Six primiparous Holstein cows were fed a Se-deficient diet, beginning at least 90 days before their first calving, and 6 other primiparous cows were given the same diet plus a supplement of 2 mg of Se/cow/d as sodium selenite. All cows were fed their diets for the duration of the experimental period. One uninfected quarter of each cow was injected with 25 microgram of Escherichia coli endotoxin at postpartum week 5. Leukocytes were isolated by centrifugation from milk collected at postinjection hour 16. Isolated cells were 92 +/- 3% neutrophils and were incubated with Staphylococcus aureus or E coli in a 1:300 ratio. Phagocytosis and intracellular killing by neutrophils were assessed after 0, 30, 60, and 90 minutes by a fluorochrome assay, using acridine orange. Viability of neutrophils was assessed by use of trypan blue. Superoxide anion production and hydrogen peroxide production by neutrophils also were determined. Cows fed Se-deficient diets had significantly (P < 0.05) lower blood Se concentration and blood glutathione peroxidase activity than cows fed Se-supplemented diets. Selenium status had no effect on the phagocytic capacity of neutrophils. Neutrophils obtained from cows fed Se-supplemented diets killed a significantly (P < 0.05) higher percentage of ingested bacteria than did neutrophils from cows fed the Se-deficient diet. Viability was significantly (P < 0.05) reduced by incubation with S aureus in neutrophils from both groups of cows, with neutrophils from Se-deficient cows having lower viability. Superoxide anion production did not differ significantly between neutrophils from the 2 groups, but extracellular hydrogen peroxide concentration was significantly (P < 0.05) higher in neutrophils harvested from milk of cows fed the Se-deficient diet.

Streptococcal species isolated from dairy cows with clinical mastitis were obtained from mastitis research workers in Florida, Louisiana, New York, Vermont, Washington, and West Virginia. Seventy-one streptococcal isolates were tested, including 39 strains of Streptococcus agalactiae, 21 strains of S dysgalactiae, and 11 strains of S uberis. The minimal inhibitory concentration of erythromycin, lincomycin, oxytetracycline, penicillin, spectinomycin, streptomycin, and tetracycline was determined for each isolate. Differences were not detected among strains with respect to geographic origin. None of the strains was resistant to penicillin. Lincomycin was the next most effective antimicrobial, with only 2 resistant strains of each streptococcal species. There were no differences among the streptococcal species with respect to resistance to either penicillin or lincomycin. Streptococcus uberis was more likely to be resistant to erythromycin than were S agalactiae and S dysgalactiae (P < 0.02). Streptococcus agalactiae and S uberis had similar distributions for resistance to oxytetracycline, tetracycline, spectinomycin, and streptomycin. Strains of S dysgalactiae were more likely to have intermediate resistance to oxytetracycline and streptomycin than were strains of S agalactiae and S uberis, which were highly resistant to oxytetracycline and streptomycin (P < 0.001). Differences were not detected among the streptococcal species with respect to resistance to spectinomycin. Resistance to multiple antimicrobials was observed in all streptococcal species tested. Although S dysgalactiae appeared to have a greater percentage of strains (73%) that were resistant to multiple antimicrobials than did S agalactiae (31%) or S uberis (45%), differences were not statistically significant.

Streptococcal species isolated from dairy cows with clinical mastitis were obtained from mastitis research workers in Florida, Louisiana, New York, Vermont, Washington, and West Virginia. Seventy-one streptococcal isolates were tested, including 39 strains of Streptococcus agalactiae, 21 strains of S dysgalactiae, and 11 strains of S uberis. The minimal inhibitory concentration of erythromycin, lincomycin, oxytetracycline, penicillin, spectinomycin, streptomycin, and tetracycline was determined for each isolate. Differences were not detected among strains with respect to geographic origin. None of the strains was resistant to penicillin. Lincomycin was the next most effective antimicrobial, with only 2 resistant strains of each streptococcal species. There were no differences among the streptococcal species with respect to resistance to either penicillin or lincomycin. Streptococcus uberis was more likely to be resistant to erythromycin than were S agalactiae and S dysgalactiae (P < 0.02). Streptococcus agalactiae and S uberis had similar distributions for resistance to oxytetracycline, tetracycline, spectinomycin, and streptomycin. Strains of S dysgalactiae were more likely to have intermediate resistance to oxytetracycline and streptomycin than were strains of S agalactiae and S uberis, which were highly resistant to oxytetracycline and streptomycin (P < 0.001). Differences were not detected among the streptococcal species with respect to resistance to spectinomycin. Resistance to multiple antimicrobials was observed in all streptococcal species tested. Although S dysgalactiae appeared to have a greater percentage of strains (73%) that were resistant to multiple antimicrobials than did S agalactiae (31%) or S uberis (45%), differences were not statistically significant.

Detection of capsular polysaccharide (CP) in milk of cows with natural intramammary infection caused by Staphylococcus aureus was attempted. Five quarters of 5 cows harboring S aureus strains that produce type-8 CP were selected. Using an ELISA with a monoclonal antibody, type-8 CP was not detected in extracts prepared from fresh milk collected aseptically. By contrast, CP was easily detectable after incubation of infected milk at 38 C for 20 hours. Quantitation of CP in extracts from incubated milk samples by use of ELISA indicated a great variation of CP expression by strains. Although an incubation step was necessary to detect CP, results of the study indicate that CP may be expressed in vivo during intramammary infection caused by S aureus.

Ten Holstein cows were fed a selenium-deficient (SeD) diet containing 0.04 mg of Se/kg of dry matter for 3 months before and throughout their first lactation. A selenium-supplemented (SeS) group of 10 cows was fed an additional 2 mg of Se/head/d to increase dietary Se concentration of the dry matter to approximately 0.14 mg/kg of body weight. An intracisternal challenge exposure of 40 to 60 colony-forming units (CFU) of Staphylococcus aureus was administered into 1 or 2 quarters of the udder of each trial cow at about the twenty-second week of lactation. Blood Se concentration (microgram/ml +/ - SEM) at the time of challenge exposure was 0.035 +/- 0.002 in SeD and 0.139 +/- 0.006 in SeS cows. Infections were established in 14/16 of the challenge-exposed quarters in SeD and 16/19 of the challenge-exposed quarters in SeS cows. The infection in 1 quarter of each Se group cleared without treatment by the end of the 8-week trial period. Log10 peak bacterial concentrations in milk from infected SeD quarters (5.04 +/- 0.25 CFU/ml) were higher (P < 0.05) than those of infected SeS quarters (4.40 +/- 0.12 CFU/ml). Log10 peak somatic cell count (SCC) in milk from infected SeD quarters (7.18 +/- 0.08 cells/ml) did not differ from that of SeS quarters (7.17 +/- 0.05 cells/ml). Peak bacterial concentrations were attained sooner (P < 0.05) in SeD quarters (9.5 +/- 4.0 days) than in SeS quarters (20.7 +/- 3.1 days). Similarly, peak SCC were reached earlier (P < 0.05) in SeD (4.3 +/- 1.1 days) than in SeS quarters (13.3 +/- 3.8 days). The Se groups did not differ significantly with respect to peak milk concentrations of bovine serum albumin or IgG1. Throughout the 8-week trial, the Se groups did not differ significantly in milk bacterial concentration, SCC, bovine serum albumin, or IgG1. Selenium status did not affect the percentage of challenge exposures resulting in infection, duration, or severity of experimentally induced S aureus mastitis.

The binding of bovine complement S protein (vitronectin) to Streptococcus dysgalactiae isolates from cattle with mastitis and the S protein's role in streptococcal adherence to bovine epithelial cells were investigated. All 25 clinical isolates of S dysgalactiae interacted with bovine S protein. None of the other streptococcal species tested bound to bovine S protein. The S protein-binding sites were saturable and highly sensitive to trypsin. The binding of bovine S protein to S dysgalactiae isolates was specific and could not be inhibited by other plasma proteins, such as fibronectin, albumin, fibrinogen, alpha 2-macroglobulin, or IgG. Similarly, streptococcal binding of bovine S protein was not influenced by the synthetic peptide Gly-Arg-Gly-Asp-Ser, which constituted the host cell attachment sequence of S protein. In adherence experiments, prior binding of bovine S protein to S dysgalactiae enhanced streptococcal adherence to bovine epithelial cells. The enhancing effects by bovine S protein were abolished when the respective binding sites on the streptococci were digested by trypsin. Thus, bovine S protein could be an important mediator of adherence of S dysgalactiae to bovine epithelial cells.

Alterations in the various leukocyte populations in milk, blood, and mammary lymph were studied by use of the flow cytometric method during acute mastitis episodes induced by endotoxin infusion (50 microgram of lipopolysaccharide of Salmonella typhimurium SH 4809) via the teat canal. Lymph samples were collected via a semipermanent catheter from an afferent duct to the supramammary lymph node. Milk somatic cell count increased at 4 hours after infusion of endotoxin. Neutrophils were the predominant cell population for up to 59 hours after infusion. Numbers of lymphocytes and monocytes-macrophages in milk also increased after the endotoxin infusion. The total cell count in milk started to decrease during the third postinfusion day and returned to preinfusion values during the fourth day. Lymphocyte numbers remained high for about 1 week after the infusion, and lymphocytes were the predominant cell population between postinfusion days 4 and 8. Total blood leukocyte count decreased during the first 6 hours after infusion, followed by an increase until postinfusion hour 31. The proportion of neutrophils in blood increased during the first day, whereas that of lymphocytes decreased. Lymph flow rate and leukocyte numbers in lymph increased after endotoxin infusion. The proportion of neutrophils in the lymph increased during the first 6 hours, whereas that of lymphocytes decreased. After postinfusion hour 6, the inverse course of events was seen.

Six primiparous Holstein cows were fed a Se-deficient diet, beginning at least 90 days before their first calving, and 6 other primiparous cows were given the same diet plus a supplement of 2 mg of Se/cow/d as sodium selenite. All cows were fed their diets for the duration of the experimental period. One uninfected quarter of each cow was injected with 25 microgram of Escherichia coli endotoxin at postpartum week 5. Leukocytes were isolated by centrifugation from milk collected at postinjection hour 16. Isolated cells were 92 +/- 3% neutrophils and were incubated with Staphylococcus aureus or E coli in a 1:300 ratio. Phagocytosis and intracellular killing by neutrophils were assessed after 0, 30, 60, and 90 minutes by a fluorochrome assay, using acridine orange. Viability of neutrophils was assessed by use of trypan blue. Superoxide anion production and hydrogen peroxide production by neutrophils also were determined. Cows fed Se-deficient diets had significantly (P < 0.05) lower blood Se concentration and blood glutathione peroxidase activity than cows fed Se-supplemented diets. Selenium status had no effect on the phagocytic capacity of neutrophils. Neutrophils obtained from cows fed Se-supplemented diets killed a significantly (P < 0.05) higher percentage of ingested bacteria than did neutrophils from cows fed the Se-deficient diet. Viability was significantly (P < 0.05) reduced by incubation with S aureus in neutrophils from both groups of cows, with neutrophils from Se-deficient cows having lower viability. Superoxide anion production did not differ significantly between neutrophils from the 2 groups, but extracellular hydrogen peroxide concentration was significantly (P < 0.05) higher in neutrophils harvested from milk of cows fed the Se-deficient diet.

The activity of leukocytes, determined by chemiluminescence (CL) emission, was compared with the somatic cell count (SCC) in 4,883 quarter-milk samples from 132 dairy cows. The presence of bacteria was determined by bacteriologic culture of samples in which SCC and CL were high. Chemiluminescence was measured with an automated illuminometer system at 37 C after separating the leukocytes from milk by allowing them to adhere to cotton-wool swabs. Chemiluminescence emission was induced by opsonized zymosan and enhanced by luminol. After luminol and zymosan were added to the measuring vials containing the swabs, CL emission increased rapidly, reaching its maximum usually at about 15 minutes of reaction time, and decreasing slowly thereafter. In general, good correlation was found between CL and SCC (r = 0.876; P less than or equal to 0.001; n = 4,883). Even milk samples with low SCC gave reliably measurable CL signals. Minor pathogens in the milk caused about a sevenfold increase in both SCC and CL, whereas major pathogens caused 14- and 25-fold increases in SCC and CL, respectively. The diagnostic situation that requires both sensitivity and specificity to be at least 90% was attained only by the CL assay for major pathogens. These results suggest that the measurement of milk leukocyte activity by CL assay applies well to the diagnosis of mastitis, and has the potential to become a large-scale laboratory test, as well as a simple cowside test.