Antimicrobial resistance profiles of 5 common bovine mastitis pathogens in large Chinese dairy herds

The prevalence of antimicrobial resistance (AMR) is increasing in human and animal pathogens, becoming a concern worldwide. However, prevalence and characteristics of AMR of bovine mastitis pathogens in large Chinese dairy herds are still unclear. Therefore, our objective was to determine the AMR profile of bacteria isolated from clinical mastitis in large (>500 cows) Chinese dairy herds. A total of 541 isolates of the 5 most common species, Staphylococcus aureus (n = 103), non-aureus staphylococci (NAS; n = 107), Streptococcus species (n = 101), Klebsiella species (n = 130), and Escherichia coli (n = 100), isolated from bovine clinical mastitis on 45 dairy farms located in 10 provinces of China were included. Presence of AMR was determined by minimum inhibitory concentrations using the microdilution method. Prevalence of multidrug resistance (resistance to >2 antimicrobials) was 27% (148/541). A very wide distribution of minimum inhibitory concentrations was screened in all isolates, including Staph. aureus isolates, which were resistant to penicillin (66%). In addition, NAS (30%) were more resistant than Staph. aureus to oxacillin (84%), penicillin (62%), tetracycline (34%), and clindamycin (33%). Prevalence of resistance to tetracycline was high (59%) in Streptococcus spp. Additionally, prevalence of resistance of both E. coli and Klebsiella spp. was high to amoxicillin/clavulanate potassium (81 and 38%, respectively), followed by tetracycline (only Klebsiella spp. 32%). A high proportion (27%) of isolates were multidrug resistant; the most frequent combinations were clindamycin-cefalexin-tetracycline or enrofloxacin-cefalexin-penicillin patterns for Staph. aureus; enrofloxacin-oxacillin-penicillin-tetracycline patterns for NAS; clindamycin-enrofloxacin-tetracycline patterns for Streptococcus spp.; amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for Klebsiella spp.; and amoxicillin/clavulanate potassium-ceftiofur-polymyxin B patterns for E. coli. Resistance for 4 kinds of antimicrobials highly critical for human medicine, including daptomycin, vancomycin, imipenem, and polymyxin B, ranged from 0 to 24%. In conclusion, prevalence of AMR in mastitis pathogens was high on large Chinese dairy farms, potentially jeopardizing both antimicrobial efficacy and public health. Results of this study highlighted the need for improvements in antimicrobial stewardship and infection control programs in large Chinese dairy farms to reduce emergence of AMR.     

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.     

Pathogen-specific production losses in bovine mastitis

Reduction in long-term milk yields represents a notable share of the economic losses caused by bovine mastitis. Efficient, economic, and safe measures to prevent these losses require knowledge of the causal agent of the disease. The aim of this study was to investigate pathogen-specific impacts of mastitis on milk production of dairy cows. The materials consisted of milk and health recording data and microbiological diagnoses of mastitic quarter milk samples of 20,234 Finnish dairy cows during 2010, 2011, and 2012. The 6 most common udder pathogens were included in the study: Staphylococcus aureus, non-aureus staphylococci (NAS), Escherichia coli, Corynebacterium bovis, Streptococcus uberis, and Streptococcus dysgalactiae. We used a 2-level multilevel model to estimate curves for lactations with and without mastitis. The data on lactation periods to be compared were collected from the same cow. To enable comparison among lactations representing diverse parities, the estimated lactation curves were adjusted to describe the cow's third lactation. Mastitis caused by each pathogen resulted in milk production loss. The extent of the reduction depended on the pathogen, the timing of mastitis during lactation, and the type of mastitis (clinical vs. subclinical). The 2 most commonly detected pathogens were NAS and Staph. aureus. Escherichia coli clinical mastitis diagnosed before peak lactation caused the largest loss, 10.6% of the 305-d milk yield (3.5 kg/d). The corresponding loss for Staph. aureus mastitis was 7.1% (2.3 kg/d). In Staph. aureus mastitis diagnosed between 54 and 120 d in milk, the loss was 4.3% (1.4 kg/d). The loss was almost equal in both clinical and subclinical mastitis caused by Staph. aureus. Mastitis caused by Strep. uberis and Strep. dysgalactiae resulted in losses ranging from 3.7% (1.2 kg/d) to 6.6% (2.1 kg/d) depending on type and timing of mastitis. Clinical mastitis caused by the minor pathogens C. bovis and NAS also had a negative effect on milk production: 7.4% (2.4 kg/d) in C. bovis and 5.7% (1.8 kg/d) in NAS when both were diagnosed before peak lactation. In conclusion, minor pathogens should not be underestimated as a cause of milk yield reduction. On single dairy farms, control of E. coli mastitis would bring about a significant increase in milk production. Reducing Staph. aureus mastitis is the greatest challenge for the Finnish dairy sector.     

Antimicrobial activity of crude extracts from actinomycetes against mastitis pathogens

The emergence of antimicrobial resistance to commonly used antibiotics has necessitated the development of new antimicrobial products. Crude extracts produced by actinomycetes contain antimicrobial metabolites that can inhibit bacterial growth. The objective of our study was to evaluate the antimicrobial activity of crude extracts (Caat1–54 and CaatP5–8) produced by actinomycetes against isolates of Staphylococcus aureus, Staphylococcus chromogenes, Streptococcus dysgalactiae, and Streptococcus uberis, which were obtained from the milk of cows affected by mastitis in 23 dairy herds. Twenty isolates of each bacterial species were used to define minimum inhibitory concentrations (MIC) of both crude extracts and ceftiofur (positive control). The MIC₅₀ and MIC₉₀ were defined at the concentration required to inhibit the growth of 50 and 90% of bacterial isolates tested, respectively. The MIC results were evaluated by survival analysis. Staphylococcus aureus isolates presented MIC₉₀ of Caat 1–54 ≥6.25 µg/mL, ceftiofur ≥12.5 µg/mL, and Caat P5–8 ≥100 µg/mL. Streptococcus uberis presented MIC₉₀ of ceftiofur ≥0.39 µg/mL, Caat 1–54 ≥50 µg/mL, and Caat P5–8 ≥100 µg/mL. Staphylococcus chromogenes isolated from subclinical mastitis presented MIC₉₀ of Caat 1–54 ≥0.78 µg/mL and ceftiofur and Caat P5–8 of ≥6.25 and ≥100 µg/mL, respectively. Streptococcus dysgalactiae isolated from clinical mastitis presented similar MIC₉₀ values between antimicrobials tested (ceftiofur, Caat 1–54, and Caat P-58), but these values (≥100 µg/mL) were higher than the values obtained from other pathogens evaluated in the present study. Our results indicate that Caat 1–54 and Caat P5–8 crude extracts present in vitro antimicrobial activity against isolates of Staph. aureus, Staph. chromogenes, Strep. dysgalactiae, and Strep. uberis isolated from clinical and subclinical mastitis.     

Relationships among superantigen toxin gene profiles,genotypes, and pathogenic characteristics of Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is one of the major etiological agents of bovine mastitis, harboring a wide variety of staphylococcal superantigen (SAg) toxin genes. The SAg toxin genes are reported to be closely associated with the pathogenicity of the Staph. aureus causing the bovine mastitis. This study was conducted to investigate SAg toxin gene profiles and to assess the relationships among SAg toxin genes, genotypes of Staph. aureus, and their pathogenic properties. A total of 327 quarter milk samples were collected from bovine mastitis cases for isolation and identification of pathogens. In total, 35 isolates were identified as Staph. aureus, and the prevalence of Staph. aureus in milk samples was 13.6% (35/256). Polymerase chain reaction (PCR) and randomly amplified polymorphic DNA (RAPD) assays were used to detect the SAg toxin genes and to genotype Staph. aureus strains isolated from milk samples of bovine mastitis in 10 dairy herds located in Ningxia, China, respectively. The results showed that among the Staph. aureus isolates (n = 35), 71.4% (n = 25) of isolates carried at least one SAg toxin gene. In total, 18 SAg genes and 21 different gene combination patterns were detected among these isolates. The most common SAg genes in Staph. aureus isolates were sei, sen, and seu (44.0% each), followed by seo, tst, and etB (28.0% each), etA (24.0%), sem and sep (16.0% each), seb, sec, sed, and sek (12.0% each), and sea and seh genes (8.0% each); the seg, sej, and ser genes were present in 4.0% of the isolates. Three gene combinations were found to be related to mobile genetic elements that carried 2 or more genes. The egc-cluster of the seg-sei-sem-sen-seo genes, located on the pathogenicity island Type I υSaβ, was detected in 16% of isolates. Interestingly, we observed 6 RAPD genotypes (I to VI) in Staph. aureus isolates, and 2 of these genotypes were strongly associated with the severity of bovine mastitis; there was a close relationship between the RAPD genotypes and SAg genes. Isolates of RAPD type III were more frequently associated with clinical and subclinical mastitis, whereas strains of type VI were mostly related to subclinical mastitis. In addition, SAg genes were related to severity of bovine mastitis. We conclude that an obvious relationship exists among RAPD genotypes, SAg toxin genes, and severity of bovine mastitis.     

Short communication: Lack of intramammary niche recolonization during a sanitation program for the contagious mastitis pathogen Staphylococcus aureus genotype B

In Switzerland, sanitation programs of dairy herds infected with the contagious mastitis pathogen Staphylococcus aureus genotype B (GTB) have been established for several years. In recent years, Streptococcus uberis and non-aureus staphylococci have emerged as the bacteria most frequently isolated from bovine milk samples. The latter cause subclinical mastitis, and some species are more persistent or pathogenic than others. The present study aimed to investigate the developments in the intramammary colonization spectrum of 5 dairy herds undergoing a sanitation program for Staph. aureus GTB. We collected single-quarter milk samples aseptically from all lactating cows at 3-mo intervals during the sanitation period; after classical bacteriological analysis, MALDI-TOF mass spectrometry was used to identify the isolates to the species level. Non-aureus staphylococci were found to be the bacterial group most frequently occurring on the selected farms, with Staphylococcus chromogenes and Staphylococcus xylosus being predominant. The present study demonstrated that GTB-infected cows treated with antibiotics lacked systematic recolonization with other bacteria during herd sanitation for the contagious Staph. aureus GTB.     

Minimum inhibitory concentrations of Staphylococcus aureus recovered from clinical and subclinical cases of bovine mastitis

Antimicrobials are often used for treatment of bovine mastitis and the possibility of selection for resistant bacteria must be considered. The objectives of this study were to determine the minimum inhibitory concentration of Staphylococcus aureus recovered from cases of clinical and subclinical bovine mastitis, and to determine the prevalence of multidrug resistance in this population. Milk samples were collected from cows on commercial dairy herds (n=13), including quarters (n=1,574) of cows with subclinical mastitis cases, and cows experiencing clinical mastitis cases (n=608). Selected Staph. aureus isolates, obtained from clinical (n=58) and subclinical (n=58) mastitis cases, were used to determine minimum inhibitory concentrations of 12 selected antimicrobials. Of Staph. aureus isolates tested, 87 (75%) did not exhibit resistance to any antimicrobial, 28 (24.1%) exhibited resistance to 1 (n=21) or 2 (n=7) classes of antimicrobials, and 1 (0.9%) exhibited multidrug resistance. All Staph. aureus (clinical and subclinical cases) were inhibited by the range of concentrations tested for ceftiofur and oxacillin. Moreover, no isolates obtained from clinical mastitis cases exhibited resistance to cephalothin, penicillin-novobiocin, or sulfadimethoxine. Of isolates, 3 exhibited resistance to enrofloxacin. Of isolates exhibiting resistance to more than 1 antimicrobial, independent of antimicrobial class, the combination of erythromycin and tetracycline, and ampicillin and penicillin accounted for the majority of resistance. Of isolates tested, 19% were resistant to tetracycline and 14% were resistant to penicillin. Survival curves of Staph. aureus relative to minimum inhibitory concentration demonstrated heterogeneity among case types for ceftiofur, cephalothin, and erythromycin. Multidrug resistance was identified in only 1 isolate obtained from a single farm.     

Evaluating a commercial PCR assay against bacterial culture for diagnosing Streptococcus uberis and Staphylococcus aureus throughout lactation

The performance of a commercial, real-time PCR assay was compared with traditional bacterial culture for the identification of Streptococcus uberis and Staphylococcus aureus in bovine milk collected at different stages of lactation. Initial validation tests using fresh and frozen quarter milk samples identified factors that affected the success of the PCR. Therefore, the standard protocol was adjusted for samples collected at the first milking postpartum (colostrum) and from clinical mastitis cases. The adjustment involved PCR testing both undiluted and diluted (1 in 10 with sterile water) DNA extracts. The performance comparison between culture and the PCR assay used milk samples collected aseptically from individual quarters of mixed-age spring-calving dairy cows, during early, mid, and late lactation. Bacterial culture results were used to select a subset of samples for PCR testing (n = 315) that represented quarters with a current or prior Strep. uberis or Staph. aureus infection. Compared with culture, PCR had a sensitivity of 86.8% and specificity of 87.7% for detecting Strep. uberis (kappa = 0.74) and 96.4% and 99.7%, respectively, for detecting Staph. aureus (kappa = 0.96). The dilution of DNA extracts for colostrum and clinical samples increased the relative sensitivity from 79.2% to 86.8% for Strep. uberis detection and from 92.9% to 96.4% for Staph. aureus, presumably through diluting unidentified PCR inhibitors. The sensitivity for detecting Strep. uberis using PCR, relative to culture, was similar throughout lactation (85–89%), whereas relative specificity was lowest immediately postcalving (64%) but improved in mid and late lactation (98%). Specificity estimates for samples collected in early lactation can be optimized by reducing the cutoff cycle threshold (Ct) value from the recommended value of 37 to 34. Although using this value improved specificity (77%), it reduced test sensitivity (77%). The PCR assay lacked agreement with culture in early lactation, specifically for diagnosing Strep. uberis. Thus, PCR should not be used as the only tool for diagnosing mastitis in early lactation.     

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.     

Detection of mastitis pathogens by analysis of volatile bacterial metabolites

The ability to detect mastitis pathogens based on their volatile metabolites was studied. Milk samples from cows with clinical mastitis, caused by Staphylococcus aureus, coagulase-negative staphylococci, Streptococcus uberis, Streptococcus dysgalactiae, and Escherichia coli were collected. In addition, samples from cows without clinical mastitis and with low somatic cell count (SCC) were collected for comparison. All mastitis samples were examined by using classical microbiological methods, followed by headspace analysis for volatile metabolites. Milk from culture-negative samples contained a lower number and amount of volatile components compared with cows with clinical mastitis. Because of variability between samples within a group, comparisons between pathogens were not sufficient for classification of the samples by univariate statistics. Therefore, an artificial neural network was trained to classify the pathogen in the milk samples based on the bacterial metabolites. The trained network differentiated milk from uninfected and infected quarters very well. When comparing pathogens, Staph. aureus produced a very different pattern of volatile metabolites compared with the other samples. Samples with coagulase-negative staphylococci and E. coli had enough dissimilarity with the other pathogens, making it possible to separate these 2 pathogens from each other and from the other samples. The 2 streptococcus species did not show significant differences between each other but could be identified as a different group from the other pathogens. Five groups can thus be identified based on the volatile bacterial metabolites: Staph. aureus, coagulase-negative staphylococci, streptococci (Strep. uberis and Strep. dysgalactiae as one group), E. coli, and uninfected quarters.     

Antimicrobial susceptibilities in dairy herds that differ in dry cow therapy usage

Intramammary infusion of antimicrobials at the end of lactation (dry cow therapy; DCT) is a central part of mastitis control programs and is one of the major indications for antimicrobial use in dairy cows. However, with increasing focus on prudent use of antimicrobials and concerns about emergence of antimicrobial resistance, the practice of treating every cow at the end of lactation with DCT is in question. This cross-sectional, observational study determined the minimum inhibitory concentrations (MIC) of 10 antimicrobials for coagulase-negative staphylococci (CNS), Staphylococcus aureus, Streptococcus dysgalactiae, and Streptococcus uberis isolates from milk samples from dairy cows with somatic cell counts >200,000 cells/mL in herds that had been organic for >3 yr (n = 7), or had used either ampicillin-cloxacillin DCT (n = 11) or cephalonium DCT (n = 8) in the preceding 3 yr. The organic herds were certified under the United States Department of Agriculture National Organic Program, meaning that there was no blanket DCT, and minimal use of antimicrobials in general, with a loss of organic status of the animal if treated with antimicrobials. Breakpoints (where available) were used to categorize isolates as resistant, intermediate, or susceptible to antimicrobials. The MIC distributions of isolates from different herd types were compared using binomial or multinomial logistic regression. Of 240 CNS isolates, 12.9, 0.8, 7.1, 32.6, and 1.2%, were intermediate or resistant to ampicillin, cephalothin, erythromycin, penicillin, and tetracycline, respectively. Of 320 Staph. aureus isolates, 29.0, 2.5, 1.2, and 34.9% were intermediately resistant or resistant to ampicillin, penicillin, erythromycin, and oxacillin, respectively. Of 184 Strep. uberis isolates, 1.1, 25.0, 1.6, and 1.6% were intermediately resistant or resistant to erythromycin, penicillin, pirlimycin, and tetracycline, respectively. Generally, the MIC of CNS and streptococcal isolates from organic herds were lower than isolates from herds using DCT. However, the differences in MIC distributions occurred at MIC below clinical breakpoints, so that the bacteriological cure rates may not differ between isolates of differing MIC. Bimodal distributions of MIC for ampicillin and penicillin were found in Staph. aureus isolates from organic herds, suggesting that isolates with a higher MIC are a natural part of the bacterial population of the bovine mammary gland, or that isolates with higher MIC have persisted within these organic herds from a time when antimicrobials had been used. Given these observations, further work is required to determine if exposure to DCT is causally associated with the risk of elevated MIC, and whether reduction or removal of DCT from herds would reduce the risk of elevated MIC of mastitis pathogens.     

Genetic correlations between pathogen-specific mastitis and somatic cell count in Danish Holsteins

The aim of this study was to estimate genetic correlations (r_a) between 2 lactation average somatic cell count (LASCC) traits and 6 different mastitis traits in 226,482 first-parity Danish Holstein cows that calved between 1998 and 2008. The LASCC traits were defined from 5 to either 170 d (LASCC_170) or 300 d (LASCC_300) after calving, and the mastitis traits were unspecific mastitis (all mastitis treatments, both clinical and subclinical, regardless of the causative pathogen) and mastitis caused by either Streptococcus dysgalactiae, Escherichia coli, coagulase-negative staphylococci (CNS), Staphylococcus aureus, or Streptococcus uberis. Variance components were estimated using bivariate threshold-Gaussian models via Gibbs sampling. The posterior means of r_a between LASCC_170 and the mastitis traits were greatest for unspecific mastitis (r_a = 0.71), followed by CNS, Strep. dysgalactiae, Strep. uberis, and E. coli (r_a = 0.54 to 0.69) and were lowest for Staph. aureus mastitis (r_a = 0.44). The genetic correlation between LASCC_300 and the mastitis traits were generally smaller (r_a = 0.47 to 0.69). Caution should be taken when interpreting the results, however, because some posterior density intervals for r_a were large (between 0.14 and 0.47 units). Phenotypically, Staph. aureus is known to be associated with high SCC and especially with subclinical mastitis through chronic infections, so the low r_a between Staph. aureus mastitis and LASCC, compared with r_a for the other pathogens, was not expected. Subclinical cases are usually submitted to dry cow therapy (not included in the present study), not treated at all, or wrongly recorded as clinical cases. Thus, the incidence of Staph. aureus mastitis is likely too low, and the genetic correlation between Staph. aureus mastitis and LASCC may therefore be underestimated in the present study. The results for the remaining pathogens were as expected, smallest for E. coli and larger but similar for Strep. dysgalactiae, Strep. uberis, and CNS. Selection for lower LASCC is expected to decrease the incidence of pathogen-specific mastitis, especially for Strep. uberis, Strep. dysgalactiae, and CNS and, to a lesser extent, for Staph. aureus and E. coli. Data recording should preferably be improved, and economic weights for the pathogen-specific mastitis traits should be estimated before implementing an udder health index that includes pathogen-specific mastitis traits.     

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.     

Real-time polymerase chain reaction-based identification of bacteria in milk samples from bovine clinical mastitis with no growth in conventional culturing

In more than 30% of milk samples from clinical and subclinical bovine mastitis, bacteria fail to grow even after 48 h of conventional culture. The “no-growth” samples are problematic for mastitis laboratories, veterinarians, and dairy producers. This study provides the first investigation of the bacteriological etiology of such samples, using a real-time PCR-based commercial reagent kit. The assay targets the DNA of the 11 most common bacterial species or groups in mastitis and the staphylococcal blaZ gene (responsible for penicillin resistance) and can identify and quantify bacterial cells even if dead or growth-inhibited. A study was made of 79 mastitic milk samples with no-growth bacteria in conventional culture, originating from cows with clinical mastitis. Of the 79 samples, 34 (43%) were positive for 1 (32 samples) or 2 (2 samples) of the target bacteria. The positive findings included 11 Staphylococcus spp. (staphylococci other than Staphylococcus aureus), 10 Streptococcus uberis, 2 Streptococcus dysgalactiae, 6 Corynebacterium bovis, 3 Staph. aureus, 1 Escherichia coli, 1 Enterococcus, and 1 Arcanobacterium pyogenes. The positive samples contained as many as 10³ to 10⁷ bacterial genome copies per milliliter of milk. This study demonstrates that in nearly half of the clinical mastitis cases in which conventional culture failed to detect bacteria, mastitis pathogens were still present, often in substantial quantities. The clearly elevated N-acetyl-β-d-glucosaminidase activity values of the milk samples, together with clinical signs of the infected cows and quarters, confirmed the diagnosis of clinical mastitis and indicated that real-time, PCR-based bacterial findings are able to reveal bacteriological etiology. We conclude that all common mastitis bacteria can occur in large quantities in clinical mastitis samples that exhibit no growth in conventional culture, and that the real-time PCR assay is a useful tool for bacteriological diagnosis of such milk samples. Low bacterial concentration is commonly speculated to explain the no-growth milk samples. This hypothesis is not supported by the results of the current study.     

Milk culture results in a large Norwegian survey--effects of season, parity, days in milk, resistance, and clustering

A nationwide random computerized assignment survey that included 3,538 sets of 4 quarter milk samples from 2,834 dairy cows was conducted during 2000. Every fifth cow from every 50th herd was randomly selected for sampling and culture during each quarter of the year. Milk culture results of pathogens known to be related to mastitis were recorded regardless of whether mastitis had been indicated by any inflammatory measure or not. Farmers were blinded to all test results to minimize any potential interventions that might be prompted by the results. The most prevalent isolate was Staphylococcus aureus, which was identified in 8.2% of the quarter milk samples. More than 15 colony-forming units/0.01 mL of Staph. aureus were found in 4.3% of the quarter milk samples, whereas 3.5% had only 1 to 3 colony-forming units/0.01 mL. Streptococcus dysgalactiae, coagulase-negative staphylococci (CNS), and Streptococcus uberis were isolated from 1.2, 3.3, and 0.4% of quarter milk samples, respectively. No isolates were found in 76.6% of the quarter milk samples tested. Among individual cows, 22.2% had an isolate of Staph. aureus in ≥ 1 quarter. Only Strep. dysgalactiae exhibited a higher prevalence with increased parity. Prevalence of Staph. aureus decreased throughout days in milk, but prevalence of Strep. dysgalactiae increased. There was a strong seasonal effect; the highest prevalence of Strep. dysgalactiae and CNS was observed during April and May (late indoor season), and the highest prevalence of Staph. aureus and Strep. uberis was observed during June and July (the outdoor season). A substantial within-cow clustering effect was found for Strep. dysgalactiae, Staph. aureus, and CNS. Additionally, a within-herd effect was found for Strep. uberis, penicillin-resistant Staph. aureus, total Staph. aureus, and CNS. No within-county cluster effect was found. Lastly, both Staph. aureus and CNS exhibited a surprisingly high seasonal effect regarding the prevalence of resistance to penicillin G. Penicillin resistance of Staph. aureus was likely due to higher prevalence of Staph. aureus as a whole, but for CNS, there was also an additional increase caused by a higher proportional rate of penicillin resistance during the late indoor season.     

Short communication: Staphylococcus aureus isolated from colostrum of dairy heifers represent a closely related group exhibiting highly homogeneous genomic and antimicrobial resistance features

In heifers, intramammary infections caused by Staphylococcus aureus affect milk production and udder health in the first and subsequent lactations, and can lead to premature culling. Not much is known about Staph. aureus isolated from heifers and it is also unclear whether or not these strains are readily transmitted between heifers and lactating herd mates. In this study, we compared phenotypic characteristics, spa types, and DNA microarray virulence and resistance gene profiles of Staph. aureus isolates obtained from colostrum samples of dairy heifers with isolates obtained from lactating cows. Our objective was to (1) characterize Staph. aureus strains associated with mastitis in heifers and (2) determine relatedness of Staph. aureus strains from heifers and lactating cows to provide data on transmission. We analyzed colostrum samples of 501 heifers and milk samples of 68 lactating cows within the same herd, isolating 48 and 9 Staph. aureus isolates, respectively. Staphylococcus aureus strains from heifers, lactating herd mates, and an unrelated collection of 78 strains from bovine mastitis milk of mature cows were compared. With 1 exception each, characterization of all strains from heifers and lactating cows in the same herd yielded highly similar phenotypic and genotypic results. The strains were Staphaurex latex agglutination test negative (Oxoid AG, Basel, Switzerland) and belonged to agr type II, CC705, and spa types tbl 2645 and t12926. They were susceptible to all antimicrobial agents tested. In contrast, the strains from mature cows in other herds were spread across different clonal complexes, spa types, and SplitsTree clusters (http://www.splitstree.org/), thus displaying a far higher degree of heterogeneity. We conclude that strains isolated from colostrum of heifers and mastitis milk of lactating cows in the same herd feature highly similar phenotypic and genomic characteristics, suggesting persistence of the organism during the first and potentially subsequent lactations or transmission between heifers and mature herd mates.     

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.     

Non-aureus staphylococci in fecal samples of dairy cows: First report and phenotypic and genotypic characterization

The aims of this study were to determine whether non-aureus staphylococci (NAS) are present in rectal feces of healthy dairy cows, and if so, to delineate species to which they belong and to study several phenotypic and genotypic traits as a first step toward determining the potential impact of fecal shedding of NAS on bovine udder health. Fecal samples were aseptically collected from the rectum of 25 randomly selected clinically healthy dairy cows in a commercial dairy herd using an automated milking system. Fecal NAS were isolated and then identified at the species level using transfer RNA-intergenic spacer PCR and sequencing of the 16S rRNA housekeeping gene. Strain typing was performed using random amplification of polymorphic DNA (RAPD)-PCR. The antimicrobial resistance profiles, biofilm formation, and growth and inhibitory characteristics of all NAS isolates were evaluated. Half of the cows were shedding NAS, resulting in 31 NAS isolates belonging to 11 different species. The most prevalent species were Staphylococcus rostri (23%, n = 7), Staphylococcus cohnii (16%, n = 5), and Staphylococcus haemolyticus (13%, n = 4) with all Staphylococcus agnetis, Staphylococcus chromogenes, and Staph. rostri isolates belonging to the same strain according to RAPD banding patterns. Acquired antimicrobial resistance was observed in 28 of the 31 NAS isolates, mainly due to β-lactamase production. Most of the isolates (84%, n = 27) had a weak biofilm-forming potential, but only 2 contained the bap gene. The ica and aap genes were not detected in any of the isolates. In vitro growth of Staphylococcus aureus and Streptococcus dysgalactiae was inhibited by Staph. agnetis isolates, and Staph. chromogenes isolates were able to inhibit the growth of Strep. dysgalactiae and Streptococcus uberis. All fecal isolates were able to grow when oxygen and iron were limitedly available, mimicking the growth conditions in the mammary gland.     

Clinical outcomes and molecular genotyping of Staphylococcus aureus isolated from milk samples of dairy primiparous Mediterranean buffaloes (Bubalus bubalis)

Staphylococcus aureus is one of the most important pathogens causing mastitis in dairy cows and in Mediterranean buffaloes. Genotype B (GTB) is contagious in dairy cows and may occur in up to 87% of cows of a dairy herd. It was the aim of this study to evaluate genotypes present, clinical outcomes, and prevalence of Staph. aureus in milk samples of primiparous Mediterranean dairy buffaloes. Two hundred composite milk samples originating from 40 primiparous buffaloes were collected from May to June 2012, at d 10, 30, 60, 90, and 150 d in milk (DIM) to perform somatic cell counts and bacteriological cultures. Daily milk yields were recorded. Before parturition until 40 to 50 DIM, all primiparous animals were housed separated from the pluriparous animals. Milking was performed in the same milking parlor, but the primiparous animals were milked first. After 50 DIM, the primiparous were mixed with the pluriparous animals, including the milking procedure. Individual quarter samples were collected from each animal, and aliquots of 1 mL were mixed and used for molecular identification and genotyping of Staph. aureus. The identification of Staph. aureus was performed verifying the presence of nuc gene by nuc gene PCR. All the nuc-positive isolates were subjected to genotype analysis by means of PCR amplification of the 16S-23S rRNA intergenic spacer region and analyzed by a miniaturized electrophoresis system. Of all 200 composite samples, 41 (20.5%) were positive for Staph. aureus, and no genotype other than GTB was identified. The prevalence of samples positive for Staph. aureus was 0% at 10 DIM and increased to a maximum of 22/40 (55%) at 90 DIM. During the period of interest, 14 buffaloes tested positive for Staph. aureus once, 6 were positive twice, and 5 were positive 3 times, whereas 15 animals were negative at every sampling. At 90 and 150 DIM, 7 (17.5%) and 3 buffaloes (7.5%), respectively, showed clinical mastitis (CM), and only 1 (2.5%) showed CM at both samplings. At 60, 90, and 150 DIM, 1 buffalo was found with subclinical mastitis at each sampling. At 30, 60, 90, and 150 DIM, 2.5 (1/40), 22.5 (9/40), 35 (14/40), and 10% (4/40) were considered affected by intramammary infection, respectively. Buffaloes with CM caused by Staph. aureus had statistically significantly higher mean somatic cell count values (6.06 ± 0.29, Log₁₀ cells/mL ± standard deviation) and statistically significantly lower mean daily milk yields (7.15 ± 1.49, liters/animal per day) than healthy animals (4.69 ± 0.23 and 13.87 ± 2.64, respectively), buffaloes with IMI (4.82 ± 0.23 and 11.16 ± 1.80, respectively), or with subclinical mastitis (5.47 ± 0.10 and 10.33 ± 0.68, respectively). Based on our knowledge, this is the first time that Staph. aureus GTB has been identified in milk samples of dairy Mediterranean buffaloes.