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 10(3) to 10(7) 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-beta-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.

Fast and reliable identification of the microorganisms causing mastitis is important for management of the disease and for targeting antimicrobial treatment. Methods based on PCR are being used increasingly in mastitis diagnostics. Comprehensive field comparisons of PCR and traditional milk bacteriology have not been available. The results of a PCR kit capable of detecting 11 important etiological agents of mastitis directly from milk in 4h were compared with those of conventional bacterial culture (48h). In total, 1,000 quarter milk samples were taken from cows with clinical or subclinical mastitis, or from clinically healthy quarters with low somatic cell count (SCC). Bacterial culture identified udder pathogens in 600/780 (77%) of the clinical samples, whereas PCR identified bacteria in 691/780 (89%) of the clinical samples. The PCR analysis detected major pathogens in a large number of clinical samples that were negative for the species in culture. These included 53 samples positive for Staphylococcus aureus by PCR, but negative by culture. A total of 137 samples from clinical mastitis, 5 samples from subclinical mastitis, and 1 sample from a healthy quarter were positive for 3 or more bacterial species in PCR, whereas culture identified 3 or more species in 60 samples from clinical mastitis. Culture identified a species not targeted by the PCR test in 44 samples from clinical mastitis and in 9 samples from subclinical mastitis. Low SCC samples provided a small number of positive results both in culture (4/93; 4.3%) and by PCR (7/93; 7.5%). In conclusion, the PCR kit provided several benefits over conventional culture, including speed, automated interpretation of results, and increased sensitivity. This kit holds much promise as a tool to complement traditional methods in identification of pathogens. In conventional mastitis bacteriology, a sample with 3 or more species is considered contaminated, and resampling of the cow is recommended. Further study is required to investigate how high sensitivity of PCR and its quantitative features can be applied to improve separation of relevant udder pathogens from likely contaminants in samples where multiple species are detected. Furthermore, increasing the number of species targeted by the PCR test would be advantageous.

Intramammary infection (IMI), also known as mastitis, is the most frequently occurring and economically the most important infectious disease in dairy cattle. This study provides a validation of the analytical specificity and sensitivity of a real-time PCR-based assay that identifies 11 major pathogen species or species groups responsible for IMI, and a gene coding for staphylococcal beta-lactamase production (penicillin resistance). Altogether, 643 culture isolates originating from clinical bovine mastitis, human, and companion animal samples were analyzed using the assay. The isolates represented 83 different species, groups, or families, and originated from 6 countries in Europe and North America. The analytical specificity and sensitivity of the assay was 100% in bacterial and beta-lactamase identification across all isolates originating from bovine mastitis (n = 454). When considering the entire culture collection (including also the isolates originating from human and companion animal samples), 4 Streptococcus pyogenes, 1 Streptococcus salivarius, and 1 Streptococcus sanguis strain of human origin were identified as Streptococcus uberis, and 3 Shigella spp. strains were identified as Escherichia coli, decreasing specificity to 99% in Strep. uberis and to 99.5% in E. coli. These false-positive results were confirmed by sequencing of the 16S rRNA gene. Specificity and sensitivity remained at 100% for all other bacterial targets across the entire culture collection. In conclusion, the real-time PCR assay shows excellent analytical accuracy and holds much promise for use in routine bovine IMI testing programs. This study provides the basis for evaluating the assay's diagnostic performance against the conventional bacterial culture method in clinical field trials using mastitis milk samples.

Results of a commercial real-time PCR analysis for 11 mastitis pathogens from bulk tank milk (BTM) samples from all 4,258 Danish dairy herds in November 2009 to January 2010 were compared with somatic cell count (SCC) and total bacteria count (TBC) estimates in BTM. For Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis, a low real-time PCR cycle threshold (Ct) value (corresponding to high bacterial DNA quantity) was correlated with higher SCC and higher TBC. For Staphylococcus aureus, low Ct values were correlated only with higher SCC. For the environmental mastitis pathogens Klebsiella spp., Enterococcus spp., and Escherichia coli, low Ct values had a correlation with higher TBC. Staphylococcus spp. were found in the BTM from all herds, Strep. uberis in 95%, Staph. aureus in 91%, and Strep. dysgalactiae in 86%, whereas E. coli, Klebsiella, and Strep. agalactiae were found in 61, 13, and 7% of the herds. It is concluded that the real-time PCR used provides results that are related to the milk quality in the herds. Real-time PCR can be used in the same way as culture for monitoring BTM samples, and is especially useful for bacteria with low prevalence (e.g., Strep. agalactiae).

Penicillin resistance identification tests are important in veterinary medicine. Six enzyme assays and a PCR test were compared for the detection of ␤-lactamase production or the ␤-lactamase gene in 175 staphylococcal isolates. We conclude that the PCR test and two nitrocefin-based assays can be recommended for routine clinical use.Staphylococci are common causes of a wide variety of diseases in animals. The most important staphylococcal pathogens include Staphylococcus aureus and coagulase-negative staphylococci (CNS), which cause bovine mastitis; Staphylococcus intermedius, which causes otitis externa and pyoderma in dogs; and Staphylococcus hyicus, which is responsible for exudative dermatitis in pigs (see, e.g., references 4, 8, and 9).In veterinary medicine, penicillin is recommended as the first choice for bacteria that are inherently sensitive to it. In contrast to human isolates (3), the prevalence of penicillin resistance in staphylococci causing animal diseases can be relatively low (1) and is most commonly due to the blaZ gene, which encodes the production of ␤-lactamase (7). The aim of this study was to compare the performance and evaluate the practicality of various alternative methods for the determination of ␤-lactamase production or the ␤-lactamase gene in staphylococci.A total of 175 staphylococcal isolates were used in this study, including 95 Staphylococcus aureus, 50 Staphylococcus intermedius, and 30 CNS isolates (Table 1). S. aureus and CNS isolates were taken from mastitis samples from cows. S. intermedius isolates were obtained from clinical samples of dogs. The CNS included eight S. epidermidis, five S. xylosus, four S. chromogenes, three S. cohnii, three S. haemolyticus, two S. hyicus, two S. saprophyticus, two S. simulans, and one S. warneri isolate. Hence, the number of CNS available for this study was relatively low, and results with them should be considered preliminary.