The relative sensitivity of bovine blood monocytes and macrophages isolated from milk to lipopolysaccharide, with respect to interleukin 1 (IL-1) production, was evaluated. Addition of lipopolysaccharide (0 to 30 microgram/ml) to theculture medium resulted in increases in secreted and intra-cellular IL-1 activity for monocytes and milk macrophages, with maximal stimulation achieved at 30 microgram oflipopolysaccharide/ml of medium. At this concentration of lipopolysaccharide, monocytes released 76% of the total IL-1, whereas milk macrophages released only 26% of the total IL-1 produced within the cell. Secretion of a small quantity of IL-1 was a common property of macrophages isolated from healthy and mastitic quarters. We concluded that limited secretion of IL-1 may render the milk macrophages less efficient in promoting lymphocyte activation.

Polymorphonuclear cells have a critical role in the pathogenesis of bovine mastitis. We have documented that experimentally induced Staphylococcus aureus mastitis is associated with cyclic increase and decrease in the quantity of viable bacteria shed in the milk. Concomitant with this cycling of bacteria is an inverse cycling of the hosts cells within the milk. Such somatic cells were determined to be greater than or equal to 95% polymorphonuclear cells. The quality of these cells was evaluated by measuring their relative efficiency of bacterial killing and phagocytosis at various times during an infection. Host polymorphonuclear cells had as much as 10,000-fold variation in the bactericidal failure rate for staphylococci during cell cycling. The most efficient bactericidal effect was observed at or near the peak of the somatic cell count (SCC). The ability of these cycling cells to ingest fluorescent beads was also quantitated by use of flow cytometry. The percentage of phagocytic polymorphonuclear cells that ingested fluorescent latex beads ranged from 15 to 80% of the total cell population during cell cycling, and tended to be optimal at or near peak SCC. In addition, the average number of beads ingested varied between 1 and 2 particles/polymorphonuclear cell, with as many as 17% of the phagocytic cells ingesting 4 or more beads at maximal efficiency. Polymorphonuclear cells from quarters infected with S aureus varied quantitatively (total SCC) and qualitatively (bactericidal activity and phagocytic ability) during the course of an infection. Not only is the quantity of host's phagocytic cells in the mammary gland central to the defense mechanism against infection, but the biological activation state appears to be equally important. The role of these cells in the pathogenesis of a cycling infection is presented in a model to explain the cyclic nature of mastitis.

Bovine whey samples were evaluated by use of lymphocyte-transformation tests to determine their effect on lymphocyte blastogenesis. Whey samples from mammary glands with clinical mastitis strongly inhibited DNA synthesis and blastogenesis in lymphocytes stimulated with mitogens or dividing because of bovine leukemia virus infection. Whey samples from apparently healthy glands either did not inhibit lymphocyte DNA synthesis or inhibited it to a lesser degree than did whey from mastitic glands. Degree of inhibition was dose-dependent. The molecules causing inhibition were noncytotoxic and underwent minimal binding to the lymphocytes. Inhibitory molecules were susceptible to various proteolytic and glycolytic enzymes, indicating a glycoprotein-like structure. Whey inhibited incorporation of thymidine if it was in the cell cultures during the early stages of stimulation. Incubation of lymphocytes in whey that inhibited thymidine incorporation did not affect DNA synthesis in subsequent culturing of the same cells without whey. Degree of inhibition was affected by the method of whey preparation.

Naturally acquired gram-negative bacterial intramammary infections (n = 160) were studied in 99 cows over a 2-year period. Escherichia coli, Klebsiella spp, Serratia spp, Enterobacter spp, and unidentified gram-negative bacteria were isolated from 28.8, 39.4, 9.4, 5.0, and 11.2%, respectively, of infected mammary glands. A majority (61%) of intramammary infections were first detected during the nonlactating period. Gram-negative bacteria isolated during the first half of the nonlactating period were predominantly Klebsiella spp, Serratia spp, and Enterobacter spp. Onset of E coli intramammary infections was more prevalent during the second half of the nonlactating period and during the first 7 days of lactation. The majority (59%) of infections were <28 days in duration, but Klebsiella spp and Serratia spp infections were of significantly (P <0.05) greater duration than infections with E coli. The greatest percentage (47%) of gram-negative bacterial intramammary infections were first detected during the summer.

Milk samples were collected at onset of 508 episodes of clinical mastitis on a 1,700-cow dairy farm in Michigan. Daily milk production and disease events were recorded for all cows in the herd. Despite statistical association with severity of mastitis, this association was too weak for N-acetyl-beta-D-glucosaminidase (NAGase) activity to be of great value as a prognostic test for clinical mastitis. High milk NAGase activity was significantly (P < 0.0001) associated with: increased duration of treatment; increased duration of clinical signs of mastitis; decreased daily milk production; and increased risk of the cow being culled because of mastitis. The NAGase value was combined with days in milk production, baseline milk production before mastitis onset, parity, and season of onset to predict the outcome of clinical cases as measured by the first 3 aforementioned variables. Statistical models explained little of the variability among cows in duration of treatment (R2 = 0.11), duration of clinical signs of infection (R2 = 0.11), and milk production change (R2 = 0.09).

The type of plasminogen activator (PA) produced by bovine milk macrophages has been determined. Macrophages produce a PA protein with molecular weight of 28,000 and isoelectic point of 8.5, and with enzymatic activity independent of fibrin. These characteristics are identical to those reported for bovine urokinase-PA. Although blood monocytes and milk macrophages produce PA after stimulation with lipopolysaccharide, mammary macrophages are clearly limited in their ability to release PA. At maximal stimulation, 78% of the PA produced by milk macrophages remained cell-associated. In marked contrast, blood monocytes released 76% of the PA produced into the culture medium. Macrophages isolated from mastitic quarters produced higher (2.5 times) amounts of PA, compared with those produced by macrophages isolated from healthy quarters. However, in both cases, macrophages were unable to secrete the protein already produced. The limited PA secretion by milk macrophages might be a residual function of a differentiated macrophage population.