The effects of increasing concentrations of retinol, retinal and retinoic acid on proliferation of bovine mammary epithelial cells were investigated in collagen gel cultures. All retinoids significantly inhibited proliferation of mammary epithelial cells. The relative inhibitory potency of the retinoids was: retinoic acid > retinal > retinol. Maximal inhibition at 10 μg/ml corresponded to a 75–95% inhibition of proliferation obtained in basal medium. Retinol, retinal and retinoic acid also inhibited proliferation of cells growth-stimulated with insulin-like growth factor-I (IGF-I). Retinoids in highest concentrations (10 μg/ml) inhibited 68–85% of proliferation of cells obtained in culture medium containing 25 ng IGF-I/ml. Retinol and retinoic acid also inhibited proliferation of cells growth-stimulated by insulin and other growth factors from the IGF growth factor family (des(1-3)IGF-I and IGF-II), as well as growth factors from the epidermal growth factor family (EGF and TGF-α), with retinoic acid being more effective than retinol. At a concentration of 100 ng/ml, retinol and retinoic acid inhibited respectively 24–38 and 44–52% of mammary cell proliferation stimulated by growth factors of the IGF family, and at 10000 ng/ml, 61–71% of cell proliferation was inhibited. The growth-stimulating effect of insulin, EGF and TGF-α was inhibited 42–64% by retinol and retinoic acid at 100 ng/ml, and 64–84% at 10000 ng/ml. The present results show that retinol, retinal and retinoic acid are potent inhibitors of bovine mammary epithelial cell proliferation. It is suggested that retinoids may have concentration-dependent roles in regulation of pubertal mammary growth and development, indicating that the milk yield potential of heifers may be affected by vitamin A status.

Variations in mammary glucose uptake were measured during the normal pregnancy-lactation cycle in dairy goats. In addition mammary glucose uptake was studied in response to somatotropin (ST) treatment in mid-lactation and acute increases in glucose concentration induced by sodium-propionate challenge in early lactation. Mammary glucose uptake was independent of arterial glucose, insulin and Insulin-like Growth Factor-1 (IGF-1) concentrations during lactation and during acute increases in arterial glucose concentration. Glucose uptake in the lactating mammary gland of the goat must therefore be carried out by an insulin-independent carrier, possible GLUT1, and glucose supply is not a limiting factor for uptake under in vivo conditions. Extraction of glucose uptake changed markedly during the normal course of lactation, following the overall changes in milk yield. Concentrations of glucose in skimmed milk, believed to reflect intracellular glucose concentration, changed in opposite directions, resulting in decreasing ratios of arterialratioskimmed milk glucose concentration with progressing lactation. Thus, mammary synthetic capacity also involves a capacity for glucose uptake, which may be influenced by variations in glucose carrier numbers, as well as mammary metabolic activity (intracellular glucose concentration). In contrast to the situation during the normal course of lactation, ST stimulated milk yield, despite less efficient glucose extraction.

Keratinization of the epidermal cells of the bovine claw generates the horn that gives the tissue its mechanical strength. Disruption of keratinization is likely to have a detrimental effect on the strength and integrity of the horn, and could lead to solar lesions and lameness. As part of a wider investigation of the cell biological causes of lameness in dairy animals, we have compared keratin synthesis and distribution in healthy bovine claw tissue with those in hooves with solar ulcers. Protein synthesis was measured by [35S]-labelled amino acid incorporation in claw tissue explant cultures. [35S]-labelled protein synthesis was higher in tissue from diseased claws than in healthy claws, and highest at the ulcer site. The identity of proteins synthesised in vitro did not differ between healthy and diseased tissue. DNA synthesis indicative of cell proliferation was also elevated in diseased tissue. Immunoblotting after one- or two-dimensional electrophoresis showed cytokeratins (CK) 4, 5/6, 10 and 14 to be amongst those expressed in healthy claw tissue. The relative abundance of these keratins was not altered in healthy regions of ulcerated hooves, nor at the ulcer site, but CK16, not usually found in healthy tissue, was detected in the sole of diseased claws. CK5/6 and CK14 were shown by immunohistochemistry to be present in the basal epidermis of healthy tissue, whereas CK10 was found in supra-basal layers. In healthy tissue from ulcerated claws, this distribution was unaltered, but at the site of solar ulcers, CK5/6 and CK14 were each found in both basal and supra-basal epidermis. The study suggests that solar ulceration of the bovine claw is not associated with gross alteration in the keratin composition of the tissue, but causes abnormal distribution of cytokeratins, perhaps as a result of loss of positional cues from the basement membrane. Ulceration did, however, stimulate cell repair involving epidermal protein synthesis (including keratins), and keratinocyte proliferation.

A major problem in camel productivity is the high mortality rate of camel calves in the first 3 months. The causes for mortality are mainly poor management practice and infectious diseases. The purpose of this research, carried out on a ranch in Kenya, was to determine the immunoglobulin G (IgG) concentration in camel colostrum as well as the extent of the calves' passive immunization by maternal antibodies. IgG concentration in colostrum and in the serum of the calf were measured during the first 3 d of life. Evaluation was carried out by comparing the respective values with those for horses and cattle. The average IgG concentration in the camel colostrum was higher than that found in literature for horses and cattle. IgG concentration in the serum of the camel calves reached its maximum 24 h after birth. In 39% of the examined calves, this maximum concentration was below 4 g/l, which is considered to be the critical value in horses and cattle. 61% of the calves achieved an IgG concentration of over 4 g/l. Since there is no correlation between IgG level in colostrum and early mortality, the results indicate that low colostrum intake during the first 24 h of life and not low IgG concentration in colostrum is presumably one of the main causes of early calf mortality. Therefore, it was recommended that the care of the newborn calves by herdsmen should be improved.

The somatic cell count of milk is routinely determined by the fluoro-opto-electronic method and sometimes by the direct epifluorescent filter technique (DEFT). This paper investigates the potential of solid phase cytometry (SPC), a novel technique combining aspects of both the fluoro-opto-electronic method and epifluorescence microscopy for somatic cell counting. In SPC, cells are retained on a membrane filter, fluorescently labelled and automatically detected on the entire membrane filter by means of a laser scanning instrument (ChemScan). Fluorescent spots can be visually inspected by an epifluorescence microscope with a computer-driven moving stage. The performance of SPC was compared with that of the fluoro-opto-electronic method using a Fossomatic 360 instrument for 68 milk samples with varying somatic cell counts (103–106/ml). The sample throughput and repeatability of SPC were inferior to those of the Fossomatic method and statistical analysis of the method comparison data using the approach of J. M. Bland & D. G. Altman (The Lancet 1986 February 8 pp 307–310) revealed a poor comparability between the two methods. Moreover, problems of milk filterability and the interference of fluorescent particles presently hamper the routine application of SPC. Nevertheless, this method represents the first example of the application of SPC to milk.

Phagocytic and bactericidal activity of polymorphonuclear neutrophil leukocytes (PMN) isolated from blood and milk, against Staphylococcus aureus, was compared between groups of six healthy dairy cows in early, mid- and late lactation using a bacteriological assay. PMN were isolated from blood with a high degree of purity, but the cells isolated from milk contained variable amounts of macrophages (MΦ) and lymphocytes (L). The results were therefore calculated using the percentage PMN in order to evaluate phagocytosis and killing by PMN only. Blood PMN phagocytosed 82% Staph. aureus and milk PMN 43% on average and there was no significant difference between the different stages of lactation. The bactericidal activity of blood PMN against Staph. aureus was 36±8% in early lactation (significantly different from mid lactation, P<0·05), 64±10% in mid lactation and 53±6% in late lactation. Milk PMN killed only 6±3% Staph. aureus in early lactation (significantly different from mid lactation, P<0·01), 27±3% in mid lactation and 20±9% Staph. aureus in late lactation. The ratio of the bactericidal activity of milk to blood PMN was 0·08, 0·43 and 0·22 in early, mid- and late lactation, respectively. In addition to the decreased function, the number of cells in milk (somatic cell count, SCC) was also 60% lower in early lactation than in mid lactation cows (P<0·01). Our results suggest an impairment of blood and milk-resident PMN bactericidal activity against Staph. aureus and a decreased number of milk-resident PMN in dairy cows at the onset of lactation.

Milk producers in Malaysia make extensive use of crossbred Sahiwal Friesian dairy cattle. These animals have, however, been found susceptible to lactation failure. A survey of cows in an experimental herd of F1 Sahiwal Friesian animals indicated that, in 30% of animals, milk yield decreased to negligible levels within the first 8 weeks post partum. Lactation failure was associated with a progressive increase in the amount of residual milk left in the udder after normal milking. By week 3 of lactation, residual milk volume was significantly greater than that in animals that, based on previous lactation history, were not susceptible to lactation failure, and accounted for up to 30% of milk available at the morning milking. The cellular consequences of residual milk accumulation were evident in the activities of acetyl-CoA carboxylase, fatty acid synthetase and galactosyltransferase, key enzyme markers of cellular differentiation, which decreased in glands undergoing lactation failure and were lower than values measured in tissue of control cows. Mammary cell number, estimated by tissue DNA content, was also reduced in animals undergoing lactation failure. These indices of mammary development indicate that lactation failure is the result of premature involution in susceptible animals. Premature involution is a predictable consequence of progressive milk stasis in failing lactation, and attributable to an increase in autocrine feedback by inhibitory milk constituents. The progressive increase in residual milk is, on the other hand, unlikely to be attributable to impaired mammary development. Measurements of milk storage during milk accumulation showed no differences between control and lactation failure cows in the distribution of milk between alveolar and cisternal storage compartments. We conclude that lactation failure in Sahiwal Friesian cows is due to a failure of milk removal, and probably the result of an impaired milk ejection reflex rather than to the glands' milk storage characteristics.

Prolactin significantly increased the rate of fatty acid synthesis in explants of mid-pregnant rat mammary gland cultured for 96 h with insulin plus corticosterone. Under these conditions, prolactin increased the specific activity of total acetyl-CoA carboxylase in nuclear-free homogenates of explants by 2·6, and increased the proportion of the enzyme in the active polymeric form from 0·44 to 0·89. Removal of prolactin after 48 h in culture decreased the specific activity of the total enzyme by about half, and decreased the proportion as polymer to 0·52. The results show that prolactin plays a major role in mid-pregnant rat mammary gland in the polymerization which accompanies increased activity of the total enzyme and increased rate of fatty acid synthesis.

Cows managed for extended lactations of 16 months duration were milked on a half-udder basis twice or thrice daily, commencing in lactation week 9. Mammary epithelial integrity (assessed by milk sodium[ratio ]potassium ratio) was greater in the half-udder which was milked thrice daily. This difference was evident throughout the lactation but became greater after week 41. Milk protein composition was assessed during late lactation (52±3 weeks). Casein number (casein as a proportion of total protein) was significantly higher in half-udders milked thrice daily, as were the relative amounts of α- and β-caseins, whilst those of κ- and γ-caseins were reduced. Two days of inverted milking frequency (i.e. thrice-milked udder halves now milked twice, and vice versa) only partly reversed these differences. We concluded that thrice-daily milking will help to prevent or ameliorate the usual decline in milk processing quality associated with late lactation. Part of this effect is due simply to reduced exposure to proteolytic enzymes as a result of decreased storage time in the udder, but part is due to a better maintenance of epithelial tight junction integrity as lactation advances, which restricts leakage of proteolytic enzymes from serum into milk.

We evaluated the effect of a commercial bovine colostral whey on the complement-mediated immune responses of calves. Two groups of neonatal calves were fed, in addition to whole milk (WM) and pooled colostrum (PC), different amounts of a commercial immunoglobulin concentrate made from pooled colostral whey (Ig-C) for the first two feedings post natum. The control group was fed WM and PC only. Serum samples were obtained at the ages of 2, 7, 14 and 30 d. Bacteriolytic activity against complement-sensitive Escherichia coli JM103 and opsonic activity against complement-lysis-resistant E. coli IH3080 strains were studied, as well as the levels of C3 complement component and E. coli JM103 specific antibodies in the sera.

Groups fed Ig-C had 2–3 times higher bacteriolytic activity than the control group of both the classic (P < 0·005) and alternative pathways (P < 0·0001) at days 2 and 7 post natum. This effect is obviously not caused solely by the antibodies ingested but also involves other unknown colostral factors, possibly lectins. The opsonisation capacities of the sera correlated well with the amounts of immunoglobulins ingested (P < 0·05) at days 2–14. The levels of C3 component in sera did not differ between the groups. In the group fed the largest amount of immunoglobulins levels of E. coli JM103-specific antibodies were highest (P < 0·0001). It can thus be concluded that the antibody independent complement activities of serum can be increased substantially by feeding colostral whey concentrate to calves during their first days of life.

The aim of the present study was to examine how different types of early interaction between dairy cows and their calves influence milking/suckling-related hormone release in the cows and sucking/bucket-drinking related hormone release in calves. Eighteen cows of the Swedish Red and White breed were studied during the first week after parturition. The cows were machine milked twice daily, and allotted to one of three treatments: [1] cow and calf were kept together and the cow was allowed to suckle the calf for 30 min about 1 h before each milking; [2] cow and calf were kept together and the calf was bucket fed twice daily; and [3] cow and calf were separated immediately after parturition, and the calf was kept in a single box and was bucket fed twice daily. Blood samples were collected around day 7 from both cows and calves. The plasma levels of oxytocin, prolactin, cortisol, gastrin, cholecystokinin (CCK), somatostatin and insulin were analysed. In the cows the levels of oxytocin, prolactin and cortisol were influenced by all three treatments, except for the level of cortisol which did not respond to suckling. The main finding was that the release of oxytocin was significantly greater during suckling compared with machine milking. In the calves, the hormone levels were also influenced by the different milk feeding routines. The plasma concentrations of oxytocin, gastrin, CCK and insulin increased in response to milk ingestion in all treatments. However, during sucking, the increase of oxytocin was significantly greater than during bucket drinking. In addition, a strong correlation between oxytocin and insulin was found in response to sucking. Further, significant increases in prolactin and somatostatin, and a decrease in cortisol were found during sucking. The level of somatostatin also increased in response to bucket feeding when calves were kept separately. During bucket feeding, no significant correlation was found with oxytocin, but strong correlations between the gastrointestinal hormones gastrin, CCK, somatostatin and insulin were seen. Together these data suggest that different hormonal patterns were triggered in the cows by suckling and milking, and in the calves by sucking and bucket drinking. This is further supported by different correlation patterns observed in the calves in response to sucking and bucket feeding. The present findings imply that management routines for cows and calves during the first week after parturition have consequences for the physiology of the animals.

An experiment using three New Zealand herds and a total of 632 cows, examined the effect of localised prophylactic treatments with antibiotic at drying-off on the incidence of new intramammary infection during the dry period and at calving. Antibiotic was infused either into the teat canal (0·22 g of dry-cow formulation) or the teat sinus (3·1 g of lactating-cow formulation) of uninfected quarters to eliminate any bacteria present in these locations at the last milking of lactation. These treatments were compared with a negative control (nil treatment) and a positive antibiotic control (infusion of 3·6 g of dry-cow formulation). All antibiotic formulations used the same active ingredient, sodium cloxacillin. No significant reduction in new dry period clinical mastitis was observed for the two localised treatments whereas the positive control treatment achieved 100% reduction in new clinical mastitis compared with untreated control quarters. A 41% reduction (P<0·05) in new Streptococcus uberis infections at calving was associated with the teat canal antibiotic treatment, compared with an 82% reduction (P<0·001) for the positive antibiotic control. Both localised treatments showed a reduced incidence of new intramammary infection (P<0·001) when pooled across periods and pathogens. Teats receiving either the teat canal antibiotic treatment or a full infusion of long acting dry-cow antibiotic had a lower incidence of open teat canals (P<0·05) at 3 weeks after drying-off.

Occurrence of milk ejection and course of milk removal were investigated in 18 dairy cows at milking intervals of 4, 8 and 12 h in early, mid or late lactation. Milk ejection occurred fastest in early lactation at a milking interval of 12 h and was delayed at short milking intervals and in late lactation. Storage capacity of the udder was estimated and the actual milk yields of experimental milkings were calculated as a percentage of storage capacity, i.e. degree of udder filling. It was shown that the occurrence of milk ejection after the start of teat stimulation is a function of udder filling. The relationship between the degree of udder filling and the delay from the start of milking until commencement of milk ejection followed a linear regression curve. Changes in occurrence and course of milk ejection have to be considered in practical milking, mainly in late stages of lactation and after short milking intervals. In automatic milking systems where variable and sometimes extremely short milking intervals occur, the duration of pre-milking udder preparation should be adapted to the expected milk yield at each individual milking procedure.

To test the hypothesis that the availability of glucose or its precursors can influence the response of milk protein concentration to the intravenous infusion of amino acids, five cows were used in a 5 × 5 Latin square design with period lengths of 7 d. The five treatments were the basal diet of grass silage ad lib. plus 5 kg/d of a cereal-based supplement containing feather meal (Basal); Basal plus 4 g/d histidine, 8 g/d methionine and 26 g/d lysine (4H); Basal plus 8 g/d histidine, 8 g/d methionine and 26 g/d lysine (8H); and these two amino acid mixtures together with 600 g/d of glucose (4HG and 8HG respectively). Earlier experiments with this basal diet had shown that histidine was first-limiting for secretion of milk protein, followed by methionine and lysine. The yield of milk protein was increased progressively with the amount of histidine infused. The efficiency of transfer of histidine into milk protein was 0·42 for the 4H and 4HG and 0·35 for the 8H and 8HG treatments, and the concentration of milk protein was increased over Basal by all infusion treatments. However, milk protein concentrations were higher, and lactose concentrations in the milk were lower, in the absence of added glucose. Concentrations of insulin in blood plasma were not affected by treatment. It is concluded that, with the treatments without added glucose, a shortage of glucose prevented an increase in lactose secretion, and hence limited the increase in milk yield, leading to an increased concentration of protein in the milk.

The contamination of pasteurized milk by Bacillus cereus during the filling process was studied in two dairy plants. Samples of pasteurized milk were taken at four different sites along the production line. The samples were stored at 7 °C for 7 d, or at 10 °C for 5 d, before plate counting and random selection of B. cereus isolates. Isolates of B. cereus were typed by the polymerase chain reaction (PCR)-based method randomly amplified polymorphic DNA (RAPD). Samples taken at three different sites between the pasteurizer and the filling machine were all holding similar low concentrations of B. cereus, while an increase of the B. cereus count was seen in the consumer packages. More B. cereus of different RAPD types was growing in the consumer packages than in samples taken just before the filling machine. Several RAPD types found in the consumer packages were not detected in the samples taken just before the filling machine.

About 10% of primiparous cows have no milk ejection during the first milkings after delivery. Therefore, 17 Brown Swiss dairy cows in their first lactation were used to evaluate the extent of disturbed milk let-down and the corresponding oxytocin (OT) plasma values in the 1st 5 days after delivery. The first milking was 9–22 h after parturition and served for classification of the cows to groups with inhibited (INH), bimodal (BIMO) or normal (NOR) milk let-down. The OT plasma levels before the start of manual teat stimulation and machine milking were comparably high during the first milking especially in NOR and BIMO cows. Ten minutes before the second milking (M2), 300 mg of the opioid antagonist naloxone was injected to test whether the disturbance was affected by the action of endogenous opioids on the neurohypophysis. The milk yield was not influenced by the naloxone treatment, and the INH cows had milk ejection only after a vaginal stimulation. Afterwards, the cows were milked twice every day, until the milk let-down and the OT release were unaffected (equal to control milking). Then, at the next milking, the cows were injected with 300 mg morphine 10 min before milking. The central OT release in response to manual teat stimulation and machine milking was completely blocked in all cows, but a vaginal stimulation was able to abolish this block, at least partially, in 16 cows. Thus, morphine produced a milk let-down characteristic as in the INH cows during the first three milkings. For the following milking, the cows were pre-treated with 300 mg naloxone (−15 min) plus 300 mg morphine (−10 min) before milking. The OT release and the milk yields were unaffected when compared with the control milking. This experiment demonstrates that exogenous opioids can affect the central release of OT in a naloxone-reversible manner even very soon after parturition. However, endogenous opioids are probably not the main mediators of disturbed central OT release and alveolar milk ejection in post-partum primiparous cows.

The efficiency of reversed-phase HPLC, capillary electrophoresis (CE), PAGE and isoelectric focusing with immunoblotting in separating ovine caseins has been evaluated. The assessment was carried out by employing electrospray ionization–mass spectrometry (ESI–MS) and matrix-assisted laser desorption ionization–time of flight as reference tools for identifying protein components. Ovine casein was fractionated by HPLC into four major peaks. With ESI–MS, each peak contained components belonging to only one of the four casein families. On-line liquid chromatography–ESI–MS allowed us to determine each fraction's composition by detecting thirteen αs1-, eleven αs2-, seven β-, and three κ-casein (CN) components. The αs1-CN and αs2-CN consisted of eight and two protein chains respectively of lengths differing through the deletion of one or more peptide sequences; they were also discretely phosphorylated as κ-CN and β-CN. By CE at pH 2·5, each casein fraction was as heterogeneous as that resulting from ESI–MS for the single HPLC-derived fractions. The separation of αs1-CN and αs2-CN proved to be excellent, with the exception of a co-migration of κ0-CN with a minor αs1-CN component and of a glycosylated κ-CN form with low-phosphorylated αs1-CN and β-CN components. Dephosphorylation of whole casein was used to reduce the heterogeneity of the native fractions and by applying currently used analytical techniques it was possible to visualize the protein moiety difference along the CE profile. CE, HPLC, and immunoblotting were all equally capable of effecting an accurate separation of the four dephosphorylated casein families. The spectra obtained by ESI–MS directly on dephosphorylated whole ovine casein samples contained the signals of the four casein families and the relative αs1-CN variants, the non-allelic αs1-CN and αs2-CN forms, dimeric κ-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.

Two groups of 15 multiparous cows in mid-lactation were used in a Latin square design experiment with 4-week experimental periods. The genetic milk protein concentration level was high in the first group and low in the second. Each group of cows was given in a random order three feeding levels that covered 85, 100 and 115% of energy requirements and 90, 110 and 125% of nitrogen requirements, respectively. In both groups, increasing level of feeding induced a significant increase in milk yield (+2·4 kg/d between lowest and highest levels) and in protein concentration (+1·7 g/kg). The proportion of paracasein in total proteins was not altered by either genetics or nutrition. The proportion of casein in total proteins was slightly increased by 0·5 percentage points (P<0·05) with the intermediate level of feeding. Plasmin and plasminogen activities were not significantly modified by the genetic milk concentration level. Plasmin activity significantly increased with nutrient supplementation, but only in animals of low genetic potential (+21% between low and high levels, P<0·01). Casein composition was not significantly altered by the genetics or level of nutrition. Over the whole range of individual measurements taken (n = 90), the relationships between casein or paracasein and total protein concentrations were linear and very narrow (R2 = 0·92 and 0·95, respectively). The proportion of casein or paracasein in total proteins significantly decreased as plasmin activity increased.

The heterogeneity of caprine caseinmacropeptide (CMP) was determined by means of treatments with neuraminidase and acid phosphatase and analyses by anion exchange FPLC and reversed-phase (RP)-HPLC, with on-line and off-line electrospray ionization mass spectrometry. The main CMP components were two non-glycosylated and di-phosphorylated forms, as well as two other mono-phosphorylated species, each corresponding to a genetic variant of caprine κ-casein due to the silent substitution Ile/ Val at position 119. Asialo-aglyco mono- and di-phosphorylated forms were found in the ratios 8–14% and 86–92%, respectively. Approximately 36% of caprine CMP was glycosylated. Based on the obtained molecular masses, the occurrence of tri-, di- and monosaccharide-containing di-phosphorylated CMP are reported, assuming that N-acetylgalactosamine, galactose, N-acetyl and N-glycolylneuraminic acids would constitute the main monosaccharides of caprine CMP. CMP microheterogeneity due to the genetic polymorphism was also observed in the glycosylated forms.

Restriction enzyme fragmentation pattern (REFP) analysis was used to recognise Staphylococcus aureus strain variation in naturally occurring bovine subclinical mastitis. Multiple colony REFP analysis identified eight distinct strains of S. aureus in addition to the original strains A and B that were infused via the intramammary route, indicating that individual quarters of the udder may be colonised simultaneously by more than one strain of S. aureus. Examination of multiple colonies per milk sample may benefit bacterial strain recognition as an epidemiological tool in mastitis investigations. The dynamics of intramammary infection were determined using a novel double crossover experimental challenge. Quarters remained persistently infected for several weeks following challenge in all four cows, irrespective of the challenge strain. This indicated that no alteration of the original subclinical infection, including the possible induction of clearance of the quarters infected with S. aureus, or replacement of the original strain by the infused strain was induced by challenge. The persistent subclinical infection in all four animals supports previous reports on the chronicity of S. aureus intramammary infection in dairy cows.