A survey of mastitis pathogens including antimicrobial susceptibility in southeastern Australian dairy herds

The objectives for this study were to (1) describe the pathogen profile in quarters from cows with clinical mastitis and in cows with subclinical mastitis in southeastern Australia; and (2) describe antimicrobial susceptibility among isolated pathogens. As a secondary objective, we aimed to compare antimicrobial resistance prevalence in pathogens isolated from clinical and subclinical mastitis samples. A convenience sample of dairy herds (n = 65) from 4 regions in southeastern Australia (Gippsland, Northern Victoria, Tasmania, Western Victoria) were invited to submit milk samples from cows with clinical and subclinical mastitis over a 14-mo period (January 2011 to March 2012). Farmers were instructed to collect aseptic quarter milk samples from the first 10 cases of clinical mastitis for each month of the study. In addition, farmers submitted composite milk samples from cows with subclinical mastitis at 1 or 2 sampling occasions during the study period. Aerobic culture and biochemical tests were used to identify isolates. Isolates were classified as susceptible, intermediate, or resistant to a panel of antimicrobial agents based on the zone of growth inhibition around antimicrobial-impregnated disks, with antimicrobial resistance (AMR) classified as nonsusceptibility by combining intermediate and resistant groups into a single category. Generalized linear mixed models were used to compare the prevalence of AMR between clinical and subclinical mastitis isolates. For clinical mastitis samples (n = 3,044), 472 samples (15.5%) were excluded for contamination. Of the remaining samples (n = 2,572), the most common results were Streptococcus uberis (39.2%), no growth (27.5%), Staphylococcus aureus (10.6%), Escherichia coli (8.4%), and Streptococcus dysgalactiae (6.4%). For subclinical mastitis samples (n = 1,072), 425 (39.6%) were excluded due to contamination. Of the remaining samples (n = 647), the most common results were no growth (29.1%), Staph. aureus (29.1%), and Strep. uberis (21.6%). The prevalence of AMR among common isolates was low for the majority of antimicrobial agents. Exploratory analysis found that the probability of Staph. aureus demonstrating resistance to penicillin was 5.16 times higher (95% confidence interval: 1.68, 15.88) in subclinical isolates relative to clinical Staph. aureus isolates. A similar association was observed for amoxicillin with subclinical Staph. aureus isolates being 4.70 times (95% confidence interval: 1.49, 14.75) more likely to be resistant than clinical Staph. aureus isolates. We concluded that the most common bacteria causing clinical mastitis in dairy herds in Australia is likely to be Strep. uberis, whereas Staph. aureus is likely to be the most common cause of subclinical mastitis. Despite decades of antimicrobial use to control these organisms, AMR appears to be uncommon.     

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.     

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.     

Relationships between milk culture results and milk yield in Norwegian dairy cattle

Associations between test-day milk yield and positive milk cultures for Staphylococcus aureus, Streptococcus spp., and other mastitis pathogens or a negative milk culture for mastitis pathogens were assessed in quarter milk samples from randomly sampled cows selected without regard to current or previous udder health status. Staphylococcus aureus was dichotomized according to sparse (≤1,500 cfu/mL of milk) or rich (>1,500 cfu/mL of milk) growth of the bacteria. Quarter milk samples were obtained on 1 to 4 occasions from 2,740 cows in 354 Norwegian dairy herds, resulting in a total of 3,430 samplings. Measures of test-day milk yield were obtained monthly and related to 3,547 microbiological diagnoses at the cow level. Mixed model linear regression models incorporating an autoregressive covariance structure accounting for repeated test-day milk yields within cow and random effects at the herd and sample level were used to quantify the effect of positive milk cultures on test-day milk yields. Identical models were run separately for first-parity, second-parity, and third-parity or older cows. Fixed effects were days in milk, the natural logarithm of days in milk, sparse and rich growth of Staph. aureus (1/0), Streptococcus spp. (1/0), other mastitis pathogens (1/0), calving season, time of test-day milk yields relative to time of microbiological diagnosis (test day relative to time of diagnosis), and the interaction terms between microbiological diagnosis and test day relative to time of diagnosis. The models were run with the logarithmically transformed composite milk somatic cell count excluded and included. Rich growth of Staph. aureus was associated with decreased production levels in first-parity cows. An interaction between rich growth of Staph. aureus and test day relative to time of diagnosis also predicted a decline in milk production in third-parity or older cows. Interaction between sparse growth of Staph. aureus and test day relative to time of diagnosis predicted declining test-day milk yields in first-parity cows. Sparse growth of Staph. aureus was associated with high milk yields in third-parity or older cows after including the logarithmically transformed composite milk somatic cell count in the model, which illustrates that lower production levels are related to elevated somatic cell counts in high-producing cows. The same association with test-day milk yield was found among Streptococcus spp.-positive pluriparous cows.     

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.     

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.     

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.     

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.     

Prevalence,antimicrobial susceptibility,and molecular characterization of Staphylococcus aureus isolated from dairy herds in northern China

Staphylococcus aureus is one of the main pathogens involved in dairy cow mastitis. Monitoring of antibiotic use would prove useful to assess the risk of Staph. aureus in raw milk. The objective of this work was to investigate the prevalence of Staph. aureus strais isolated from raw milk in northern China, and to characterize antimicrobial susceptibility of these strains and their key virulence genes. In total, 195 raw milk samples were collected from 195 dairy farms located in 4 cities of northern China from May to September 2015. Out of 195 samples, 54 (27.7%) were positive for Staph. aureus. Among these 54 samples, 54 strains of Staph. aureus were isolated, and 16 strains were identified as methicillin-resistant Staph. aureus. The strains exhibited high percentages of resistance to penicillin G (85.2%), ampicillin (79.6%), and erythromycin (46.3%). Moreover, 72% of the strains showed resistance to more than one antimicrobial agent. Overall, 63% of penicillin-resistant strains possessed the blaZ gene, and 60% of the erythromycin-resistant strains possessed erm(A), erm(B), erm(C), msr(A), or msr(B) genes with 8 different gene patterns. All isolates resistant to gentamicin, kanamycin, and oxacillin carried the aac6'-aph2”, ant(4')-Ia, and mecA genes, respectively. Two tet(M)-positive isolates carried specific genes of the Tn916-Tn1545 transposon. The most predominant virulence genes were sec, sea, and pvl, which encode staphylococcal enterotoxins (sec and sea) and Panton-Valentine leukocidin, respectively. Thirty-two isolates (59.2%) harbored one or more virulence genes. The majority of Staph. aureus strains were multidrug resistant and carried multiple virulence genes, which may pose a risk to public health. Our data indicated that antimicrobial resistance of Staph. aureus was prevalent in dairy herds in northern China, and that antibiotics, especially penicillin G and ampicillin, to treat mastitis caused by Staph. aureus should be used with caution in northern China.     

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.     

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.     

A longitudinal study investigating the prevalence of Staphylococcus aureus genotype B in seasonally communal dairy herds

Staphylococcus aureus is a major mastitis-causing pathogen. Various genotypes have been recently identified in Switzerland but Staph. aureus genotype B (GTB) was the only genotype associated with high within-herd prevalence. The risk of introducing this Staph. aureus genotype into a herd may be increased by frequent animal movements. This may also be the case when cows from different herds of origin are commingled and share their milking equipment for a limited period of time. The aim of the present study was to determine the prevalence of Staph. aureus GTB in seasonally communal dairy herds before and after a summer period when dairy farming is characterized by mixing cows from different herds of origin in 1 communal operation. In addition, the environment was investigated to identify potential Staph. aureus GTB reservoirs relevant for transmission of the disease. A total of 829 cows from 110 herds of origin in 9 communal operations were included in the study. Composite milk samples were collected from all cows during the first or second milking after arrival at the communal operation and again shortly before the end of the season. Swab samples from the environment, involved personnel, and herding dogs present were collected before the cows arrived. At the end of the season, sampling of personnel was repeated. All samples were analyzed for the presence of Staph. aureus GTB using an established quantitative PCR. At the beginning of the season, Staph. aureus GTB-positive cows were identified in 7 out of 9 communal operations and the within-communal operation prevalence ranged from 2.2 to 38.9%. At the second sampling, all communal operations were Staph. aureus GTB positive, showing within-communal operation prevalence from 1 to 72.1%. The between-herd of origin prevalence increased from 27.3 to 56.6% and the cow-level prevalence increased from 11.2% at the beginning of the season to 29.6% at the end of the season. On 3 different communal operations, Staph. aureus GTB-positive swabs from seasonally employed personnel were identified at the end of the season. The results indicate that Staph. aureus GTB can easily spread in communal operations when cows from different herds of origin are mixed during the summer season. Effective management measures need to be designed to prevent the spread of Staph. aureus GTB in seasonally communal herds.     

DNA carryover in milk samples from routine milk recording used for PCR-based diagnosis of bovine Staphylococcus aureus mastitis

Real-time PCR techniques are increasingly used to detect udder pathogens from milk samples collected non-aseptically at routine milk recording. The objectives of this study were (1) to estimate the statistical associations between cycle threshold (Ct) values for Staphylococcus aureus in non-aseptically collected composite samples taken at routine milk recording from cows milked consecutively with the same milking unit and milk meter; and (2) to formulate practical and plausible guidelines for understanding the diagnostic implications of PCR testing for Staph. aureus intramammary infection at routine milk recording. The study included 4 herds with conventional milking parlors and repeatedly low Ct-values for Staph. aureus (representing a high DNA load) in bulk tank milk. Composite milk samples were collected from all cows at all milking units during routine milk recording using the Tru-Test electronic milk meter (Tru-Test Group, Auckland, New Zealand) and analyzed using the PathoProof PCR (Thermo Fisher Scientific, Vantaa, Finland) assay. Milking clock times were retrieved at each milk meter to establish the milking order of the cows at each unit. A multinomial logistic regression was applied to estimate the association between Ct-values from cows milked consecutively with the same milking unit and milk meter. The following groups were selected based on Ct-values: (1) 0–31.3, (2) 31.4–33.9, (3) 34.0–37, (4) 37.1–39.9, and (5) 40 (negative result). The association between groups from cows milked consecutively with the same milking unit and milk meter was statistically significant. Approximately 60% of cows were in Ct group 5 if the antecedent cow was also in Ct group 5, but only 20% of cows were in Ct group 5 if the antecedent cow was in Ct group 1. The probability of cows being in Ct group 1 was not markedly influenced by the group of the antecedent cow. Statistical relationships in the intermediate range gave a plausible indication of a dose-response relationship. Carryover of bacterial DNA via the milking unit and milk meter is very likely to affect PCR results for Staph. aureus. Therefore, information about milking order must be considered in mastitis control efforts. We suggest a practical interpretation of PCR results: cows with a Ct-value <32 can be labeled “very likely to be infected with Staph. aureus,” but cows with Ct-values of >37 and 32–37 can be labeled “very likely to be negative for Staph. aureus” and “uncertain Staph. aureus status,” respectively.     

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.     

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.     

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.     

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.     

Relationships between milk culture results and composite milk somatic cell counts in Norwegian dairy cattle

Associations between test-day composite milk somatic cell counts (CMSCC) and results from quarter milk cultures for various pathogens associated with mastitis, including Staphylococcus aureus, Streptococcus spp., coagulase-negative staphylococci (CNS), were investigated. S. aureus was dichotomized according to sparse (≤1,500 colony forming units/mL of milk) or rich (>1,500 colony forming units/mL of milk) growth of the bacteria. Quarter milk samples were obtained on between 1 and 4 occasions from 2,714 cows in 354 Norwegian dairy herds, resulting in a total of 3,396 samples. Cows included in the study were randomly selected, without regard to current or previous udder health status. Measures of test-day CMSCC were obtained every second month, and related to 3528 microbiological diagnoses at the cow level. Mixed linear regression models incorporating a compound symmetry covariance structure accounting for repeated test-day CMSCC within cow, and a random effect variable on herd level, was used to quantify the relationship between a positive milk culture and the natural logarithm of test-day CMSCC (LnCMSCC). The material was stratified in time periods before 151 d in milk (DIM) and after 150 DIM. A positive diagnosis for any category of mastitis pathogen was significantly associated with elevated CMSCC. Pathogen positive cows sampled for microbiological diagnosis during the first 150 DIM had higher levels of CMSCC throughout lactation than cows with a positive diagnosis after 150 DIM. Streptococcus spp.-positive milk cultures were associated with steadily elevated values for CMSCC throughout lactation both when sampled before and after 150 DIM. Cows diagnosed with rich growth of S. aureus after 150 DIM experienced a characteristic and sharp increase in CMSCC, but this effect was not observed in cows with a positive diagnosis for rich growth of S. aureus during the first 150 DIM. A considerable increase in CMSCC in cows positive for CNS during the first part of the lactation period was also observed. The practicability of using CMSCC in a diagnostic test to identify cows with a positive milk culture for mastitis pathogens was also assessed. The sensitivity, specificity, and positive predictive values of the tests were regarded as low when sampling for milk culture was conducted, irrespective of cow level characteristics.     

Intramammary antimicrobial treatment of subclinical mastitis and cow performance later in lactation

The aim of this study was to evaluate long-term therapeutic effects of antimicrobial treatment of recently acquired subclinical mastitis (RASCM) during lactation. Quarter-level clinical mastitis (CM) follow-up, composite somatic cell counts (SCC), and cow-level milk yield later in lactation were evaluated using follow-up data from 2 previously published linked randomized field trials. The first trial randomly assigned antimicrobial treatment with any intramammary product or negative control to culture-positive quarters of cows having a first elevated composite SCC after 2 consecutive low composite SCC measurements. Untreated cows that had a second elevated composite SCC at the next measurement and were staphylococci-positive (i.e., Staphylococcus aureus or non-aureus staphylococci) were randomly assigned to treatment or control. Quarter-level CM cases were reported by the participating herd personnel, and milk yield and composite SCC data were obtained from the regular test-day recording. Frailty survival models were used to evaluate the long-term therapeutic effects of antimicrobial treatment of RASCM on quarter-level CM follow-up. Mixed linear regression models were applied to quantify the effect on milk yield and composite SCC. Data of 638 quarters from 486 cows in 38 herds were available for statistical analyses, of which 229 quarters of 175 cows received antimicrobial treatment for RASCM. Antimicrobial treatment culminated in reduced composite SCC levels later in lactation but did not result in different milk yield levels or CM follow-up compared with control cows. Antimicrobial treatment of cows with RASCM should therefore only be considered in exceptional situations given the current focus on antimicrobial usage reduction in animal husbandry.     

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.