Bovine milk microbiome: a more complex issue than expected.

The aim of this study was to analyze bacterial profiles of bovine mastitic milk samples and samples from healthy quarters using Next Generation Sequencing of amplicons from 16S rRNA genes and to compare results with microbiological results by PCR assays of the same samples. A total of 49 samples were collected from one single dairy herd during the same day. The samples were divided in two sample sets, which were used in this study. The DNA extraction as well as the library preparation and sequencing of these two sets were performed separately, and results of the two datasets were then compared. The vast majority of genera detected appeared with low read numbers and/or in only a few samples. Results of PCR and microbiome analyses of samples infected with major pathogens Staphylococcus aureus or Streptococcus uberis were consistent as these genera also covered the majority of reads detected in the microbiome analysis. Analysis of alpha diversity revealed a much higher species richness in set 1 than in set 2. The dominating bacterial genera with the highest read numbers clearly differed between datasets, especially in PCR negative samples and samples positive for minor pathogens. In addition to this, linear discriminant analysis (LDA) was conducted between the two sets to identify significantly different genera/family level microbes. The genus Methylobacterium was much more common in set 2 compared to set 1, and genus Streptococcus more common in set 1. Our results indicate amplification of contaminating bacteria in excess in samples with no or minor amounts of pathogen DNA in dataset 2. There is a need for critical assessment of results of milk microbiome analyses.

Elimination of experimentally induced bovine intramammary infection assessed by multiplex real-time PCR and bacterial culture.

Diagnosis of bovine intramammary infection (IMI) has traditionally been based on bacterial culture, but currently IMI can also be detected with DNA based methods, such as multiplex real-time PCR. The aim of this study was to describe the elimination of bacteria in experimentally induced IMI on the quarter level, using conventional bacterial culture (BC) and multiplex real-time PCR. Two coagulase-negative staphylococcal species, Staphylococcus epidermidis and Staphylococcus simulans, were experimentally inoculated into 14 healthy quarters of 8 dairy cows during 4 consecutive study periods. Intramammary infections were followed with 20 milk samplings per each quarter. Milk somatic cell count was monitored to evaluate the inflammation process in the quarters. Four quarters cured spontaneously during the study period according to the culture. The PCR detected staphylococcal DNA from these quarters for several days after they were defined as cured in BC. Agreement between BC and PCR results varied from substantial to almost perfect agreement for the first 36 h postchallenge, decreasing to moderate levels toward the end of the sampling period. Based on this study, we recommend collecting possible follow-up samples to assess the bacteriological cure from IMI not until 2 to 3 wk after the onset of mastitis or after the quarter milk somatic cell count has normalized when PCR is used.

Determination of the intramammary dose of benzylpenicillin required to maintain an adequate concentration in the milk to inhibit Gram-positive bacteria in the clinically normal udder for 24 hr.

The aim of this study was to determine the intramammary dose of benzylpenicillin required to maintain a concentration in the milk above the MIC for the Gram-positive bacteria that cause mastitis. The product used in this study was a commercially available procaine benzylpenicillin in an oily suspension with micronized particles. Three dose levels were used: 200,000, 300,000, and 600,000 IU. Concentrations of benzylpenicillin in cow milk and plasma were determined after a single intramammary dose was administered into one quarter of each of the five cows in each treatment group. Samples were analyzed using an HPLC-MS/MS method, which was validated during the study. Concentrations in the milk were well above the MIC for the target pathogens for all doses tested. There was a linear dose-dependent increase in the mean AUCs of benzylpenicillin concentrations in plasma and milk. At the first milking, 12 hr after dosing, there was a significant difference between the mean milk benzylpenicillin concentrations in cows treated with a dose of 600,000 IU, and those treated with 200,000 or 300,000 IU. Although this study shows a linear relationship between the dose of procaine benzylpenicillin administered and the concentration in the milk in the healthy udder, it would be useful to conduct studies on cows with mastitis to define the optimum dose and duration of intramammary treatment with benzylpenicillin.

Public health risk of antimicrobial resistance transfer from companion animals.

Antimicrobials are important tools for the therapy of infectious bacterial diseases in companion animals. Loss of efficacy of antimicrobial substances can seriously compromise animal health and welfare. A need for the development of new antimicrobials for the therapy of multiresistant infections, particularly those caused by Gram-negative bacteria, has been acknowledged in human medicine and a future corresponding need in veterinary medicine is expected. A unique aspect related to antimicrobial resistance and risk of resistance transfer in companion animals is their close contact with humans. This creates opportunities for interspecies transmission of resistant bacteria. Yet, the current knowledge of this field is limited and no risk assessment is performed when approving new veterinary antimicrobials. The objective of this review is to summarize the current knowledge on the use and indications for antimicrobials in companion animals, drug-resistant bacteria of concern among companion animals, risk factors for colonization of companion animals with resistant bacteria and transmission of antimicrobial resistance (bacteria and/or resistance determinants) between animals and humans. The major antimicrobial resistance microbiological hazards originating from companion animals that directly or indirectly may cause adverse health effects in humans are MRSA, methicillin-resistant Staphylococcus pseudintermedius, VRE, ESBL- or carbapenemase-producing Enterobacteriaceae and Gram-negative bacteria. In the face of the previously recognized microbiological hazards, a risk assessment tool could be applied in applications for marketing authorization for medicinal products for companion animals. This would allow the approval of new veterinary medicinal antimicrobials for which risk levels are estimated as acceptable for public health.

The effect of sampling technique on PCR-based bacteriological results of bovine milk samples.

The aim of the study was to evaluate the effect of sampling technique on the microbiological results of bovine milk samples using multiplex real-time PCR. Comparison was made between a technique where the milk sample was taken directly from the udder cistern of the udder quarter using a needle and vacuum tube and conventional sampling. The effect of different cycle threshold (Ct) cutoff limits on the results was also tested to estimate the amount of amplified DNA in the samples. A total of 113 quarters from 53 cows were tested pairwise using both techniques, and each sample was studied with real-time PCR. Sampling from the udder cistern reduced the number of species per sample compared with conventional sampling. In conventional samples, the number of positive Staphylococcus spp. results was over twice that of samples taken with the needle technique, indicating that most of the Staphylococcus spp. originated from the teat or environmental sources. The Ct values also showed that Staphylococcus spp. were present in most samples only in low numbers. Routine use of multiplex real-time PCR in mastitis diagnostics could benefit from critical evaluation of positive Staphylococcus spp. results with Ct values between 34.0 and 37.0. Our results emphasize the importance of a careful aseptic milk sampling technique and a microbiologically positive result for a milk sample should not be automatically interpreted as an intramammary infection or mastitis.

N-acetyl -ß-D-glucosaminidase activity in cow milk as an indicator of mastitis.

Activity of lysosomal N-acetyl-ß-d-glucosaminidase (NAGase) in milk has been used as an indicator of bovine mastitis. We studied NAGase activity of 808 milk samples from healthy quarters and quarters of cows with spontaneous subclinical and clinical mastitis. Associations between milk NAGase activity and milk somatic cell count (SCC), mastitis causing pathogen, quarter, parity, days in milk (DIM) and season were studied. In addition, the performance of NAGase activity in detecting clinical and subclinical mastitis and distinguishing infections caused by minor and major bacteria was investigated. Our results indicate that NAGase activity can be used to detect both subclinical and clinical mastitis with a high level of accuracy (0·85 and 0·99). Incomplete correlation between NAGase activity and SCC suggests that a substantial proportion of NAGase activity comes from damaged epithelial cells of the udder in addition to somatic cells. We therefore recommend determination of NAGase activity from quarter foremilk after at least six hours from the last milking using the method described. Samples should be frozen before analysis. NAGase activity should be interpreted according to DIM, at least during the first month of lactation. Based on the results of the present study, a reference value for normal milk NAGase activity of 0·1-1·04 pmoles 4-MU/min/µl for cows with ≥30 DIM (196 samples) could be proposed. We consider milk NAGase activity to be an accurate indicator of subclinical and clinical mastitis.

Performance of a real-time PCR assay in routine bovine mastitis diagnostics compared with in-depth conventional culture.

Reliable identification of the aetiological agent is crucial in mastitis diagnostics. Real-time PCR is a fast, automated tool for detecting the most common udder pathogens directly from milk. In this study aseptically taken quarter milk samples were analysed with a real-time PCR assay (Thermo Scientific PathoProof Mastitis Complete-12 Kit, Thermo Fisher Scientific Ltd.) and by semi-quantitative, in-depth bacteriological culture (BC). The aim of the study was to evaluate the diagnostic performance of the real-time PCR assay in routine use. A total of 294 quarter milk samples from routine mastitis cases were cultured in the national reference laboratory of Finland and examined with real-time PCR. With BC, 251 out of 294 (85.7%) of the milk samples had at least one colony on the plate and 38 samples were considered contaminated. In the PCR mastitis assay, DNA of target species was amplified in 244 samples out of 294 (83.0%). The most common bacterial species detected in the samples, irrespective of the diagnostic method, was the coagulase negative staphylococci (CNS) group (later referred as Staphylococcus spp.) followed by Staphylococcus aureus. Sensitivity (Se) and specificity (Sp) for the PCR assay to provide a positive Staph. aureus result was 97.0 and 95.8% compared with BC. For Staphylococcus spp., the corresponding figures were 86.7 and 75.4%. Our results imply that PCR performed well as a diagnostic tool to detect Staph. aureus but may be too nonspecific for Staphylococcus spp. in routine use with the current cut-off Ct value (37.0). Using PCR as the only microbiological method for mastitis diagnostics, clinical relevance of the results should be carefully considered before further decisions, for instance antimicrobial treatment, especially when minor pathogens with low amount of DNA have been detected. Introducing the concept of contaminated samples should also be considered.

Comparative proteome profiling of bovine and human Staphylococcus epidermidis strains for screening specifically expressed virulence and adaptation proteins.

The present study reports a comparative proteome cataloging of a bovine mastitis and a human-associated Staphylococcus epidermidis strain with a specific focus on surfome (cell-wall bound and extracellular) proteins. Protein identification by 1DE coupled with LC-MS/MS analyses resulted in 1400 and 1287 proteins from the bovine (PM221) and human (ATCC12228) strains, respectively, covering over 50% of all predicted and more than 30% of all predicted surfome proteins in both strains. Comparison of the identification results suggests elevated levels of proteins involved in adherence, biofilm formation, signal transduction, house-keeping functions, and immune evasion in PM221, whereas ATCC12228 was more effective in expressing host defense evasion proteases, skin adaptation lipases, hemagglutination, and heavy-metal resistance proteins. Phenotypic analyses showed that only PM221 displays protein- and DNA-mediated adherent growth, and that PM221 was more efficient in cleaving tributyrin, a natural compound of milk fat under low CO2 conditions. These findings are in line with the identification data and suggest that distinct expression of lipases and adhesive surfome proteins could lead to the observed phenotypes. This study is the first extensive survey of S. epidermidis proteomes to date, providing several protein candidates to be examined for their roles in adaptation and virulence in vivo. All MS data have been deposited in the ProteomeXchange with identifier PXD000404 (http://proteomecentral.proteomexchange.org/dataset/PXD000404).

Genomics and Proteomics Provide New Insight into the Commensal and Pathogenic Lifestyles of Bovine- and Human-Associated Staphylococcus epidermidis Strains.

The present study reports comparative genomics and proteomics of Staphylococcus epidermidis (SE) strains isolated from bovine intramammary infection (PM221) and human hosts (ATCC12228 and RP62A). Genome-level profiling and protein expression analyses revealed that the bovine strain and the mildly infectious ATCC12228 strain are highly similar. Their genomes share high sequence identity and synteny, and both were predicted to encode the commensal-associated fdr marker gene. In contrast, PM221 was judged to differ from the sepsis-associated virulent human RP62A strain on the basis of distinct protein expression patterns and overall lack of genome synteny. The 2D DIGE and phenotypic analyses suggest that PM221 and ATCC12228 coordinate the TCA cycle activity and the formation of small colony variants in a way that could result in increased viability. Pilot experimental infection studies indicated that although ATCC12228 was able to infect a bovine host, the PM221 strain caused more severe clinical signs. Further investigation revealed strain- and condition-specific differences among surface bound proteins with likely roles in adhesion, biofilm formation, and immunomodulatory functions. Thus, our findings revealed a close link between the bovine and commensal-type human strains and suggest that humans could act as a reservoir of bovine mastitis-causing SE strains.

Pleuromutilins: use in food-producing animals in the European Union, development of resistance and impact on human and animal health.

Pleuromutilins (tiamulin and valnemulin) are antimicrobial agents that are used mainly in veterinary medicine, especially for swine and to a lesser extent for poultry and rabbits. In pigs, tiamulin and valnemulin are used to treat swine dysentery, spirochaete-associated diarrhoea, porcine proliferative enteropathy, enzootic pneumonia and other infections where Mycoplasma is involved. There are concerns about the reported increases in the MICs of tiamulin and valnemulin for porcine Brachyspira hyodysenteriae isolates from different European countries, as only a limited number of antimicrobials are available for the treatment of swine dysentery where resistance to these antimicrobials is already common and widespread. The loss of pleuromutilins as effective tools to treat swine dysentery because of further increases in resistance or as a consequence of restrictions would present a considerable threat to pig health, welfare and productivity. In humans, only one product containing pleuromutilins (retapamulin) is authorized currently for topical use; however, products for oral and intravenous administration to humans with serious multidrug-resistant skin infections and respiratory infections, including those caused by methicillin-resistant Staphylococcus aureus (MRSA), are being developed. The objective of this review is to summarize the current knowledge on the usage of pleuromutilins, resistance development and the potential impact of this resistance on animal and human health.

Somatic cell count in bovine quarter milk samples culture positive for various Staphylococcus species.

BACKGROUND: Non-aureus staphylococci (NAS) are the most prevalent group of bacteria isolated in bovine mastitis milk in Finland and many other countries. They usually cause subclinical or mild clinical mastitis. The increase in milk somatic cell count (SCC) during NAS intramammary infection varies from slight to marked, reflecting the severity of infection in the quarter. Limited evidence has indicated that NAS species may have different impact on milk SCC. We used a large data set originating from a prevalence study, including isolates from quarter milk samples and the SCCs of the respective quarters, to study the effect of different NAS species on quarter milk SCC. RESULTS: Staphylococcal species of a total of 1265 isolates, originally identified as NAS, were analysed with MALDI-TOF MS. The most prevalent NAS species were S. epidermidis, S. simulans, S. chromogenes and S. haemolyticus. Forty-two isolates appeared to be S. aureus. Geometric mean milk SCC of all quarter samples was 114,000 cells/mL and median 126,000 cells/mL. Staphylococcus species had a significant effect on the SCC of the quarter. The highest SCCs were caused by S. aureus, S. agnetis/S. hyicus (these two species cannot be distinguished with MALDI-TOF MS) and S. simulans. The mean SCCs of milk samples that were culture positive for these three species did not differ significantly from each other but were significantly higher than the mean SCCs of milk samples positive for any other species. The mean SCC of milk samples positive for S. chromogenes was significantly higher than those of milk samples positive for S. epidermidis or S. warneri. CONCLUSION: Our results confirm that different Staphylococcus species have different impacts on milk SCC, as shown in previous studies. S. aureus caused the highest SCC, as expected, but the SCCs caused by S. agnetis/S. hyicus and S. simulans did not differ significantly from that of S. aureus. Other Staphylococcus species may also cause high SCC but are often isolated also from quarters with SCC on the level of healthy quarters.

Review on methicillin-resistant Staphylococcus pseudintermedius.

Staphylococcus pseudintermedius is an important opportunistic pathogen of companion animals, especially dogs. Since 2006 there has been a significant emergence of methicillin-resistant S. pseudintermedius (MRSP) mainly due to clonal spread. This article reviews research on MRSP with a focus on occurrence, methods used for identification, risk factors for colonization and infection, zoonotic potential and control options. Potential areas for future research are also discussed.

Innate immune response in experimentally induced bovine intramammary infection with Staphylococcus simulans and S. epidermidis.

Coagulase-negative staphylococci (CNS) are in several countries the most common bacteria isolated in subclinical mastitis. To investigate the innate immune response of cows to infections with two common mastitis-causing CNS species, Staphylococcus epidermidis and Staphylococcus simulans, experimental intramammary infection was induced in eight cows using a crossover design. The milk somatic cell count (SCC), N-acetyl-ß-D-glucosaminidase (NAGase) activity, milk amyloid A (MAA), serum amyloid A (SAA) and proinflammatory cytokines interleukin (IL)-1ß, IL-8, and tumor necrosis factor α (TNF-α) were determined at several time points before and after challenge. All cows became infected and showed mild to moderate clinical signs of mastitis. The spontaneous elimination rate of the 16 infections was 31.3%, with no difference between species. Infections triggered a local cytokine response in the experimental udder quarters, but cytokines were not detected in the uninfected control quarters or in systemic circulation. The innate local immune response for S. simulans was slightly stronger, with significantly higher concentrations of IL-1ß and IL-8. The IL-8 response could be divided into early, delayed, or combined types of response. The CNS species or persistency of infection was not associated with the type of IL-8 response. No significant differences were seen between spontaneously eliminated or persistent infections.

Concentrations of bovine lactoferrin and citrate in milk during experimental endotoxin mastitis in early- versus late-lactating dairy cows.

Lactoferrin (Lf) is a molecule naturally present in bovine milk that affects the availability and transport systems of iron. Lf also binds endotoxin (lipopolysaccharide, LPS) of Gram-negative bacteria and modulates the immunological response. In the present study, concentrations of bovine Lf (bLf) and citrate in milk were determined in early (EL) and late (LL) lactating dairy cows, using an experimentally induced endotoxin mastitis model and a crossover design. Nine clinically healthy Finnish Ayrshire cows were challenged twice with 100 µg endotoxin infused into one udder quarter. Milk samples were collected from the challenged and control quarters of each cow before and after endotoxin infusion during 3 d, and bLf and citrate concentrations were measured. In all cows, clinical signs of mastitis were seen at both times of challenge, but the response was more severe in EL than in LL. Concentration of bLf in the milk started to rise approximately 8 h after endotoxin infusion and was still higher than normal on the third day, especially in the late-lactating cows. In milk of the LL group, concentrations of bLf were significantly higher than in the EL group. In contrast, concentrations of citrate were higher in milk of the EL cows compared with the LL cows. Concentration of bLf and citrate varied substantially among cows. The molar ratio of citrate to bLf before and after challenge was significantly higher during the EL period. The results of this study partly explain why cows in early lactation are more susceptible to intramammary infections and why mastitis is more severe in them.

Coagulase-negative staphylococci-emerging mastitis pathogens.

Coagulase-negative staphylococci (CNS) have become the most common bovine mastitis isolate in many countries and could therefore be described as emerging mastitis pathogens. The prevalence of CNS mastitis is higher in primiparous cows than in older cows. CNS are not as pathogenic as the other principal mastitis pathogens and infection mostly remains subclinical. However, CNS can cause persistent infections, which result in increased milk somatic cell count (SCC) and decreased milk quality. CNS infection can damage udder tissue and lead to decreased milk production. Staphylococcus simulans and Staphylococcus chromogenes are currently the predominant CNS species in bovine mastitis. S. chromogenes is the major CNS species affecting nulliparous and primiparous cows whereas S. simulans has been isolated more frequently from older cows. Multiparous cows generally become infected with CNS during later lactation whereas primiparous cows develop infection before or shortly after calving. CNS mastitis is not a therapeutic problem as cure rates after antimicrobial treatment are usually high. Based on current knowledge, it is difficult to determine whether CNS species behave as contagious or environmental pathogens. Control measures against contagious mastitis pathogens, such as post-milking teat disinfection, reduce CNS infections in the herd. Phenotypic methods for identification of CNS are not sufficiently reliable, and molecular methods may soon replace them. Knowledge of the CNS species involved in bovine mastitis is limited. The dairy industry would benefit from more research on the epidemiology of CNS mastitis and more reliable methods for species identification.

Coagulase-negative staphylococci as cause of bovine mastitis- not so different from Staphylococcus aureus?

In this review of the literature, mastitis-causing coagulase-negative staphylococci (CNS) and Staphylococcus aureus are compared. Staphylococci are the bacteria most commonly isolated from bovine mastitis, and CNS are now predominant over S. aureus in most countries. CNS include various species, but only a few prevail in bovine mastitis. S. aureus can cause clinical mastitis, but often causes subclinical mastitis, which remains persistent and increases milk somatic cell count. CNS, traditionally regarded as minor pathogens, seem to lack the ability to cause severe mastitis. CNS can, however, persist in the mammary gland and moderately increase milk somatic cell count. Resistance to various antimicrobials is more common in CNS than in S. aureus, but CNS mastitis responds much better to antimicrobial treatment than S. aureus mastitis.

Effect of bovine lactoferrin on the internalization of coagulase-negative staphylococci into bovine mammary epithelial cells under in-vitro conditions.

Coagulase-negative staphylococci (CNS) have emerged as bovine mastitis pathogens in many countries. CNS mastitis is generally mild but can persist in the udder for long periods. Pathogenesis of CNS intramammary infection is not well understood. In the present study, adhesion, invasion and intracellular replication of twenty-two CNS strains isolated from bovine mastitis and the effect of bovine lactoferrin (bLf) on the internalization were studied in vitro in a bovine mammary epithelial (BME) cell model. The CNS strains were of Staphylococcus chromogenes, Staph. simulans, Staph. epidermidis, Staph. haemolyticus and Staph. cohnii urealyticus; two strains of Staph. aureus were used as controls. Seven of the CNS strains originated from persistent and five from transient mastitis infections. The in-vitro susceptibility of the strains to bLf was also investigated. All CNS species examined had an adhesive ability equal to that of Staph. aureus, but internalization varied among staphylococcal strains. The antagonistic effect of bLf on the adhesion and invasion of CNS strains was weak, but bLf significantly decreased intracellular replication and replication rates of CNS. No correlation between the in-vitro susceptibility of the strain to bLf or internalization among clinical signs of mastitis was established. No difference between the persistent and transient CNS strains in adhesion, invasion or replication rate was recorded. This in-vitro BME cell model can be used to study the virulence potential of mastitis pathogens, although the severity and persistence of eventual infections shall be further investigated in vivo. The role of bLf in intramammary infection caused by CNS may be limited.

Cross-sectional prospective survey to study indication-based usage of antimicrobials in animals: results of use in cattle.

BACKGROUND: Indication-based data on the use of antimicrobials in animals were collected using a prospective cross-sectional survey, similarly as for surveys carried out in human medicine, but adapting the questionnaire to include veterinary-specific issues. The participating veterinarians were randomly selected from a sample population of practising veterinarians. The sampling was stratified to take into account the proportions of different types of veterinary practice in the country. All patients consulting the veterinary practice during a 1-week period were included in the study and veterinarians returned a completed questionnaire for each patient receiving antimicrobial treatment. As cattle received most of the treatments, results from the survey are given using cattle as an example species. RESULTS: The survey was sent to 681 veterinarians, of whom 262 (39%) responded. In total 2850 questionnaires were completed. The largest quantities of antimicrobials, measured in kilograms, were used for cattle, followed by pigs, dogs and horses. The species that were treated most were cattle (n = 1308), dogs (n = 989) and cats (n = 311). For cattle, the most common reason for treatment was acute mastitis (52%), followed by dry-cow therapy (21%), subclinical mastitis (6%) and treatment for acute enteritis (4%). The remaining treatments covered 17% of cattle patients and 15 different indications. For acute mastitis, parenteral or intramammary treatment was used in 36% and 34% of the cases, respectively. The remaining 30% received both treatments simultaneously. Of the parenteral treatments (n = 459), benzyl penicillin was used in 83% of the treated animals (n = 379), while fluoroquinolones were used in 49 cases (11%). Of the 433 cows receiving intramammary treatment, ampicillin combined with cloxacillin was most commonly used (n = 157; 36%), followed by cephalexin+streptomycin (n = 113; 26%). CONCLUSION: This cross-sectional prospective survey provided a useful method for the collection of information on the indication-based use of antimicrobials in different animal species. Cattle were the most commonly treated animal species during the study period. The most common indication for antimicrobial use in cows was mastitis. Benzyl penicillin was the drug most frequently used for the treatment of mastitis, which seems appropriate according to the national guidelines on the use of antimicrobials in cattle in Finland.

Bacteriological etiology and treatment of mastitis in Finnish dairy herds.

BACKGROUND: The Finnish dairy herd recording system maintains production and health records of cows and herds. Veterinarians and farmers register veterinary treatments in the system. Milk samples for microbiological analysis are routinely taken from mastitic cows. The laboratory of the largest dairy company in Finland, Valio Ltd., analyzes most samples using real-time PCR. This study addressed pathogen-specific microbiological data and treatment and culling records, in combination with cow and herd characteristics, from the Finnish dairy herd recording system during 2010-2012. RESULTS: The data derived from 240,067 quarter milk samples from 93,529 dairy cows with mastitis; 238,235 cows from the same herds served as the control group. No target pathogen DNA was detected in 12% of the samples. In 49% of the positive samples, only one target species and in 19%, two species with one dominant species were present. The most common species in the samples with a single species only were coagulase-negative staphylococci (CNS) (43%), followed by Staphylococcus aureus (21%), Streptococcus uberis (9%), Streptococcus dysgalactiae (8%), Corynebacterium bovis (7%), and Escherichia coli (5%). On average, 36% of the study cows and 6% of the control cows had recorded mastitis treatments during lactation. The corresponding proportions were 16 and 6% at drying-off. For more than 75% of the treatments during lactation, diagnosis was acute clinical mastitis. In the milk samples from cows with a recorded mastitis treatment during lactation, CNS and S. aureus were most common, followed by streptococci. Altogether, 48% of the cows were culled during the study. Mastitis was reported as the most common reason to cull; 49% of study cows and 18% of control cows were culled because of mastitis. Culling was most likely if S. aureus was detected in the milk sample submitted during the culling year. CONCLUSIONS: The PCR test has proven to be an applicable method also for large-scale use in bacterial diagnostics. In the present study, microbiological diagnosis was unequivocal in the great majority of samples where a single species or two species with one dominating were detected. Coagulase-negative staphylococci and S. aureus were the most common species. S. aureus was also the most common pathogen among the culled cows, which emphasizes the importance of preventive measures.