Effects of Staphylococcus aureus intramammary infection on the expression of estrogen receptor α and progesterone receptor in mammary glands of nonlactating cows administered estradiol and progesterone to stimulate mammary growth.

Intramammary infections (IMI) are prevalent in nonlactating dairy cattle and are known to alter mammary structure and negatively affect the amount of mammary epithelium in the gland. Mechanisms responsible for the observed changes in mammary growth during an IMI are poorly understood, yet the importance of the key mammogenic hormones driving mammary growth is well recognized. This study's objective was to characterize the expression of estrogen receptor α (ESR1) and progesterone receptor (PGR) in mammary glands stimulated to grow and develop in the presence or absence of an IMI as well as preliminarily characterize myoepithelial cell response to IMI. Mammary growth was stimulated in 18 nonpregnant, nonlactating dairy cows using subcutaneous estradiol and progesterone injections, and 2 culture-negative quarters of each cow were subsequently infused with either saline (n = 18) or Staphylococcus aureus (n = 18). Mammary parenchyma tissues were collected 5 d (n = 9) or 10 d (n = 9) postchallenge and examined using immunofluorescence microscopy to quantify positive nuclei and characterize staining features. There tended to be a greater number of ESR1-positive nuclei observed across 8 random mammary parenchyma fields of view in saline quarters than in Staph. aureus quarters (201 vs. 163 ± 44 nuclei). Saline quarters also contained a greater number of PGR-positive nuclei (520 vs. 440 ± 45 nuclei) and myoepithelial cells (971 vs. 863 ± 48 nuclei) than Staph. aureus-challenged quarters. However, when ESR1, PGR, and myoepithelial nuclei counts were adjusted for Staph. aureus quarters containing less epithelium, differences between quarter treatments abated. The examined ESR1 and PGR staining characteristics were similar between saline and Staph. aureus quarters but were differentially affected by day of tissue collection. Additionally, nuclear staining area of myoepithelial cells was greater in Staph. aureus quarters than in saline quarters. These results indicate that IMI had little effect on the number or staining characteristics of ESR1- or PGR-positive nuclei relative to epithelial area, but myoepithelial cells appear to be affected by IMI and the associated inflammation in nonlactating mammary glands that were stimulated to grow rapidly using mammogenic hormones. Accordingly, reductions in mammary epithelium in affected glands are not suspected to be resultant of alterations in the number or staining characteristics of ESR1- or PGR-positive mammary epithelial cells.

Apoptosis and proliferation in Staphylococcus aureus-challenged, nonlactating mammary glands stimulated to grow rapidly and develop with estradiol and progesterone.

Bovine mastitis is a common and costly disease in the dairy industry and is known to negatively affect the amount of epithelium in nonlactating mammary glands. Despite this recognition, an understanding of the mechanisms contributing to reductions in epithelium is lacking. The objective of this study was to evaluate cellular apoptosis and proliferation in uninfected and Staphylococcus aureus-infected mammary glands that were stimulated to rapidly grow and develop. Estradiol and progesterone injections were administered to 18 nonlactating dairy cows to induce mammary growth, and 2 quarters from each animal were infused with saline or Staph. aureus. Mammary tissues were collected at 5 (n = 9) and 10 d (n = 9) postinfusion and examined using quantitative bright field and florescent immunohistochemistry. Staphylococcus aureus mammary glands tended to have a greater number of mammary epithelial cells undergoing apoptosis than saline quarters. In the stromal compartment, challenged quarters contained a lower proportion of cells undergoing apoptosis than saline quarters overall; however, cell types undergoing apoptosis were differentially affected. Staphylococcus aureus quarters contained a lesser percentage of apoptotic fibroblasts while also containing more nonapoptotic immune cells than saline quarters in the intralobular stroma compartment. A similar number of proliferating epithelial cells were present in Staph. aureus and saline mammary tissues, but more proliferating cells were present in the intralobular stroma compartment of Staph. aureus-infused quarters than those infused with saline. When these cellular responses are considered together, it indicates that changes in cellular apoptosis and proliferation contribute to changes in the gland structure by potentiating the expansion of the intralobular stromal compartment, via cellular accumulation, and limiting the amount of epithelium due to increases in cellular apoptosis in affected glands. Reductions in mammary epithelium are expected to reduce future milk yields and productive herd life.

Identification of factors influencing teat dip efficacy trial results by meta-analysis.

Two meta-analyses were conducted using data from peer-reviewed natural exposure (NE) and experimental challenge (EC) teat dip efficacy trials to identify factors influencing the new intramammary infection (IMI) rate. A NE data set containing 16 studies and an EC data set containing 21 studies were created. New IMI rate was calculated based on the percentage of new quarter infections per month (PNQI/mo) for each observation, in both data sets, and used as the dependent variable for model derivation. A linear, mixed-effects model with a random study effect, weighted by number of quarters eligible for infection, was derived for each data set. The final NE model included the effects of experimental design (split herd or split udder), mastitis pathogen group (Staphylococcus aureus, Streptococcus agalactiae, environmental streptococci, gram-negative species, Corynebacterium spp., or coagulase-negative staphylococci), postmilking treatment (iodine, chlorhexidine, linear dodecyl benzene sulfonic acid, chlorine compounds, phenol compounds, or undipped negative controls), and the interaction between mastitis pathogen group and postmilking treatment. Overall, Corynebacterium spp. had the highest new IMI rate (0.0139±0.0018 PNQI/mo), and environmental streptococci and gram-negative species had the lowest (0.0023±0.0022 PNQI/mo). Additionally, trials utilizing a split herd experimental design had a 2-fold higher new IMI rate than trials using a split udder design. The final EC model included the effects of mastitis pathogen (Staph. aureus and Strep. agalactiae), postmilking treatment (iodine, chlorine compounds, "other" active ingredients, or undipped negative controls), geographic region of study (Eastern, Southern, and Pacific Northwest), and the 2-way interactions of region and pathogen group and postmilking treatment and pathogen group. Overall, Staph. aureus and Strep. agalactiae had similar new IMI rates. Quarters dipped postmilking in either iodine (0.0127±0.0099 PNQI/mo), chlorine compounds (0.0258±0.0095 PNQI/mo), or "other" active ingredient teat dips (0.0263±0.0106 PNQI/mo) had lower new IMI rates than undipped quarters (0.0859±0.0087 PNQI/mo). These results indicate that experimental design influences the new IMI rate of teat dip efficacy trials and that using an effective postmilking teat dip has a greater effect on controlling the new Staph. aureus and Strep. agalactiae IMI rate than the teat dip's active ingredient.

Technical note: Development of a challenge model for Streptococcus uberis mastitis in dairy heifers.

A challenge model for experimentally inducing Streptococcus uberis mastitis in bred dairy heifers was developed. Qualifying heifers (n=7) exhibited antibody titers of <1:10,000 against Strep. uberis antigens and were free of intramammary infections (IMI). Two contralateral quarters of each heifer were assigned to receive an infusion of Strep. uberis (1,000 to 2,000 cfu); remaining quarters served as unchallenged controls. For a successful challenge and infection, 3 of 4 consecutive mammary secretion samples had to culture positive for Strep. uberis. Six of the 7 heifers were challenged successfully in both infused quarters with a mean dose of 1,080 cfu; once confirmed, infections were treated with a one-time infusion of nonlactating cow therapy. Before challenge, mammary secretion leukocyte counts averaged 8.4×10(6)/mL in all quarters. At 24h after challenge, leukocyte count increased to 18.4×10(6)/mL in challenged quarters, peaking on d 5 at 24.3×10(6)/mL; unchallenged quarters remained at ≤10.4×10(6)/mL, but increased to 15.2×10(6)/mL on d 7 and then decreased. Before challenge, macrophages predominated (81%) in mammary secretions followed by lymphocytes (15.3%) and neutrophils (3.7%). By 24h after challenge, neutrophils increased in challenged quarters and predominated for the duration of the trial (65.3 to 70%), whereas macrophages predominated in unchallenged control quarters (65.2 to 75.2%). The challenge model was successful in establishing Strep. uberis IMI in 85.7% of animals, and IMI were controlled (100% cure) by administering nonlactating cow therapy. All heifers calved free of IMI and antimicrobial residues, with milk production similar to that of herd mates and with somatic cell counts (SCC) <200,000 cells/mL.

Use of commercial somatic cell counters to quantify somatic cells in non-lactating bovine mammary gland secretions.

Measurement of the somatic cell count (SCC) in milk is commonly used to detect mastitis in lactating dairy cows. Many techniques and tools have been developed and adapted to quantify milk SCC, but few tools have been evaluated in their ability to enumerate somatic cells in non-lactating bovine mammary secretions. This limits the tools available for detecting mastitis in non-lactating animals. The objective of these studies was to evaluate methods of somatic cell quantification, originally developed for milk, in their ability to quantify the SCC in non-lactating bovine mammary secretions when compared to the gold standard microscopic quantification method. Two experiments were conducted. In a first experiment, 222 mammary secretions were collected and diluted 1:10 with PBS. Cells in these suspensions were quantified microscopically and with a DeLaval Cell Counter. Microscopic SCC (MSCC) ranged from 1.9 × 106 to 259.5 × 106 cells/mL while DeLaval Cell Counter SCC (DSCC) ranged from 1.8 × 106 to 27.0 × 106 cells/mL; a measurement of agreement between the 2 measures, based on the Lin's Concordance Correlation Coefficient (CCC) suggested moderate agreement between measures (CCC = 0.60). In a second experiment 72 mammary secretions were collected and diluted 1:50 in PBS. Somatic cells in these suspensions were quantified microscopically, with a DeLaval Cell Counter, and by a DHIA laboratory using a Fossomatic™ FC. MSCC ranged from 1.6 to 47.5 × 106 cells/mL, DSCC ranged from 1.0 to 35.7 × 106 cells/mL, and Fossomatic SCC (FMSCC) ranged from 1.6 to 46.7 × 106 cells/mL. CCCs of 0.81 and 0.88 resulted when DSCC and FMSCC were paired with the MSCC, respectively. The results of this work indicate that a significantly greater concentration of somatic cells exist in non-lactating mammary secretions and dilution of these mammary secretions influences accuracy of SCC estimates. Future studies seeking to quantify somatic cells in mammary secretions from non-lactating cows should identify the most appropriate dilution factors specific to each method of measure, given that these two factors will influence the accuracy of SCC estimates. Development of a standardized approach for quantifying somatic cells in non-lactating dairy animals such as heifers and cows, via a rapid automated counter, can allow for the detection of mastitis in non-lactating dairy animals.

Effects of an immunomodulatory feed additive on intramammary infection prevalence and somatic cell counts in a dairy herd experiencing major health issues.

A trial was conducted to determine if feeding OmniGen-AF® (OG) to 22 late lactation cows 60 days prior to and during the early dry period, a time of increased susceptibility to mastitis, could reduce disease incidence in a dairy herd experiencing major health issues. Treated cows (n = 11) consumed a ration containing OG [9 g/100 kg of body weight/day] beginning 60 days before dry-off, during the dry period, and through 30 days in milk (DIM). Control cows received the same ration during the dry period through 30 DIM only. Body weights, body condition scores (BCS), intramammary infection (IMI) prevalence, new IMI rates, somatic cell counts (SCC), milk yield, and adverse health events were measured. No differences were found between treatments for body weight or BCS. Adverse health event data at calving showed no differences between treatments except for percentage of cows with hyperketonemia, which was lower among treated cows (63.6% vs 100%). Prevalence of IMI from calving through 30 DIM for treated cows (6.1%) was lower than controls (11.05%); likewise, new IMI rate during this time for treated cows (0.61%) was lower than controls (5.81%). The SCC from calving through 30 DIM for treated cows (215,000/ml) was lower than controls (493,000/ml). Average production/day at the first DHIA test (~33 DIM) showed that treated cows produced more milk (39.9 kg) than controls (35.34 kg). In conclusion, feeding OG 60 days prior to dry-off reduced hyperketonemia and mastitis, lowered SCC, and numerically increased milk yield in a dairy herd experiencing major health issues.

An immunomodulatory feed additive enhances in vitro viral vaccine recall antigen responses in dairy heifers.

Enhancing immunological responses to vaccination is an important goal in many herd health management systems. OmniGen-AF®(OG) is an immunomodulatory feed additive that has been shown to enhance innate immune function in ruminants and its effects on adaptive immunity require additional study. The objective of this study was to evaluate post-vaccine antibody titers and circulating cellular memory development in heifers fed OG and administered a commercially available modified-live bovine respiratory disease (BRD) vaccine. Twenty-four Holstein heifers were assigned to one of two diets for 170 days: Control TMR (CON; n = 11), or TMR plus OG (TRT; 9 g/100 kg BW/day; n = 13). Samples for hematology, serology, and cellular assays were collected on D-110, 0, 21, 42, and 60 of the trial. Heifers were administered two priming doses of a modified-live BRD vaccine, with a third dose given on D0. There were no significant differences in total WBC and absolute number or the percentage of circulating lymphocytes, monocytes, neutrophils, RBC, or platelets on D-110 through D21. On D42 and D60, CON had significantly higher numbers of lymphocytes. On D0, mean serum neutralizing (SN) titer to BHV-1 was significantly higher for CON compared to TRT. SN titers were not significantly different between CON and TRT at any other time point for BHV-1, BVDV type 1, or BVDV type 2. TRT mounted a significantly stronger recall proliferative response to 0.5 multiplicity of infection (MOI) of BHV-1, BVDV type 1 and BVDV type 2 on D42 and D60; 0.25 MOI of BVDV type 1 on D21 and D42; and 0.25 MOI BVDV type 2 on D42 compared to CON. IL-4 production induced by 0.5 and 1.0 MOI BHV-1 (D42 and D60); 0.25 MOI of BVDV type 1 (D21); and 0.25 and 0.5 MOI of BVDV type 2 (D60) were significantly higher for TRT than CON. IL-17 production induced by 0.25 MOI of BVDV type 1 was significantly higher on D60 for TRT compared to CON. IFN-gamma and IL-10 were not significantly different between treatments. These data indicate feeding OG has a beneficial effect on responses to vaccine antigens in Holstein dairy heifers.

Curli production and genetic relationships among Escherichia coli from cases of bovine mastitis.

Curli are adhesive surface structures produced by some Escherichia coli and Salmonella strains that bind host proteins and activate inflammatory mediators. In this study, 61 E. coli isolates from 36 clinical cases of bovine mastitis were characterized using enterobacterial repetitive intergenic consensus-PCR and screened for their ability to produce curli. Effect of curli production on case recovery, based on a return to precase milk yield, was investigated for a subset of 43 isolates from 20 quarters of 19 cows. Thirty-five (57%) of 61 isolates were curli positive. Fifty-eight of the 61 isolates clustered into 2 clonal groups at 52% genetic similarity. Genetically diverse E. coli isolates were simultaneously cultured from individual cases. Twenty-three isolates from 13 cows were clustered in clonal group I, of which 5 cases (38%) were curli positive; 35 isolates from 22 cows were clustered in clonal group II, of which 15 cases (68%) were curli positive. No association was found between genetic similarity and phenotypic curli expression of isolates from cows with clinical E. coli mastitis cases. Phenotypic curli expression in isolates did not affect recovery of cows' milk yield to premastitis production levels.

Ultrastructural and biochemical response of rat mammary epithelial cells to vinblastine sulfate.

Effects of vinblastine on cell ultrastructure and synthesis and secretion of proteins were studied in rat mammary epithelial cells. At high concentrations, this alkaloid induced drastic structural alterations in secretory epithelia both in vivo and in vitro including cytoplasmic swelling, depolarization and vesiculation of rough endoplasmic reticulum. At lower concentrations, vinblastine induced structural changes similar to those induced by colchicine using the same system. Secretory vesicles accumulated in the cytoplasm, there was hypertrophy of the rough endoplasmic reticulum and portions of the rough endoplasmic reticulum became vesiculated and engorged with electron-dense material. Microtubules were absent but paracrystalline structures were frequently observed associated with Golgi components. In vitro biochemical studies with dissociated alveoli demonstrated that vinblastine inhibited both the synthesis and secretion of protein. Thus the ultrastructural effects of vinblastine in mammary epithelia are similar to those induced by other alkaloids that affect microtubule integrity, but in contrast to colchicine, this alkaloid has a pronounced effect on protein synthesis.

Growth patterns and histochemical characterization of bovine mammary corpora amylacea.

Corpora amylacea in bovine mammary tissue were quantified across a range of size differentials for histochemical properties, lactation age, and lactation stage, in an attempt to characterize amyloid nucleation and growth. At all size classifications, corpora stained positively for amyloid, calcium deposits, and glycoprotein, while staining negatively for mucopolysaccharides. Prevalence of corpora amylacea among the size differentials was unrelated to age of lactating animals, although no corpora were observed in quarters of primiparous heifers at parturition. Corpora amylacea were most abundant during the later stages of lactation for all size differentials, and least abundant during late involution and early lactation. The majority of corpora were observed in alveolar lumens at all stages of lactation. Our results suggest that corpora amylacea development is not restricted to a particular stage of lactation, although their nucleation appears to occur within the alveolar lumens. Gradual increases in both size and numbers of corpora from parturition to late lactation suggest that development of these structures accelerates as lactation progresses. Morphological relationships between corpora amylacea and mammary parenchymal tissue during the later stages of lactation suggest that these structures may have a role in the involutionary process. These findings provide the foundation for additional immunocytochemical techniques to determine the origin of amyloid fibril components.

Cytological effects of hormones and plasma on bovine mammary tissue in vitro.

Mammary explants from two heifers pretreated with oestradiol and progesterone were cultured for 96 h with various combinations of hormones and calf plasma to identify the complement causing mammary tissue development in vitro in the cow. Initial tissue histology, determined by quantitative morphological analysis, was maintained by incubation with insulin, cortisol, oestrogen and progesterone; enlarged lumina were observed after treatment with insulin and cortisol. Lactogenesis was induced in vitro by insulin, cortisol and prolactin, enhanced by adding oestrogen and progesterone at the doses used here and further stimulated by the addition of plasma. The most highly developed mammary alveoli were characterized by an increased luminal area with lipid and stainable secretion, epithelia with large lipid droplets in the apical cytoplasm and a limited stromal area. In a second experiment, samples of plasma were collected from two cows, successfully induced to lactate, on days 6, 15, 17, 19 and 21 after treatment with oestradiol and progesterone. Mammary gland explants from five heifers pretreated with oestradiol and progesterone in vivo were cultured with insulin and cortisol plus these plasma samples (30%, v/v) to test for changes in lactogenic activity. All plasma samples were found equally beneficial in promoting tissue differentiation. These experiments show that low concentrations of ovarian steroids synergize with prolactin at the level of the mammary epitherlium and suggest that other plasma components aid the development of bovine mammary epithelium in vitro.

Origin, fate, and properties of multinucleated giant cells and their association with milk-synthesizing tissues of the bovine mammary gland.

Histological study of multinucleated giant cells (MGC) in bovine mammary glands demonstrated that the cells predominated during mammary involution. Concentrations of MGC increased from the early to later stages of involution, and were more prevalent in older cows. There was no association between infection status and prevalence of MGC. A study of the relationship between MGC and other components of the mammary parenchyma indicated that these cells formed in response to substances that accumulated in mammary secretions during involution. MGC appeared to play a role in resorption of insoluble milk constituents, and removal of amyloid and effete macrophages.

A comparison of the STAPH-Ident and STAPH-Trac systems to conventional methods in the identification of staphylococci isolated from bovine udders.

The STAPH-Ident and STAPH-Trac systems (Analytab Products, Plainview, N.Y.) were compared to conventional methods for identification of staphylococci isolated from bovine udders. The STAPH-Ident system identified 80.5% of isolates correctly. An additional 7.6% of Staphylococcus hyicus strains were delineated from S. epidermidis by characterizing acetoin and pigment production. Final accuracy of the STAPH-Ident system was 88.1%. The STAPH-Trac system identified 66.1% of isolates. Negative phosphatase tests for 42.3% of S. hyicus strains resulted in misidentification as S. simulans. Consequently, only 45.5% of S. hyicus isolates were identified correctly by the STAPH-Trac system. Minor modification of each system would permit accurate, rapid identification of staphylococci isolated from bovine udders.

Evaluation of a rapid inoculum standardization system for antimicrobial susceptibility testing of bacterial isolates from the bovine mammary gland.

A commercially available rapid inoculum standardization system (RISS) was compared to the broth inoculum standardization method (BIS) for antimicrobial susceptibility testing of bacterial isolates from the bovine mammary gland. Overall agreement between RISS and the BIS method was 95.9%. RISS bypasses the 2-8 h incubation period required by BIS, thus providing antimicrobial susceptibility test data in a more timely manner. RISS was determined to be an acceptable alternative method to the BIS method for preparation of standardized inoculum for antimicrobial susceptibility testing of bovine mammary gland isolates.

A case study of Streptococcus group G infection in a dairy herd.

In this paper an outbreak of bovine mastitis in a dairy herd caused by Streptococcus group G is described. Initial identification of the organism as Streptococcus agalactiae was based upon hemolysis, esculin reaction, and CAMP reaction observed on blood agar used for bulk milk analysis. Initial therapy with a penicillin-containing, lactating cow product cured 24.4% of all streptococcal infections. Definitive serogrouping by coagglutination determined that the majority of infections were due to a weakly-hemolytic, esculin-negative Streptococcus group G. Treatment with a cephalosporin, lactating cow product was only moderately successful (54.9%). Dry-cow therapy with 300 mg cephalosporin eliminated 69.5% of refractory infections. Animals remaining infected following dry-cow therapy were culled. Histopathological study of parenchymal tissue in the lower portion of infected quarters revealed mild damage and slight involutionary changes, whereas, deep parenchymal areas appeared relatively unaffected.

Tuberculin elicited cellular immune response in the lactating bovine mammary gland vaccinated intramammarily with Mycobacterium bovis.

The development of a local antigen-specific sensitivity was monitored histologically and in secretions of the bovine mammary gland. Three cows in mid-lactation were immunized by injecting 50 microliter of a killed Mycobacterium bovis vaccine into the dorsal secretory tissue of the rear mammary glands; two cows served as unvaccinated controls. Ten weeks after vaccination, all cows were challenged by intramammary infusion of 1.0 microgram tuberculin in 5 ml phosphate-buffered saline (PBS). Three quarters of each cow received tuberculin at 72, 48, and 24 hours before slaughter; a control quarter received PBS at 72 hours. Vaccinated cows exhibited an intense, local cellular reaction to tuberculin in teat-end tissues at all times post infusion; PBS-infused glands were normal. A moderate leukocyte response in parenchymal tissues adjacent to the gland cistern of tuberculin-infused quarters was observed, but deep parenchymal tissues were normal; no effect on milk yield was found. Tuberculin-infused quarters exhibited histological responses in teat cisternal tissues similar to those in delayed-type hypersensitivity reactions. Leukocytic accumulation was primarily macrophages and lymphocytes with few neutrophils. Erythema was observed in the distal half of the cistern, and fibrin deposits were found in subepithelial connective tissues. The epithelium, although distended with leukocytes, was intact and numerous mitotic figures were present. Unvaccinated cows showed no response to challenge. Results demonstrated a marked and specific cellular response in sensitized cows to challenge with tuberculin.

Effect of a milk-derived factor on the inflammatory response to Staphylococcus aureus intramammary infection.

Daily injections of an anti-inflammatory milk-derived factor (MDF) into mice increased resistance to Staphylococcus aureus challenge, and reduced leukocyte infiltration. Intraperitoneal injection of MDF into lactating mice prior to S. aureus intramammary challenge resulted in greater milk secretory activity and less inflammation compared with untreated controls, but had little effect on the number of S. aureus recovered from mammary tissue. Infusion of MDF directly into mouse mammary glands prior to challenge reduced S. aureus recovered after challenge. Incubation of bovine mammary macrophages in medium supplemented with MDF enhanced phagocytosis of opsonized S. aureus. In addition, infusion of 5 mg MDF into uninfected bovine mammary glands 24 h prior to S. aureus challenge resulted in fewer infections (five of ten) than in control quarters (seven of nine). Repeated daily injections of 5 mg MDF into S. aureus-infected quarters increased the percent of mammary neutrophils and decreased the recovery of S. aureus, but did not eliminate infections. Intravenous injection of 8 g MDF into cows resulted in pronounced leukopenia while the accompanying effect on mammary leukocytes was less marked but followed a similar course. Results suggest that the use of MDF in mice enhanced resistance to experimental infection and was beneficial in maintaining mammary secretory activity and reducing inflammation after bacterial challenge. In the cow, MDF promoted phagocytosis in vitro and was effective against challenge when infused intramammarily.

Electron microscopic study of leucocytic infiltration of the mammary teat duct during infection with Staphylococcus aureus.

Leucocytic response to Staphylococcus aureus infection was observed in the bovine mammary teat duct using transmission electron microscopy. Leucocytes migrating across the stratified squamous epithelium were observed in close association with areas colonised by cocci. Leucocytes gained access across the epithelium to the teat duct lumen by: passage as luminal cells desquamated; migration through degenerate cell cytoplasm; and penetration of cell junctions. The results provide evidence of marked leucocytic infiltration into ductal tissue which may participate in the cellular response to mastitis.

Prevalence and effects of intramammary infection in beef cows.

Prevalence and effects of intramammary infection in 322 beef cows was determined during three calving intervals. Intramammary infection was confirmed in 37% of cows and 18.1% of quarters. Coagulase-positive staphylococci accounted for 17.9% of infections with Staphylococcus aureus isolated from 7.1% of cows. Coagulase-negative staphylococci and micrococci accounted for the remainder of infectious organisms. Butterfat and total protein levels were reduced 27.3 (P less than .05) and 25.5% (P less than .01), respectively, in milk from quarters infected with S. aureus. Somatic cell counts were elevated (P less than .001) with 3,827 X 10(3) cells/ml for S. aureus-infected quarters as compared with 555 X 10(3) cells/ml for uninfected quarters. Somatic cell counts were negatively correlated with 210-d calf weaning weights. Staphylococcus aureus-infected cows weaned calves weighing 19.1 kg less (P less than .01) than uninfected cows. At a present market value of $1.65/kg, economic losses were placed at $31.43/calf from cows infected with S. aureus in one or more quarters.

Response of heifer mammary gland macrophages and neutrophils to interferon-gamma stimulation in vitro.

The phagocytic and killing abilities of heifer mammary gland macrophages (M phi) and neutrophils were evaluated after exposure to recombinant bovine interferon-gamma (rBoIFN-gamma) stimulation in vitro. Macrophages or neutrophils were cultured for 2 h with 0, 10(2), 10(4) and 10(5) units rBoIFN-gamma/mL. Phagocytosis assays were performed by incubation with Staphylococcus aureus at a leukocyte:bacteria ratio of 1:10. After 45 min, cells were stained with acridine-orange and phagocytic and killing abilities were determined. Although rBoIFN-gamma had no effect on M phi phagocytic activity, neutrophil phagocytic activity after incubation in 10(4) units rBoIFN-gamma (41.62%) was significantly higher than 0 (25.24%) or 10(2) units rBoIFN-gamma (24.73%). Neutrophil and M Phi killing abilities were not affected by any dose of rBoIFN-gamma. Results suggested that rBoIFN-gamma promoted neutrophil phagocytic activity, but did not affect neutrophil killing or overall M phi function in vitro.