Multiplex PCR and RPLA Identification of Staphylococcus aureus enterotoxigenic strains from bulk tank milk.

Enterotoxigenic Staphylococcus aureus in raw milk poses a potential health hazard to consumers, and the identification of such strains should be used as part of a risk analysis of milk and milk products. The primary purpose of this study was to investigate the occurrence of enterotoxigenic S. aureus strains in raw milk supplied for dairy processing in the Czech Republic. A further aim was to compare the production of staphylococcal enterotoxins (SEs) with the presence of the corresponding genes. This was undertaken using multiplex polymerase chain reaction (PCR) and reversed passive latex agglutination (RPLA). Out of 440 bulk tank milk samples from 298 dairy herds, 70 proved positive for S. aureus (15.9%). Staphylococcal enterotoxin genes (ses) were detected in 39 (55.7%) isolates. The genes most commonly detected were sei (38.6%), seg (31.4%) and sea (27.1%). Genes seb, seh, sed, sej and sec were observed in 10%, 4.3%, 2.9%, 2.9% and 1.4% of strains respectively. Genes see and sel did not occur. The most frequently detected genotypes were seg, sei at 11.4%; sea at 10.0%; and sea, seg, sei at 8.6%. Toxin production was observed in nine (12.9%) S. aureus isolates. Seven strains were detected as SEB- (10%) and two as SED- (2.9%) producing. A relatively high number (32%) of discrepancies between the results with multiplex PCR and RPLA assays was obtained, particularly on account of SEA. Nineteen strains were sea positive by PCR but SEA negative by RPLA, and one strain was sec positive and SEC negative. The results of both methods were identical concerning SEB and SED. It was concluded that detection of ses by PCR was a useful additional tool to support identification of enterotoxigenic strains.

The thermal treatment effects on bovine blood neutrophil granulocytes apoptosis and necrosis in vitro.

The aim of the study was to evaluate the effect of selected temperatures on viability (apoptosis and necrosis) of bovine blood neutrophil granulocytes (neutrophils) in vitro. The following temperatures were tested: -80, -20, 4, 23, 37 degrees C. Heparinised bovine blood was incubated for 1, 4 and 24 h under respective temperature. Apoptosis and necrosis of neutrophils were detected by light microscopy, transmission electron microscopy (TEM) and flow cytometry (FCM). From selected temperatures, 4 degrees C impaired the neutrophil viability least. The proportion of apoptotic and necrotic neutrophils amounted to (mean +/- SD) 5.25 +/- 3.53% and 0.83 +/- 0.38%; 7.09 +/- 2.07% and 1.64 +/- 0.50%; 35.39 +/- 12.53% and 5.46 +/- 1.46%; after 1, 4 and 24 h incubation, respectively. The temperature (4 degrees C) is the best alternative for short-term storage.

The role of CD14 during resolution of experimentally induced Staphylococcus aureus and Streptococcus uberis mastitis.

This study was undertaken to investigate the time course of surface expression of CD14 on neutrophils and macrophages and to determine their association with resolution of inflammatory responses during Staphylococcus aureus and Streptococcus uberis experimental mastitis. Infections of the mammary gland induce a local immune response characterized by an increase in the total counts of CD14+ neutrophils and CD14+ macrophages particularly. On the other hand, resolution is accompanied by an increase in relative counts of CD14+ neutrophils, CD14+ vacuolized macrophages and apoptotic neutrophils. Following the immune reaction of mammary gland against Gram-negative/positive bacteria is very similar. Between the apoptotic and CD14+ neutrophils a high correlation was measured during the whole experimental period (S. aureus: r=0.64; S. uberis: r=0.61; P<0.05). Using anti-CD14 monoclonal antibodies in vitro suggested the involving of the CD14 surface receptor in recognition of apoptotic neutrophils by macrophages.

The image of exocytosis during neutrophils and macrophages phagocytic activities in inflammation of mammary gland triggered by experimental Staphylococcus aureus infection.

The experiments were carried out in five clinically normal virgin heifers. Before the experimental infection and at 24, 48, 72 and 168 h after the infection, respective mammary glands were rinsed with phosphate buffered saline. Neutrophils as well as macrophages underwent a classic exocytosis accompanied by translocation of lysosomal granules. The granules filled the protuberances of the plasmalemma and after the protuberances separated from the cell, they entered the extracellular space in the shape of round bodies of different sizes. After exocytosis, neutrophils displayed a smaller nucleus/cytoplasm ratio, a greater chromatin density of the nucleus, and an overall smaller size. Macrophages phagocytosed bacteria and/or neutrophils with and without signs of apoptosis (early and late apoptotic respectively) and neutrophils after exocytosis. Macrophages underwent cytolysis that was accompanied by extrusion of granules, phagosomes and phagolysosomes containing phagocytosed bacteria or neutrophils. Confluences were formed in which the process of digestion continued. Apoptosis of neutrophils gradually appeared and intensified in resolution of inflammation. The macrophages contributed to the inactivation of bacterial noxa as well as of histotoxic contents of neutrophils. Nevertheless, macrophages often underwent cytolysis at the site of inflammation.

Macrophages of the bovine heifer mammary gland: morphological features during initiation and resolution of the inflammatory response.

This work characterizes macrophage morphological features during initiation and resolution of an inflammatory response by the bovine mammary gland. The study has been carried out in 20 mammary glands of five virgin heifers by using light microscopy of natural and stained cells and by using transmission electron microscopy (TEM). The inflammatory reaction was induced by an intramammary administration of phosphate-buffered saline (PBS). It has been found that both the initial as well as the resolution phases of the inflammatory reaction are characteristic of the presence of various morphologically different macrophage forms. During the initial phase of the inflammatory response, the major proportion of the macrophage population consisted of monocyte-like macrophages, which represented newly migrated cells. These macrophages were 12-15 mum in size, with spherical or ovoidal shapes, and contained homogenous, fine-granular cytoplasm rich in Golgi complexes, numerous mitochondria, and no lysosomes. The nuclei of the macrophages were kidney-shaped, and surrounded by dark chromatin along the peripheries. Macrophages with phagocytosed apoptotic neutrophils in the cytoplasm were detected already during the initial phase. These macrophages reached the highest proportion 48-72 h after the influx induction and participated in the resolution of the inflammatory reaction. Other cells, also detected during the resolution of the inflammatory reaction, were vacuolized macrophages that formed the largest cells in the lavages of the mammary glands and that were structurally characteristic for the presence of vacuoles in the cytoplasm. In TEM the macrophage vacuoles formed both phagolysosomes with residues of pre-digested material of phagocytosed apoptotic neutrophils and vacuoles that were less electon-dense. Morphologically different forms of macrophages reflected their real-time functions in the inflammation process.

Tissue pool neutrophils of the bovine mammary gland: ultrastructural features during in vitro senescence.

The aim of the study was to verify whether the in vitro senescence process of tissue pool neutrophils of the bovine mammary gland is accompanied by similar changes of ultrastructure as typically occurs in in vivo conditions. The experiments were carried out in four clinically healthy, Holstein x Bohemian Red-Pied crossbred heifers aged 14-16 months. With the aid of transmission electron microscopy (TEM), scanning electron microscopy (SEM) and flow cytometry (FCM), neutrophil apoptosis in vivo was detected and during senescence it was monitored in vitro. The neutrophil apoptosis comprised three ultrastructurally different stages: (1) karyopyknosis, (2) zeiosis, and (3) apoptotic bodies. These stages were obvious in the apoptotic neutrophils both in vivo and in vitro. In addition to the common morphological signs, however, ultrastructural differences were also detected in apoptotic neutrophils in vitro. These in vitro ultrastructural differences mostly comprised hyper-vacuolation of the cytoplasm with mega-vacuoles and secondary necrosis of apoptotic neutrophils. Morphological features of apoptosis during in vitro senescence of tissue pool neutrophils of the bovine mammary gland were shown to be in close accordance with these in vivo signs.

Effects of certain inducers of leukocytes migration into the bovine mammary gland on neutrophil apoptosis manifestation in a subsequent in vitro cultivation.

The aim of the study was to elucidate the effects of induced leukocyte migration into the bovine mammary gland on the manifestations of early and late apoptotic features of neutrophils cultivated in vitro. The Latin square design was used in two experiments, each involving four experimental repetitions in 4 clinically healthy virgin heifers. The neutrophil early apoptotic features were detected by flow cytometric detection (FCM) of phosphatidyl-serine translocation. Late neutrophil apoptotic features were detected by ELISA quantitation of histone-complexed DNA fragments. Leukocyte influx induction was accomplished by using four inducers: i) sterile buffered saline solution (PBS); ii) 5 % glucose solution (GLU); iii) synthetic muramyl dipeptide analogue (MDP); and iv) lipopolysaccharide (LPS), administered into the mammary gland lumen. Leukocytes from mammary glands were obtained by mammary gland lumen lavages after influx induction. The total cell counts in lavages increased after treatment by all inducers in comparison to the counts before influx induction (P<0.001). Cell counts were higher and differed significantly by MDP and LPS (P<0.01) in contrast to PBS. The highest proportion of neutrophils was induced by LPS (P<0.01). After three-hour incubation, light microscopy examination revealed the highest manifestation of neutrophil apoptosis after induction by GLU (P<0.05). The lowest apoptosis manifestation, though statistically non-significant, was detected after induction by MDP and LPS. Determination of early manifestation of neutrophil apoptosis revealed the lowest manifestation of neutrophil apoptosis after induction by LPS (P<0.01). The results of late manifestation of neutrophil apoptosis revealed the highest proportion of apoptotic neutrophils after induction by GLU (P<0.05). The manifestation of secondary necrosis of apoptotic neutrophils or neutrophil lysis after 3 h of incubation was low and not significant. In conclusion, certain inducers of neutrophil migration into the lumen of bovine mammary glands (GLU and LPS in the present experiments) significantly influence the manifestation of neutrophil apoptosis during their subsequent in vitro incubation.

Staphylococcus aureus isolates from dairy cows and humans on a farm differ in coagulase genotype.

Staphylococcus aureus is a frequent cause of animal and human infections. The aim of the present study was to test diversity of the populations of S. aureus colonising cattle and humans sharing an infected environment. Eighty-six S. aureus isolates obtained from dairy cows, from people coming into contact with dairy cows on the farm and the other farm personnel were characterized by restriction fragment length polymorphism of the coagulase gene. Molecular analyses identified ten polymorphism types with prevalent presentation of type II in isolates from cow's milk and type IV in isolates from people coming into contact with dairy cows on the farm (the cattlemen) and the other farm personnel. Seven further genotypes were identified among the isolates from the cattlemen. The results indicate that the strains dominating in human population did not equate to the causative agents of bovine mastitis.

Prevalence of and resistance to anti-microbial drugs in selected microbial species isolated from bulk milk samples.

The prevalence of strains of Staphylococcus aureus, coagulase-negative (CN) staphylococci, Listeria monocytogenes, Escherichia coli, Enterococcus faecalis, E. faecium and Bacillus cereus, was investigated in 111 bulk milk samples. Staphylococcus aureus was isolated from 38 samples, CN staphylococci from 63 samples, E. coli from 49 samples, E. faecalis or E. faecium from 107 samples, and L. monocytogenes from two samples. Bacillus cereus was not found in any of the samples and three samples were free of any of the selected species. Sensitivity to the anti-microbial drugs amikacin, ampicillin, ampicillin + sulbactam, cephalothin (CLT), cephotaxime, clindamycin, chloramphenicol (CMP), co-trimoxazole, erythromycin (ERY), gentamicin, neomycin, norfloxacin, oxacillin, penicillin, streptomycin (STR), tetracycline (TTC) and vancomycin was tested using the standard dilution technique. Minimum inhibitory concentration (MIC) characteristics (MIC50, MIC90, MIC range) were determined for each microbial species. Resistance against one or more anti-microbial drugs was found in 93% of S. aureus, 40% of CN staphylococci, 73% of E. coli, 88% of E.faecalis, 55% of E.faecium, and one L. monocytogenes strain. Most of the strains, particularly enterococci, were resistant to STR, TTC, and ERY (MIC50 4 microg/ml). A high percentage of staphylococci were resistant to beta-lactam antibiotics. High resistance to CLT was found in 11 strains of E. coli (MIC 256 microg/ml) and strains resistant to CMP (MIC90 16 microg/ml) were detected. The highest numbers of resistance phenotypes were found in E. coil (16) and CN staphylococci (12). Eighteen identical resistance phenotypes were demonstrated in indicator bacteria (E. coli, E. faecalis, E. faecium) and pathogens (S. aureus, CN staphylococci) isolated from the same bulk milk sample. The obtained resistance data were matched against the herd owners' information on therapeutic use of the drugs. This confrontation could not explain the findings of strains resistant to ERY or CMP. Our findings are evidence of selection of resistant strains among not only pathogenic agents, but also among indicator bacteria which can become significant carriers of transmissible resistance genes.

Comparison of methods for the determination of antimicrobial resistance in Staphylococcus aureus from bovine mastitis.

The results of three standard methods (broth dilution, agar dilution, disk diffusion) and an experimental modification of the microdilution method for determination of resistance to ampicillin, cephalotin, cloxacillin, neomycin, novobiocin, penicillin and streptomycin were compared using 151 Staphylococcus aureus isolates obtained from cases of mastitis. The accuracy of the dilution methods was compared by determination of minimum inhibition concentrations (MIC, MIC50, MIC90 and modal MIC) and by assessment of the agreement within the tolerance of +/-1 dilution step in 2-fold dilution series. The results of the dilution methods were further compared with those of the reference disk diffusion method and the strains were classified as sensitive or resistant using the interpretation criteria for human strains. The comparisons indicated that MIC characteristics and the final classification as sensitive or resistant were method-dependent. Resistance to aminoglycoside antibiotics was observed more often when using broth dilution methods, especially when the broth was supplemented with lactose.

Variations among unbred heifers in the activities of polymorphonuclear leucocytes from the mammary gland and blood.

The phenotypic characteristics are described for the activity of polymorphonuclear leucocytes NMN) obtained by either lavage of the cavity system of juvenile mammary glands stimulated with a synthetic muramyl dipeptide analogue or isolation from the peripheral blood. Attention was paid to the variability of characteristics and its sources, and to correlations among them. The following characteristics were investigated in 27 clinically healthy, unbred Bohemian Red Pied x Holstein heifers: migration activity in situ, number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and unstimulated and zymosan-stimulated luminol-dependent chemiluminescence. Considerable individual variation was found in the characteristics. Significant differences between blood PMN and PMN from lavages after influx induction were found for bactericidal activity (P < 0.05) and chemiluminescence (P < 0.01). A significant correlation between blood PMN and mammary gland PMN was found only for the number of phagocytosing cells (r = 0.329; P < 0.01). Highly significant positive correlations (P < 0.01) were demonstrated between the number of phagocytosing PMN [a], phagocytotic index [b], and bactericidal activity [c] in both blood PMN (r(ab) = 0.602; r(ac) = 0.565; r(bc) = 0.529) and mammary gland PMN (r(ab) = 0.730, r(ac) = 0.618, r(bc) = 0.589). No significant correlation was demonstrated for non-stimulated (NS), zymosan-stimulated (ZS), or opsonized zymosan-stimulated (OZS) chemiluminescence with any of the other characteristics of phagocytotic activity, in either blood PMN or mammary gland PMN (P > 0.05). The animal was a highly significant source of variability for all the phagocytotic activity characteristics (P < 0.01). Udder quarter was a non-significant source of variability for all the characteristics of phagocytotic activity except for NS chemiluminescence (P < 0.05) and ZS or OZS chemiluminescence (P < 0.01). However, udder quarter was a non-significant source of variability of chemiluminescence indices ZS/NS and OZS/NS (P > 0.05). It has been demonstrated that in situ migration activity, the number of phagocytosing PMN, phagocytotic index, bactericidal activity of PMN and chemiluminescence indices of PMN collected from juvenile mammary glands of unbred heifers after influx induction can be regarded as candidate early markers of resistance to mammary infections.

Neutrophil apoptosis during the resolution of bovine mammary gland injury.

The role of neutrophil apoptosis in the resolution of bovine mammary gland injury induced by intramammary administration of physiological buffered saline (PBS) or lipopolysaccharide (LPS) was investigated. Twenty mammary glands of five non-pregnant heifers were used in the two studies and each animal received both stimuli. Samples of cell populations were collected by mammary gland lavages before and 24, 48, 72 and 96 hours after treatment and examined by light microscopy and staining for myeloperoxidase (MPO). A marked influx of neutrophils into the mammary gland was observed 24 hours after stimulation. At the same time, apoptotic neutrophils and MPO-positive macrophages (MAC) were identified in the samples. The numbers increased to reach maximum values at 48 hours after stimulation with PBS and at 72 hours after stimulation with LPS. The observed differences in the length of the resolution period indicate that neutrophil viability can be modulated by delaying the apoptotic process. Apoptosis of neutrophils and their subsequent phagocytosis by MAC can be regarded as a significant mechanism in the removal of neutrophils from the acutely injured mammary glands and, hence, in the resolution of bovine mastitis.

Apoptosis of polymorphonuclear leukocytes of the juvenile bovine mammary gland during induced influx.

The dynamics of apoptosis of polymorphonuclear leukocytes (PMN) during induced influx of PMN into the cavity system of the juvenile bovine mammary gland in order to investigate the role of apoptosis of PMN in the resolution of mastitis was studied. The instillation of a synthetic analogue of muramyl dipeptide into teat sinus of the sixteen mammary glands was followed by a massive influx of PMN culminating after 24 h and resolving after 96 h. Every 24 h following the influx, apoptotic PMN were microscopically detected, based on morphological characteristics. Twenty four hours after the stimulation, apoptotic PMN were already observed, and peak counts of apoptotic PMN were reached 48 h after the stimulation. The lowest differential count of apoptotic PMN, corresponding to the pre-stimulation value, was found 96 h after the stimulation. The presence of macrophages (MAC) containing phagocytized apoptotic PMN was observed by histochemical staining for myeloperoxidase (MPO) and electron microscopy. The percentage of MPO-positive macrophages increased during the resolution phase to reach peak values 48 h after the stimulation. Apoptosis of PMN and phagocytosis by macrophages may represent a removal mechanism that is important in the resolution of the induced influx of PMN in the cavity system of juvenile bovine mammary gland.

Ultrastructure of phagocytes from mammary glands of non-pregnant heifers.

Results of ultramicroscopic investigations of phagocytes isolated from non-secreting and aberrantly secreting juvenile mammary glands of non-pregnant heifers are presented. The two types of phagocytes observed in cell suspensions obtained by lavage of mammary gland cavities were polymorphonuclear leukocytes and macrophages. Polymorphonuclear leukocytes were spherical or irregular in shape and contained segmented nuclei. Azurophilic and specific electron-dense granules, mitochondria, glycogen particles, phagosomes and phagolysosomes in cytoplasma and characteristic pseudopodia on the cell surface were observed. In addition to these normal polymorphonuclear leukocytes, degenerating cells, characterized by spherical nuclei, total absence of pseudopodia, merged nuclear segments and altered granules, other cellular organelles and plasmalemma were present. Two types of macrophages, i.e. vacuolized and non-vacuolized, could be distinguished. Typical of the non-vacuolized type was a kidney-shaped nucleus, a rich Golgi complex and a large amount of lysosomes in the cytoplasm. The vacuolized macrophages contained a large amount of electron-dense vacuoles in the cytoplasm. Unlike non-secreting glands, the cell suspensions collected from aberrantly secreting juvenile mammary glands contained only vacuolized macrophages. The vacuolization results from phagocytosis of corpuscular particles of aberrant milk plasma.

Minimal inhibition concentrations of selected antibiotics for strains of Staphylococcus aureus.

Minimal inhibition concentrations (MIC) of gentamycin (Ge), neomycin (Neo), rifampicin (Rif), ampicillin (Amp), lincomycin (Lin), erythromycin (Ery), and streptomycin (STM) were determined by the agar dilution technique using 46, 130, 131, 125, 140, 139 and 142 strains of Staphylococcus aureus, respectively. The strains, selected from the collection of the authors' laboratory, were isolated from mammary gland secretions of cows affected with clinical or subclinical mastitis. The following ranges of MIC (micrograms/ml) were assessed for the antibiotics under study: Ge 0.125-0.50, Neo 0.06-0.50, Rif 0.0039-0.030, Amp 0.015-1.00, Lin 0.25-1.00, Ery 0.06-0.25, STM 0.50-64.0. Modal MIC (micrograms/ml) were as follows; Ery 0.125 (86%), Lin 0.5 (71.4%), Rif 0.007 (68.7%), Ge 0.25 (56.5%), STM 1.00 (54.2%), Neo 0.25 (53.8%), Amp 0.06 (41.6%). The order of efficiency expressed in MIC 90 (micrograms/ml) was as follows: Rif (0.015), Ery (0.125), Ge (0.25), Neo (0.25), Amp (0.5), STM (4.0).

[Effectiveness of the BR-Test and the Intest in the determination of chemotherapeutic residues in raw and preserved milk]. / Ucinnost BR-testu a INTESTU pri stanovení reziduí chemoterapeutik v syrovém a prezervovaném mléce.

Negative detection of residues of inhibitory substances has become one of the standard quality traits of raw milk also in the Czech Republic. Hence a requirement appeared in 1991 to evaluate the usability of two commercially produced microtitration tests for this purpose. The tests of BR-TEST (Enterotox) and INTEST (Mlékárenský prumysl) were performed in weekly intervals for the time longer by a week than is the declared expiration, that means seven replications in BR-TEST and five replications in INTEST. Compact and stripped microplates (STRIPS) designated BR-TEST "AS", BR-TEST 12x8 "AS" and INTEST C-96 type and INTEST 0-96/6x16 were used for the tests. The principle of inhibition demonstration in the concentration series of selected drugs was applied. The drugs were chosen with respect to the frequency of their use for clinical treatments in the CR. Tab. II shows the used drugs and concentration series. The following parameters were tested: detection limits, reproducibility, effect of milk preservation, and the declared expiration. Tab. III shows detection limits. It is apparent the BR-TEST detection responses to streptomycin and chloramphenicol were lower in BR-TEST than in INTEST in our experiments. The responses to penicillin, oxacillin and oxytetracycline were lower in some cases, and equal in other ones. On the other hand, the INTEST detection response to sulfadimidin was markedly lower in comparison with BR-TEST. Tab. IV summarizes the range of detection limits in milk with Heeschen's reagent. In our experiments the detection response of BR-TEST to the tested chemotherapeutics was lower in some cases, in other cases it was the same as in INTEST.(ABSTRACT TRUNCATED AT 250 WORDS)

[Determination of protein A in Staphylococcus aureus strains from a collection]. / Stanovení proteinu A u sbírkových kmenu Staphylococcus aureus.

The author used the haemagglutination method to estimate A protein in 153 strains of Staphylococcus aureus from collections. Protein A was detected in 66% of coagulase positive strains deposited in the course of time in the collection in 1983-1988. All strains were kept in skimmed milk at -20 degrees C. For evaluation of the reaction a semiquantitative scale was proposed. The author observed a diminished capacity to form protein A with the time of storage of the strains. In strains deposited for a shorter time in the collection (only three years) the percentage of positive results rose to 92.5%. In coagulase negative strains protein A was not detected.

[Algorithms in the evaluation of electrical conductivity in milk in screening for mastitis]. / Algoritmy vyhodnocování elektrické konduktivity mléka pro screening mastitid.

An algorithm of evaluation of diagnostic data for electronic screening of subclinical and clinical mastitis was derived from 5848 field measurements of the electric conductivity of mammary gland secretions and from the same number of clinical, bacteriological and cytological examinations of 92 dairy cows. The algorithm consists in the calculation of arithmetical mean of the two highest values of mixed, i.e. absolute, and or differential conductivity of a seven-day sliding cycle. Regardless of the intensity of disease forms, for healthy and colonized mammary glands, for mammary glands suffering from nonspecific subclinical mastitis, infectious subclinical mastitis and infectious clinical mastitis, the overall agreement with findings according to repeated clinical, bacteriological and cytological examinations made 88.8% with 4.2% of falsely negative and 7.0% of falsely positive findings. The overall agreement for persistent forms of diseases made 95.3% without falsely negative findings and with 4.7% of falsely positive findings.

[Relation of the count of somatic cells in tank samples to the incidence of contagious mastitis on large farms]. / Závislost poctu somatických bunek v bazénových vzorcích na prevalenci kontagiózních mastitid ve velkých stádech.

The relation between the prevalence of contagious mastitis with the finding of Streptococcus agalactiae and Staphylococcus aureus and the number of somatic cells in the tank samples of milk was studied on the basis of a clinical and bacteriological examination of 11 129 daily cows on four large farms, with a capacity of 600 to 1240 animals. A statistically significant correlation was demonstrated between the analyzed characteristics (r = 0.621). The relation was demonstrated at a prevalence up to 25.3%. The number of somatic cells provides a marked indication of prevalence only from 10%. We failed to demonstrate a statistically significant correlation between the prevalence of mastitis with a cultivation finding of Staphylococcus aureus and the number of somatic cells in tank samples of milk (r = 0.054). The results suggest that on large farms it will not be possible to use only the number of somatic cells in tank milk samples as a signal of diagnostic improvement and preventive mastitis-control measures, since a 10% prevalence of these diseases usually excludes the possibility of herd sanitation.