method to culture Staphylococcus aureus from bovine milk

Staphylococcus aureus is an important udder pathogen often associated with subclinical mastitis in dairy cows. Identification of Staph. aureus-positive udder quarters and cows is an important part of control programs to reduce spread of Staph. aureus within and between dairy herds. Therefore, accurate and easy-to-perform culturing methods of Staph. aureus in milk are needed. In the present study, 8 methods for isolation of Staph. aureus in bovine milk samples were investigated. The methods involved different culturing volumes, enrichment, incubation, and freezing processes as well as sedimentation and use of the Mastistrip cassette (SVA, Uppsala, Sweden). Three different sets of milk samples were collected, and 6, 5, and 4 methods were used in each subset of samples. Our results indicate an increased probability of detecting Staph. aureus in milk samples when a simple incubation step (37°C for 18 h) without additives was included before culturing. Using this incubation method, the number of Staph. aureus-positive udder quarters and cows increased by 50 and 29%, respectively, compared with using the standard method of direct culturing of 10 μL of milk. The improved method may be especially useful for detection of low concentrations of Staph. aureus in milk; for example, when screening herds for Staph. aureus.     

Genetic relatedness and virulence factors of bovine Staphylococcus aureus isolated from teat skin and milk

The objective of this study was to assess the role of teat skin colonization in Staphylococcus aureus intramammary infections (IMI) by evaluating genetic relatedness of Staph. aureus isolates from milk and teat skin of dairy cows using pulsed-field gel electrophoresis and characterizing the isolates based on the carriage of virulence genes. Cows in 4 known Staph. aureus-positive herds were sampled and Staph. aureus was detected in 43 quarters of 20 cows, with 10 quarters positive in both milk and skin (20 isolates), 18 positive only in milk, and 15 only on teat skin. Quarters with teat skin colonized with Staph. aureus were 4.5 times more likely to be diagnosed with Staph. aureus IMI than quarters not colonized on teat skin. Three main clusters were identified by pulsed-field gel electrophoresis using a cutoff of 80% similarity. All 3 clusters included both milk and skin isolates. The majority of isolates (72%) belonged to one predominant cluster (B), with 60% of isolates in the cluster originating from milk and 40% from teat skin. Genotypic variability was observed within 10 pairs (formed by isolates originating from milk and teat skin of the same quarter), where isolates in 5 out of the 10 pairs belonged to the same cluster. Forty-two virulence factors were screened using PCR. Some virulence factors were carried more frequently by teat skin isolates than by milk isolates or isolates from quarters with high somatic cell counts. Isolates in the predominant cluster B carried virulence factors clfA and clfB significantly more often than isolates in the minor clusters, which may have assisted them in becoming predominant in the herds. The present findings suggest that teat skin colonization with Staph. aureus can be an important factor involved in Staph. aureus IMI.     

Prevalence of mastitis pathogens and their resistance against antimicrobial agents in dairy cows in Brandenburg, Germany

The primary objective of this study was to determine management practices concerning mastitis in Brandenburg, Germany, the prevalence of mastitis pathogens in dairy cows, and their resistance to selected antimicrobial agents. A further objective was to study the potential effect of parity and stage of lactation on the resistance of Staphylococcus aureus isolates against ampicillin. Milk samples for microbiological culture were collected from 4 groups of clinically healthy cows (first lactation, >1 lactation, >50 d in milk, and >250 d in milk; 8 cows/group) in 80 dairy herds. Resistance of gram-positive pathogens against 6 antimicrobial agents was tested using the broth microdilution method. Mastitis pathogens were isolated from 26.4% of the milk samples. Coagulase-negative staphylococci (CNS, 9.1% of quarters) and Corynebacterium bovis (7.3%) were the pathogens most frequently isolated. Among the major pathogens, Staph. aureus (5.7%) and Streptococcus uberis (1.0%) had the highest prevalence. Streptococcus agalactiae was isolated in samples from 29% of the herds. Although the prevalence of most pathogens was higher in older cows, the prevalence of CNS was higher in primiparous cows. Results of the mastitis control questionnaire showed that cows with clinical mastitis were transferred to a sick cow pen in 70% of the herds. Cephalosporins were the drug of first choice for treatment of clinical mastitis cases followed by fixed combinations of antimicrobial agents, β-lactamase-resistant penicillins, and penicillin. Most farmers treated cows 3 to 4 times per case. Cloxacillin, alone or in combination, and penicillin were most often used for dry-cow therapy. Antimicrobial resistance of the pathogens was within the range of other reports. Resistance of Staph. aureus to ampicillin increased significantly during the first lactation. Further research is required to determine the factors that lead to the selection of Staph. aureus strains that are resistant to ampicillin during the first lactation.     

A partial budget model to estimate economic benefits of lactational treatment of subclinical Staphylococcus aureus mastitis

Subclinical Staphylococcus aureus mastitis is rarely treated during lactation because it is widely believed to be uneconomical, although there are no economic studies that support this view. Partial budgeting was used to develop a deterministic simulation model to estimate the net cost or benefit of antibiotic treatment of subclinical S. aureus mastitis during lactation. Direct and indirect effects of treatment were taken into account, including prevention of clinical flare-ups and contagious transmission. Input variables were based on literature and on 2003-2004 prices in the Netherlands. When contagious transmission of S. aureus was likely (reproductive ratio R = 5.3), 3- and 8-d treatments resulted in an average net profit of €95.62 and €142.42, respectively, compared with no treatment. When the probability of S. aureus transmission was low (R = 0.32), the average economic benefit of 3- or 8-d treatment was − €21.12 and − €57.70, respectively. On low-transmission farms, 3-d treatment was profitable when the appropriate cows were selected for treatment using known risk factors for cure. Sensitivity analysis showed that the 6 most influential input variables in the model were chance of bacteriological cure, R, probability of culling, retention pay-off, and cost of antibiotics and bacterial culture. Although the economic outcome of lactational treatment of subclinical S. aureus mastitis is highly herd-, cow-, and strain-dependent, treatment is economically justified in many situations.     

Mastitis-related subtypes of bovine Staphylococcus aureus are characterized by different clinical properties

Based on a former study from our group, one subtype of Staphylococcus aureus was associated with high within-herd prevalence of mastitis, whereas the other subtypes were associated with a low prevalence (sporadic intramammary infection). To confirm this hypothesis, a prospective study was done in 29 Swiss dairy herds. In particular, milk samples were collected from 10 herds with Staph. aureus herd problems (cases) and compared with samples from 19 herds with only sporadic cases of with Staph. aureus intramammary infection (controls). The isolates were tested for their virulence gene pattern and genotyped by PCR amplification of the 16S-23S rRNA intergenic spacer. The patterns and genotypes were then associated and compared with epidemiological and clinical data. Confirming the hypothesis, one particular subtype (genotype B) was associated with high within-herd and within-cow prevalence of intramammary infection, whereas the other subtypes were associated with low within-herd prevalence and infected single quarters. The gene patterns and genotypes were highly related, demonstrating the genetic diversity of the genotypes. The somatic cell counts were clearly increased in herds with a genotype B problem compared with herds with infections of other genotypes. Based on the different clinical properties and treatment consequences associated with these different genotypes found in Switzerland, we recommend subtyping Staph. aureus in other countries to determine if this finding is universally applicable.     

Short communication: Outbreak of methicillin-resistant Staphylococcus aureus (MRSA)-associated mastitis in a closed dairy herd

Cows are probably the main source of contamination of raw milk with Staphylococcus aureus. Mammary glands with subclinical mastitis can shed large numbers of Staph. aureus in milk. Because of the risk of this pathogen to human health as well as animal health, the aim of this paper was to describe an outbreak of mastitis caused by methicillin-resistant Staph. aureus (MRSA), oxacillin-susceptible mecA-positive Staph. aureus (OS-MRSA), and methicillin-susceptible Staph. aureus (MSSA) on a dairy farm. Milk samples were obtained from all quarters, showing an elevated somatic cell count by the California Mastitis Test. The isolates were identified by phenotypic and genotypic methods. Staphylococcus spp. were isolated from 53% (61/115) of the milk samples, with 60 isolates identified as Staph. aureus (98.4%) and 1 isolate identified as Staphylococcus epidermidis (1.6%). The presence of the mecA gene was verified in 48.3% of Staph. aureus isolates. Of the Staph. aureus isolates, 23.3% were MRSA and 25.0% were OS-MRSA. The total of mastitis cases infected with MRSA was 12.2%. The detection of this large percentage of mastitis cases caused by MRSA and OS-MRSA is of great concern for the animals’ health, because β-lactams are still the most important antimicrobials used to treat mastitis. In addition, Staph. aureus isolates causing bovine mastitis represent a public health risk.     

Short communication: Antimicrobial efficacy of intramammary treatment with a novel biphenomycin compound against Staphylococcus aureus, Streptococcus uberis, and Escherichia coli-induced mouse mastitis

Bovine mastitis undermines udder health, jeopardizes milk production, and entails prohibitive costs, estimated at $2 billion per year in the dairy industry of the United States. Despite intensive research, the dairy industry has not managed to eradicate the 3 major bovine mastitis-inducing pathogens: Staphylococcus aureus, Streptococcus uberis, and Escherichia coli. In this study, the antimicrobial efficacy of a newly formulated biphenomycin compound (AIC102827) was assessed against intramammary Staph. aureus, Strep. uberis, and E. coli infections, using an experimental mouse mastitis model. Based on its effective and protective doses, AIC102827 applied into the mammary gland was most efficient to treat Staph. aureus, but also adequately reduced growth of Strep. uberis or E. coli, indicating its potential as a broad-spectrum candidate to treat staphylococcal, streptococcal, and coliform mastitis in dairy cattle.     

Technical note: Antimicrobial susceptibility of Portuguese isolates of Staphylococcus aureus and Staphylococcus epidermidis in subclinical bovine mastitis

To evaluate the antimicrobial resistance traits of staphylococci responsible for subclinical bovine mastitis in Portugal, the minimum inhibitory concentrations (MIC) of 7 antimicrobial agents, frequently administered for mastitis treatment, were determined for 30 Staphylococcus aureus and 31 Staphylococcus epidermidis field isolates. β-Lactamase production was detected through the use of nitrocefin-impregnated discs. The MIC that inhibited 90% of the isolates tested (MIC₉₀) of penicillin, oxacillin, cefazolin, gentamicin, sulfamethoxazole/trimethoprim, oxytetracycline, and enrofloxacin were, respectively, 4, 0.5, 1, 1, 0.25, 0.25, and 0.06 μg/mL for Staph. aureus and ≥64, 8, 1, 32, ≥64, ≥64, and 0.06 μg/mL for Staph. epidermidis. All Staph. aureus isolates showed susceptibility to oxacillin, cefazolin, gentamicin, sulphamethoxazole/trimethoprim, and enrofloxacin. β-Lactamase production was detected in 20 of these isolates (66.7%), all of which were resistant to penicillin. Of the 31 Staph. epidermidis tested, 24 (77.4%) were β-lactamase positive. All isolates were susceptible to both cefazolin and enrofloxacin. Nine Staph. epidermidis isolates were resistant to oxacillin, with MIC values ranging from 4 to 8 μg/mL. The MIC values of 5 antimicrobial agents tested were higher than those reported in other countries. Enrofloxacin was the only exception, showing lower MIC values compared with other reports. Overall, the antimicrobial agents tested in our study, with the exception of penicillin, were active against the 61 isolates studied.     

Identification of potential sources of Staphylococcus aureus in herds with mastitis problems

Staphylococcus aureus is a common udder pathogen of dairy cows that often causes herd problems. Various mastitis control programs have been used to combat the problem but have not always been efficient in preventing new Staph. aureus infections, indicating the presence of possible sources of infection other than those traditionally considered. Therefore, the aim of the study was to identify potential sources of infection relevant for Staph. aureus mastitis within 5 dairy herds with udder health problems caused by Staph. aureus. Samples were collected from milk of lactating cows, from body sites, and from the environment of lactating cows, dry cows, late pregnant heifers, young heifers 4 to 12 mo old, and heifer calves 0 to 3 mo old. Isolates of Staph. aureus were identified and compared using pulsed-field gel electrophoresis. Four to 7 unique Staph. aureus pulsotypes were found within each herd, with one strain predominating in milk in each herd. The milk pulsotypes were also frequently isolated in body samples, especially on hock skin, and in the immediate environment of lactating cows, and were sometimes found in other animal groups, especially in dry cows and heifer calves 0 to 3 mo old. The prevalence of Staph. aureus in milk and other types of samples varied markedly between herds. Staphylococcus aureus isolates with genotypes indistinguishable from those found in milk also dominated in extra-mammary sites within the dairy herds studied, and hock skin was identified as an important reservoir of Staph. aureus. The results contribute new knowledge necessary to improve strategies for udder health control in herds.     

Short communication: profile of virulence factors of Staphylococcus aureus isolated from subclinical bovine mastitis in the state of Rio de Janeiro, Brazil

Staphylococcus aureus produces exoproteins that contribute to its ability to colonize the mammary gland such as hemolysins, coagulase, slime, and protein A. This study characterized phenotypically and genotypically these virulence factors in 50 Staph. aureus isolates. These isolates were obtained from milk samples from subclinical mastitis cases identified in 15 dairy cattle farms located in the state of Rio de Janeiro, Brazil. All of the confirmed Staph. aureus samples were PCR positive for the coa gene, which displayed 3 different size polymorphisms. The amplification of the spaA X region yielded a single amplicon for each isolate with the prevalent amplicon sized 315 bp. The Staph. aureus isolates were 24 and 16% positive for the hla and hlb genes, respectively, and 22 and 20% positive for the icaA and icaD genes, respectively. Amplification of the agr gene RNAIII was positive in 74% of the strains. Twenty-seven different profiles were identified among the samples, indicating a great diversity of Staph. aureus involved in the etiology of mastitis cases in the analyzed region. These findings are valuable to the comprehension of the distribution of the profiles of Staph. aureus strains isolated from subclinical mastitis cases in the state of Rio de Janeiro.     

Genotype-specific risk factors for Staphylococcus aureus in Swiss dairy herds with an elevated yield-corrected herd somatic cell count

Bovine mastitis is a frequent problem in Swiss dairy herds. One of the main pathogens causing significant economic loss is Staphylococcus aureus. Various Staph. aureus genotypes with different biological properties have been described. Genotype B (GTB) of Staph. aureus was identified as the most contagious and one of the most prevalent strains in Switzerland. The aim of this study was to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB in Swiss dairy herds with an elevated yield-corrected herd somatic cell count (YCHSCC). One hundred dairy herds with a mean YCHSCC between 200,000 and 300,000 cells/mL in 2010 were recruited and each farm was visited once during milking. A standardized protocol investigating demography, mastitis management, cow husbandry, milking system, and milking routine was completed during the visit. A bulk tank milk (BTM) sample was analyzed by real-time PCR for the presence of Staph. aureus GTB to classify the herds into 2 groups: Staph. aureus GTB-positive and Staph. aureus GTB-negative. Moreover, quarter milk samples were aseptically collected for bacteriological culture from cows with a somatic cell count ≥150,000 cells/mL on the last test-day before the visit. The culture results allowed us to allocate the Staph. aureus GTB-negative farms to Staph. aureus non-GTB and Staph. aureus-free groups. Multivariable multinomial logistic regression models were built to identify risk factors associated with the herd-level presence of Staph. aureus GTB and Staph. aureus non-GTB. The prevalence of Staph. aureus GTB herds was 16% (n = 16), whereas that of Staph. aureus non-GTB herds was 38% (n = 38). Herds that sent lactating cows to seasonal communal pastures had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 10.2, 95% CI: 1.9–56.6), compared with herds without communal pasturing. Herds that purchased heifers had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds without purchase of heifers. Furthermore, herds that did not use udder ointment as supportive therapy for acute mastitis had significantly higher odds of being infected with Staph. aureus GTB (odds ratio: 8.5, 95% CI: 1.6–58.4) or Staph. aureus non-GTB (odds ratio: 6.1, 95% CI: 1.3–27.8) than herds that used udder ointment occasionally or regularly. Herds in which the milker performed unrelated activities during milking had significantly higher odds of being infected with Staph. aureus GTB (rather than Staph. aureus non-GTB) compared with herds in which the milker did not perform unrelated activities at milking. Awareness of 4 potential risk factors identified in this study guides implementation of intervention strategies to improve udder health in both Staph. aureus GTB and Staph. aureus non-GTB herds.     

A longitudinal field study to evaluate the diagnostic properties of a quantitative real-time polymerase chain reaction-based assay to detect Staphylococcus aureus in milk

Bacteriological culture as a diagnostic tool for chronic infections with Staphylococcus aureus intramammary infection is not completely satisfactory. The cyclical shedding pattern of Staph. aureus with intervals of low excretion is considered to be the main reason. We recently developed a novel assay for Staph. aureus in milk, based on real-time quantitative PCR (QPCR). In a longitudinal study of chronically infected cows we evaluated the diagnostic properties of this test under field conditions. Diagnostic sensitivity of the novel test proved to be very high with a value of 99.4%; diagnostic specificity was 97.1%. In addition, the shedding patterns of Staph. aureus for the sampling period were analyzed. Using log₁₀-transformed QPCR data and plotting them across sampling time revealed a sinusoidal shedding pattern in 6 of 11 naturally infected quarters. Shedding patterns obtained by QPCR and by bacteriological culture were synchronous. In conclusion, the novel test has a very high diagnostic sensitivity and specificity so that quarters chronically infected with Staph. aureus are reliably detected, independent from their actual shedding quantity.