In the present study, 103 Staphylococcus aureus strains isolated from milk samples from 60 cows with mastitis from eight different farms in seven different locations in one region of Germany were compared pheno-and genotypically and by identification of various toxins. On the basis of culture and hemolytic properties and by determination of the tube coagulase reaction, all of the isolates could be identified as S. aureus. This could be confirmed by PCR amplification of species-specific parts of the gene encoding the 23S rRNA. In addition, all of the S. aureus isolates harbored the genes encoding staphylococcal coagulase and clumping factor and the genes encoding the X region and the immunoglobulin G binding region of protein A. These four genes displayed size polymorphisms. By PCR amplification, the genes for the toxins staphylococcal enterotoxin A (SEA), SEC, SED, SEG, SEI, SEJ, and TSST-1 but not those for SEB, SEE, SEH, and the exfoliative toxins ETA and ETB could be detected. To analyze the epidemiological relationships, the isolates were subjected to DNA fingerprinting by macrorestriction analysis of their chromosomal DNAs. According to the observed gene polymorphisms, the toxin patterns, and the information given by macrorestriction analysis of the isolates by pulsedfield gel electrophoresis, a limited number of clones seemed to be responsible for the cases of bovine mastitis on the various farms.

Somatic cell counts (SCC) are generally used as an indicator of udder health. In Germany, a cutoff value of 100,000 cells/mL is currently used to differentiate between healthy and diseased mammary glands. In addition to SCC, differential cell counts (DCC) can be applied for a more detailed evaluation of the udder health status. The aim of this study was to differentiate immune cells in milk of udder quarters classified as healthy based on SCC values of <100,000 cells/mL. Twenty cows were selected and 65 healthy udder quarters were compared with a control group of 15 diseased udder quarters (SCC>100,000 cells/mL). Cells were isolated from milk of all quarters to measure simultaneously percentages of lymphocytes, macrophages, and polymorphonuclear neutrophilic leukocytes (PMNL) by flow cytometric analysis. The bacteriological status of all 80 quarters was also determined. Differential cell count patterns of milk samples (n = 15) with extreme low SCC values of ≤ 6,250 cells/mL revealed high lymphocyte proportions of up to 88%. Milk cell populations in samples (n = 42) with SCC values from >6,250 to ≤ 25,000 cells/mL were also dominated by lymphocytes, whereas DCC patterns of 6 out of 41 milk samples with SCC values from ≥ 9,000 to ≤ 46,000 cells/mL indicated already inflammatory reactions based on the predominance of PMNL (56-75%). In 13 of 15 milk samples of the diseased udder quarters (SCC >100,000 cells/mL), PMNL were categorically found as dominant cell population with proportions of ≥ 49%. Macrophages were the second predominant cell population in almost all samples tested in relation to lymphocytes and PMNL. Further analysis of the data demonstrated significant differences of the cellular components between udder quarters infected by major pathogens (e.g., Staphylococcus aureus; n = 5) and culture-negative udder quarters (n = 56). Even the percentages of immune cells in milk from quarters infected by minor pathogens (e.g., coagulase-negative staphylococci; n = 19) differed significantly from those in milk of culture-negative quarters. Our flow cytometric analysis of immune cells in milk of udder quarters classified as healthy by SCC <100,000 cells/mL revealed inflammatory reactions based on DCC.

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Escherichia coli (STEC) in healthy dairy ruminants was investigated between 1996 and 1998 by a multiplex polymerase chain reaction (mPCR) technique. A total of 13 552 E. coli colonies from 726 cows, 28 sheep and 93 goats out of 112 randomly selected dairy farms in Hessia, Germany were analysed. STEC strains were recovered from 131 (18Á0%) cows, nine (32Á1%) sheep and 70 (75Á3%) goats. Further characterization of the STEC isolates showed that 89 (0Á66% of the investigated colonies) of animal ®eld strains carried stx1 gene, 64 (0Á47%) stx2 gene and 57 (0Á42%) stx1 and stx2 gene. Sixty (93Á8%) out of 64 stx2 ®eld strains were harboured by cows. In contrast, 74 (83Á1%) out of 89 stx1 dairy animal ®eld strains were from ovine or caprine origin. Only 17 (8Á1%) stxpositive isolates (13 from cattle, three from sheep and only one from goat) were positive for eaeA gene. Eight (9Á0%) of the stx1, ®ve (7Á8%) of the stx2 and four (7Á0%) of the stx1/stx2 gene-positive ®eld strains carried the eaeA gene. The prevalence of EHEC-haemolysin (EHEC-hlyA) gene sequence was 88Á8% (79 isolates) of the stx1 and 68Á8% (44 isolates) of the stx2 isolates. Out of 57 stx1-and stx2-positive ®eld-strains, 34 (59Á6%) carried the EHEC-hlyA gene. E. coli O serovars O:157 and O:111 were not found. Only one isolate was positive with O26 antiserum.

Somatic cell counts (SCC) are generally used as an indicator of udder health. Currently in Germany, 100,000 cells/mL is the threshold differentiating infected and noninfected mammary glands. The aim of our study was the detailed analysis of udder health in a representative part of the dairy cow population in Hesse, Germany. Between 2000 and 2008, 615,187 quarter foremilk samples were analyzed. In addition to evaluation of distribution of SCC and prevalence of mastitis pathogens, pathogen prevalence was also calculated depending on SCC. The data indicated that 38% of all samples had SCC >100,000 cells/mL and 62% showed SCC ≤ 100,000 cells/mL; 31% of all samples revealed SCC ≤ 25,000 cells/mL. Coagulase-negative staphylococci were the dominant pathogens in the Hessian quarter foremilk samples (17.17% of all samples) followed by Corynebacterium spp. (13.56%), Streptococcus uberis (8.7%), and Staphylococcus aureus (5.01%). Mastitis pathogens were detected in 83% of all samples with SCC >100,000 cells/mL. However, the prevalence of mastitis pathogens in the SCC range from 1,000 to ≤ 100,000 cells/mL was 8.5% (5.51% minor pathogens, 2.01% major pathogens, and 0.98% other pathogens). For farms producing high quality milk, exceptional hygiene management is compulsory. One of the farms randomly selected showed clearly different results from the Hessian survey. Fifteen percent more samples lay in the SCC range ≤ 100,000 cells/mL with a lower prevalence of mastitis pathogens of 1.91% (1.03% minor pathogens, 0.83% major pathogens, and 0.05% other pathogens). Based on these results, inflammatory processes can obviously be detected in mammary glands of udder quarters healthy according to the current definitions. However, we argue that such inflammation can be detected by examination of the relationship of immune cells in milk.