Objective-To evaluate the ability of bovine complement to kill a variety of field isolates and laboratory strains of Brucella abortus. Design-The experimental approach was to determine the sensitivity of B abortus isolates to killing by bovine serum, and to document the role of complement in brucellacidal activity. Sample population-Six laboratory isolates and 12 field isolates of B abortus were tested. Procedure-The ability of B abortus to survive exposure to undiluted bovine serum for 2 hours at 37 C was assessed. The role of complement in killing was determined by examining the ability of heat (56 C for 60 minutes) and cobra venom factor to obliterate the activity in serum, and by detecting binding of the ninth component of bovine complement to serum-sensitive target cells. Results-Isolates of B abortus that were resistant to the bactericidal activity of normal bovine serum were revealed. These included field isolates and laboratory strains. Furthermore, the study confirmed earlier reports that bovine serum-mediated killing of B abortus is caused by the complement cascade. Conclusions-Some isolates of B abortus, like other gram-negative bacteria, were resistant to complement-mediated killing. Resistance was associated with smooth colony morphology. Isolates lacking detectable O antigen were serum sensitive.

Bovine immunodeficiency virus (BIV) is prevalent in beef and dairy cattle, yet the mode(s) of BIV transmission are undefined. Using polymerase chain reaction, which specifically targeted a 235-bp, highly conserved region of the BIV pol gene, BIV-infected leukocytes were detected in the blood and milk of BIV-seropositive cows. These data confirm the presence of BIV in milk and identify the potential for lactogenic transmission of this virus.

We used monoclonal antibodies and immunohistologic examination of lymph nodes, to elucidate the pathogenesis of lymphosarcoma induced by, infection with bovine leukemia virus (BLV). The superficial cervical lymph nodes from 3 BLV-infected but apparently healthy sheep and 5 sheep with full-blown lymphosarcoma were examined. We also investigated the integration of bovine leukemia provirus by use of Southern blotting. In lymph nodes from sheep lacking clinical signs of infection, in which the provirus had been integrated at multiple sites in the genome, many large hypertrophic follicles were observed in the cortex. These follicles had germinal centers consisting of CD4+T cells and B cells that expressed surface IgM (sIgM) and major histocompatibility complex (MHC) class-II antigens, but not B cell-specific B2 molecule. The percentage of CD4+T cells in the cortex was significantly (P < 0.05) higher than that of the controls and sheep with lymphosarcoma. In all sheep with lymphosarcoma, the lymph nodes were completely destroyed by proliferating neoplastic cells, and in addition, small atrophic follicles, which consisted of normal B-cell marker-positive cells, were seen near the trabecula and the subcapsule. In these instances, neoplastic cells appeared to be a monoclonal population derived from a single CD5- B-cell lineage and to be classified as 2 types, CD5-CD4-CD8-B2+MHC class-II+sIgM+ and CD5-CD4-CD8-B2+MHC class-II+sIgM-. Moreover, CD8+T cells infiltrated diffusely throughout the tumorous lymph nodes apart from the atrophic follicles, and CD4+T cells were observed around atrophic follicles. Both types of T cells were small-size, normal lymphocytes with round and noncleaved nuclei, and were apparently non-neoplastic cells. In fact, after separation by use of a panning method, it seems that, in blood mononuclear cells from BLV-infected sheep without clinical signs of infection, but in lymphosarcomatous stages, the proviral genome was integrated only in B cells and not in T cells. Thus, we conclude that the host's immune response may be still maintained at a lymphosarcomatous stage.

Growth of Pasteurella haemolytica A1 in RPMI 1640 medium containing 0.5% bovine serum albumin (BSA) for 2.5 hours enhanced culture supernatant leukotoxic activity [30,700 +/- 12,900 toxic units/ml, compared with leukotoxic activity of culture supernatants produced in RPMI 1640 medium alone (120 +/- 40 toxic units/ml)]. Gel filtration chromatography of the leukotoxic activity from RPMI 1640 medium supernatants in buffer containing 50 mM NaCl indicated a single leukotoxic activity peak (peak I) eluting near the gel resin molecular mass exclusion limit (estimated molecular mass of approx 8,000 kd). In contrast, culture supernatants produced in RPMI 1640 plus bovine serum albumin medium (RPMI + BSA) had peak I and 2 additional leukotoxic activity peaks (peaks II and III) with estimated molecular mass of approximately 80 and < 30 kd, respectively. All leukotoxic activity peaks were composed of approximately 100-kd molecular mass leukotoxin protomer, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against leukotoxin. Subjecting culture supernatant leukotoxic activity produced in RPMI + BSA to gel filtration chromatography in buffer containing 500 mM NaCl or 6M urea resulted in detection of only a single leukotoxic activity peak with estimated approximate molecular mass of 250 and 800 kd, respectively. These findings suggest that P haemolytica exists as a high molecular mass aggregate with low leukotoxic activity which, in the presence of BSA, partially disaggregates to multiple toxin forms with enhanced leukotoxic activity. Some of these leukotoxin forms interact with dextran-based gel resins at low ionic strength.

Twenty-four 10-month-old Polled Hereford heifers were inoculated sc with live cells of one of the following strains of Brucella abortus: S19 delta 31K (n = 4), S19 delta SOD (n = 4), RB51 (n = 4), and strain 19 (n = 6); controls (n = 6) were given saline solution. Heifers given the deletion mutants S19 delta 31K and S19 delta SOD, and those given strain 19 developed antibody responses to B abortus and cutaneous reactions to brucellin. Heifers given strain RB51 did not develop antibodies that reacted in the standard tube agglutination test, but sera reacted in tests, using an antibody dot-blot assay containing RB51 antigen. The S19 delta 31K and S19 delta SOD strains of B abortus isolated from lymph node tissue after vaccination did not differ genetically from the master stock strain. All heifers were bred naturally at 16 to 17 months of age, and were challenge-exposed intraconjunctivally with virulent B abortus strain 2308 during the fifth month of pregnancy. All vaccinated heifers were protected (ie, none aborted and none had B abortus isolated from their tissues after parturition). Calves born from vaccinated dams were free of B abortus. Antibody responses in heifers after challenge exposure were an indicator of immunity. All 5 control heifers (nonvaccinated) developed serum antibodies after challenge exposure; 3 aborted, and 1 delivered a small, weak calf at 8.5 months of gestation. Thus, live mutant strains of B abortus can induce protective immunity when given at 10 months of age, and strain RB51 is a strong candidate for further testing.

The role of interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor a during endotoxin-induced mastitis in cows was characterized. Six cows had 10 microgram of Escherichia coli lipopolysaccharide infused into 1 mammary gland. Three other cows served as nontreated controls. Within 1.5 to 2.5 hours after infusion, endotoxin caused obvious edema of the mammary gland and increased serum albumin concentration in milk of infused glands 6 times. Milk somatic cell count began to increase 3 to 5 hours after infusion in all treated glands. At 7 hours after infusion, somatic cell counts were increased > 10 times, compared with counts in milk from control cows. Pyrexia of > 1 C developed in only 1 cow, but all treated cows had serum cortisol concentrations > 50 ng/ml in response to endotoxin treatment. High concentrations of IL-1 (10 to 600 U/ml) and IL-6 (2 to 22 U/ml) were detected in milk of infused glands beginning 2.5 to 4 hours after infusion. Endotoxin did not induce detectable amounts of tumor necrosis factor activity in milk or serum. Swelling and mammary gland permeability changes preceded any detectable increase in IL-1 and IL-6 activity, indicating that these clinical signs of inflammation were not mediated by these cytokines. Systemic responses and the leukocytic influx into endotoxin-infused glands developed after or concurrently with initial increases in IL-1 and IL-6 activities in milk. These results suggested that IL-1 and IL-6 may have a role in mammary gland defenses and in the pathophysiologic changes during endotoxin-induced mastitis.

In vitro transferability of penicillin, streptomycin, tetracycline, and erythromycin resistance from coagulase-negative staphylococci to Staphylococcus aureus and among the former species of bovine mammary gland origin was examined by bacterial mating on filters and by mixed-culture matings in broth and in skim milk. One hundred twenty-six (42 each on filter, in broth, and in skim milk) matings were performed among 37 isolates of different Staphylococcus species. Transfer of resistance to penicillin, tetracycline, or erythromycin was not detected. Of 51 matings performed to determine streptomycin-resistance transfer, 9 (3 each on filters, in broth, and skim milk) were successful. Nine strains representing 3 species of coagulase-negative staphylococci were tested as prospective donors of streptomycin resistance. Of these, 2 strains could transfer streptomycin resistance. A double-resistant donor, S hominis, not only transferred its streptomycin resistance to an S chromogenes strain lacking resistance, but also to an S aureus strain already carrying penicillin and tetracycline resistance. The transfer of streptomycin resistance from the donor S hominis, harboring 2 plasmids, to a plasmidless S chromogenes recipient strain was associated with apparent acquisition of the smaller plasmid of the donor by the recipient. The single-resistant donor, S epidermidis 681A, transferred streptomycin resistance to a tetracycline-resistant S aureus recipient. This strain however, failed to transfer its streptomycin resistance to another S aureus, 2 S hyicus, and 1 S xylosus recipient. Frequency of transfer of streptomycin resistance ranged from 1.1 X 10(-5) to 1 X 10(-4). When transfer of resistance was successful, attempts were made to characterize the transfer process. Conjugation appeared to be the mode of streptomycin-resistance transfer. Transfer of resistance between staphylococci of bovine mammary gland origin appears to be fairly uncommon. However, in view of the limitations of the procedures used, additional in vitro and in vivo work is needed to further assess the role of coagulase-negative staphylococci in dissemination of antibiotic resistance.

Neutrophil migration through bovine teat tissues into the teat cistern, after endotoxin infusion into the teat cistern, was determined in vivo by 2 experimental procedures, indium-111 labeling of blood neutrophils, and obtaining multiple biopsy specimens from the teat cistern tissues. In both experiments, the number of leukocytes in the teat cistern flushing samples was continuously measured. A lag phase of approximately 1 hour was required between endotoxin infusion into the teat cistern and the first observed neutrophil accumulation in the teat tissues. The rate of neutrophil accumulation in the teat tissues was highest between postinfusion (pI) hours 1 and 2, and the accumulation process ceased after PI hour 3. Neutrophils migrated toward the epithelium, and intraepithelial neutrophils were observed beginning approximately 2 hours after infusion, which coincided with the first influx of cells into the teat cistern. The cell influx into the teat cistern increased continuously up to PI hour 3, peaked between PI hours 3 and 5, and was close to preinfusion value at PI hour 22. Use of indium-111-labeled neutrophils in the study of the inflammatory process provides a reliable noninvasive method to quantify cell migration in vivo. Use of biopsies allows quantification of the number of cells in different tissue areas, but has the disadvantage of being invasive. These 2 procedures complement each other, and could be of use in future studies of the local inflammatory process.

Sixteen Holstein cows were used to test the effect of postmilking teat treatment on colonization and intramammary infection by Staphylococcus aureus on chapped teats. Treatments were (1) chapping the teat and using 1% I2/10% glycerin postdip solution, (2) 1% I2/10% glycerin postdip solution on nonchapped teats, (3) chapping the teat and using 10% glycerin postdip solution, (4) chapping the teat and not using a postdip solution. All mammary glands were free of S aureus teat skin colonization and intramammary infection at the start of the study. Teats selected for chapping were dipped in 1N NaOH prior to 3 applications of S aureus broth culture; cultures were applied at 12-hour intervals on all teats. Treatments were applied after each milking for 30 days and were initiated after the second broth dip. Teat skin swab specimens and milk samples were collected before treatment application. Teat skin condition was scored daily. Nonchapped teats (treatment 2) did not support skin or orifice colonization by S aureus. Treatment-1 teats healed most rapidly and supported less colonization in skin and orifice than did treatment-3 and -4 teats. Teat skin scores and skin colonization were lower for treatment-3 than treatment-4 teats. A correlation between teat skin colonization and teat skin conditions was found. Two intramammary infections were found in treatment-4 quarters and 1 in a treatment-3 quarter. On the basis of our findings, we concluded that poor teat skin condition will more readily support S aureus colonization, that a dip of 1% I2 with glycerin helped reduce S aureus colonization and was associated with faster healing, and that glycerin in teat dips may be of value in preventing colonization by S aureus and in promoting healing.

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.