Acute phase response elicited by experimental bovine diarrhea virus (BVDV) infection is associated with decreased vitamin D and E status of vitamin-replete preruminant calves.

Studies in young animals have shown an association between vitamin deficiencies and increased risk of infectious disease; however, there is a paucity of information regarding the effect of acute infection on the vitamin status of the vitamin-replete neonate. To characterize the effects of acute infection on vitamin D and E status of the neonate, 6 vitamin-replete preruminant Holstein bull calves were experimentally infected with bovine viral diarrhea virus (BVDV; strain BVDV2-1373). Six mock-inoculated calves served as controls. Sustained pyrexia, leukopenia, and asynchronous increases in serum haptoglobin and serum amyloid A characterized the response of calves to infection with BVDV. Infection was also associated with increased serum IFN-γ, IL-2, and IL-6 concentrations. During the last 8 d of the 14-d postinoculation period, serum 25-hydroxyvitamin D and α-tocopherol concentrations in infected calves decreased by 51 and 82%, respectively. The observed inverse association between vitamin D and E status and serum amyloid A in infected calves suggests that the infection-induced acute phase response contributed to the reduced vitamin status of these animals. Additional studies are necessary to determine if the negative effect of infection on status are unique to this specific infection model or is representative of preruminant calf's response to acute infection. Studies are also needed to characterize mechanisms underlying infection-related changes in vitamin D and E status and to determine whether additional vitamin D or E supplementation during an acute infection diminishes disease severity and duration in the young animal.

Effects of chronic environmental cold on growth, health, and select metabolic and immunologic responses of preruminant calves.

The physiological response of the preruminant calf to sustained exposure to moderate cold has not been studied extensively. Effects of cold on growth performance and health of preruminant calves as well as functional measures of energy metabolism, fat-soluble vitamin, and immune responsiveness were evaluated in the present study. Calves, 3 to 10 d of age, were assigned randomly to cold (n = 14) or warm (n = 15) indoor environments. Temperatures in the cold environment averaged 4.7 degrees C during the study. Frequent wetting of the environment and the calves was used to augment effects of the cold environment. Temperatures in the warm environment averaged 15.5 degrees C during the study. There was no attempt to increase the humidity in the warm environment. Preventative medications or vaccinations that might influence disease resistance were not administered. Nonmedicated milk replacer (20% crude protein and 20% fat fed at 0.45 kg/d) and a nonmedicated starter grain fed ad libitum were fed to all calves. Relative humidity was, on average, almost 10% higher in the cold environment. Warm-environment calves were moderately healthier (i.e., lower respiratory scores) and required less antibiotics. Scour scores, days scouring, and electrolyte costs, however, were unaffected by environmental temperature. Growth rates were comparable in warm and cold environments, although cold-environment calves consumed more starter grain and had lower blood glucose and higher blood nonesterified fatty acid concentrations. The nonesterified fatty acid and glucose values for cold-stressed calves, however, did not differ sufficiently from normal values to categorize these calves as being in a state of negative-energy balance. Levels of fat-soluble vitamin, antibody, tumor necrosis factor-alpha, and haptoglobin were unaffected by sustained exposure to moderate cold. These results support the contention that successful adaptation of the dairy calf to cold is dependent upon the availability of adequate nutrition.

Development of an adult-like cell-mediated immune response in calves after early vaccination with Mycobacterium bovis bacillus Calmette-Guérin.

Effects of neonatal vaccination on antigen-specific cellular and humoral immune responses of dairy calves have not been well described. The purpose of this study was to characterize the ontogeny of the adaptive immune response in calves sensitized to the attenuated strain of Mycobacterium bovis, bacillus Calmette-Guerín. Holstein bull calves were nonvaccinated (n = 6, vaccination controls) or vaccinated subcutaneously (n = 6) with bacillus Calmette-Guerín at 1 and 7 wk of age. Composition and functional capacities of blood mononuclear cell populations from calves were evaluated at 1 (prevaccination), 3, 6, 7, 8, 9, and 12 wk of age. Young adults (nulliparous heifers, n = 4) vaccinated in an identical manner were sampled concurrently to evaluate effects of animal maturity on the development of the adaptive immune response. Responses of nonvaccinated calves to recall antigen (Mycobacterium bovis purified protein derivative) ex vivo and in vivo (i.e., cutaneous delayed-type hypersensitivity) were minimal or nonexistent. Responses of cells from vaccinated calves and young adults to recall antigen, however, were evident as early as wk 2 after primary vaccination. Antigen-induced T cell subset proliferation, and secretion of interferon-gamma, nitric oxide, and tumor necrosis factor-alpha by cells from vaccinated calves were comparable to or greater than responses of vaccinated adults during the 11-wk study. Eleven weeks after primary vaccination, cutaneous responses of vaccinated calves and young adults to intradermal administration of antigen were pronounced and comparable, demonstrating the capacity of the bovine neonate to develop a vigorous cell-mediated immune response in vivo. Antibody responses (i.e., antibody concentrations in sera and in supernatants from antigen-stimulated cultures of blood mononuclear cells) of vaccinated calves, in contrast, were markedly lower than parallel responses of vaccinated adults. In conclusion, these results suggest that the bovine neonate can mount a vigorous, adult-like cell-mediated immune response when vaccinated at an early age.

Use of recombinant ESAT-6:CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis.

Immunological diagnosis of Mycobacterium bovis infection of cattle is often confounded by cross-reactive responses resulting from exposure to other mycobacterial species, especially Mycobacterium avium. Early secretory antigenic target 6 (ESAT-6) and culture filtrate protein 10 (CFP-10) are dominant gamma interferon (IFN-gamma)-inducing antigens of tuberculous mycobacteria, and they are absent from many environmental nontuberculous mycobacteria. Because M. avium exposure is the primary confounding factor in the diagnosis of M. bovis-infected animals, in vitro responses to a recombinant ESAT-6:CFP-10 (rESAT-6:CFP-10) fusion protein by blood leukocytes from cattle naturally exposed to M. avium or experimentally challenged with Mycobacterium avium subsp. avium or Mycobacterium avium subsp. paratuberculosis were compared to responses by M. bovis-infected cattle. Responses to heterogeneous mycobacterial antigens (i.e., purified protein derivatives [PPDs] and whole-cell sonicates [WCSs]) were also evaluated. Tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and nitric oxide responses by M. bovis-infected cattle to rESAT-6:CFP-10 exceeded (P < 0.05) the corresponding responses by cattle naturally sensitized to M. avium. Experimental infection with M. bovis, M. avium, or M. avium subsp. paratuberculosis induced significant (P < 0.05) IFN-gamma and nitric oxide production to WCS and PPD antigens, regardless of the mycobacterial species used for the preparation of the antigen. Responses to homologous crude antigens generally exceeded responses to heterologous antigens. Nitric oxide and IFN-gamma responses to rESAT-6:CFP-10 by blood leukocytes from M. bovis-infected calves exceeded (P < 0.05) the corresponding responses of noninfected, M. avium-infected, and M. avium subsp. paratuberculosis-infected calves. Despite the reported potential for secretion of immunogenic ESAT-6 and CFP-10 proteins by M. avium and M. avium subsp. paratuberculosis, it appears that use of the rESAT-6:CFP-10 fusion protein will be useful for the detection of tuberculous cattle in herds with pre-existing sensitization to M. avium and/or M. avium subsp. paratuberculosis.

Effect of lipopolysaccharide on indices of peripheral and hepatic metabolism in lactating cows.

Four multiparous lactating cows (175 to 220 d in milk [DIM]) were used in a 4 x 4 Latin square design to assess the effects of four doses (0.0, 0.5, 1.0, and 1.5 microg/kg of body weight) of lipopolysaccharide (LPS; Escherichia coli 0111:B4) on performance and plasma metabolite and hormone concentrations. In addition, effects of immune activation on in vitro hepatic metabolic capacity were evaluated in 12 multiparous lactating cows (150 to 220 DIM) infused with 0 (n = 6), 1.0 (n = 4) or 2.0 (n = 2) microg of LPS/kg. Milk production and DMI decreased linearly with LPS dose for 24 h after LPS infusion. Overall mean plasma tumor necrosis factor-alpha, insulin, glucagon, and cortisol concentrations increased linearly with LPS dose, and plasma beta-hydroxybutyrate decreased linearly by dose after LPS infusion. Infusion of LPS decreased the insulin:glucagon molar ratio, but did not affect plasma concentrations of growth hormone, insulin-like growth factor-1, leptin, or L-(+)-lactate. Plasma concentrations of glucose tended to increase initially and subsequently decrease, and there was a quadratic tendency for increased plasma nonesterified fatty acid concentrations after LPS administration. In vitro hepatic capacity for conversion of [1-(14)C]L-(+)-lactate and [1-(14)C]palmitate, but not [1-(14)C]propionate or [1-(14)C]L-alanine, to CO2 increased after LPS administration. Hepatic capacity to convert [1-(14)C]propionate to glucose tended to increase, but neither esterification nor the conversion of palmitate to acid soluble products was altered by LPS. The LPS infusion resulted in significant changes of endocrine mediators responsible for regulation of energy metabolism of lactating cows and tended to alter subsequent in vitro hepatic metabolic capacity.

Diagnostic implications of antigen-induced gamma interferon, nitric oxide, and tumor necrosis factor alpha production by peripheral blood mononuclear cells from Mycobacterium bovis-infected cattle.

Bovine tuberculosis in the United States has proven costly to cattle producers as well as to government regulatory agencies. While in vivo responsiveness to mycobacterial antigens is the current standard for the diagnosis of tuberculosis, in vitro assays are gaining acceptance, especially as ancillary or complementary tests. To evaluate in vitro indices of cellular sensitization, antigen-induced gamma interferon (IFN-gamma), nitric oxide (NO), and tumor necrosis factor alpha (TNF-alpha) responses by blood mononuclear cells from Mycobacterium bovis-infected cattle were quantified and compared. Using an aerosol model of infection, two doses of each of two strains of M. bovis (95-1315 and HC-2045T) were used to induce a range of IFN-gamma, NO, and TNF-alpha responses. Infection-specific increases in NO, but not in IFN-gamma or TNF-alpha, were detected in nonstimulated cultures at 48 h, a finding that is indicative of nonspecific activation and spontaneous release of NO. The infective dose of M. bovis organisms also influenced responses. At 34 days postinfection, IFN-gamma, NO, and TNF-alpha responses in antigen-stimulated cells from cattle receiving 10(5) CFU of M. bovis organisms were greater than responses of cells from cattle infected with 10(3) CFU of M. bovis organisms. The NO response, but not the IFN-gamma and TNF-alpha responses, was influenced by infective strains of M. bovis. The TNF-alpha, NO, and IFN-gamma responses followed similar kinetics, with strong positive associations among the three readouts. Overall, these findings indicate that NO and TNF-alpha, like IFN-gamma, may prove useful as indices for the diagnosis of bovine tuberculosis.

Effects of the mammary gland on functional capacities of blood mononuclear leukocyte populations from periparturient cows.

The composition and functional capacity of peripheral blood mononuclear leukocyte populations from dairy cows are altered substantially during the peripartal period. These changes are associated with a heightened susceptibility of the mammary gland to infection. It has been postulated that the metabolic demands associated with lactogenesis may impact negatively leukocyte function during the periparturient period. In the present study, serum immunoglobulin G1 concentration and functional capacities of peripheral blood mononuclear leukocytes from intact (n = 6) and mastectomized (n = 6) periparturient Jersey cows were evaluated and compared. Cell function assessments included lymphocyte proliferation, immunoglobulin M secretion, and interferon-gamma secretion by unstimulated and pokeweed mitogen stimulated mononuclear leukocytes. Data were summarized as mean responses for 5-d periods beginning 21 d prepartum and concluding at 19 d postpartum. The progressive decrease in serum immunoglobulin G in intact but not mastectomized cows before parturition likely was attributable to the selective uptake of this isotype by the mammary gland. Lymphocyte proliferation and secretion of interferon-gamma and polyclonal IgM by mitogen-stimulated leukocytes from intact cows decreased during the 15-d period before calving, reaching a nadir at 0 to 4 d postpartum. From 5 to 19 d postpartum, these functions often were comparable to those observed 2 to 3 wk prepartum. Functions of leukocytes from mastectomized cows did not change during the study period, although they often were of lower magnitude than those of cells from nonlactating cows. These results reconfirm the occurrence of a generalized reduction in blood mononuclear leukocyte function during the periparturient period. They also suggest that the reduction in leukocyte function during the period may be, in part, due to the physiologic demands imposed on the dairy cow by the lactating mammary gland.

Expression of L-Selectin (CD62L), CD44, and CD25 on activated bovine T cells.

Mycobacterium bovis infection of cattle represents a natural host-pathogen interaction and, in addition to its economic and zoonotic impact, represents a model for human tuberculosis. Extravasation and trafficking of activated lymphocytes to inflammatory sites is modulated by differential expression of multiple surface adhesion molecules. However, effects of M. bovis infection on adhesion molecule expression have not been characterized. To determine these changes, peripheral blood mononuclear cells from M. bovis-infected cattle were stimulated with M. bovis purified protein derivative (PPD) or pokeweed mitogen (PWM) and evaluated concurrently for proliferation and activation marker expression. Stimulation with PPD or PWM increased CD25 and CD44 mean fluorescence intensity (MFI) and decreased CD62L MFI on CD4(+) cells from infected animals. CD62L MFI on PPD- and PWM-stimulated gammadelta T-cell receptor-positive (TCR(+)) and CD8(+) cells was also reduced compared to that of nonstimulated gammadelta TCR(+) and CD8(+) cells. Using a flow cytometry-based proliferation assay, it was determined that proliferating cells, regardless of lymphocyte subset, exhibited increased expression of CD25 and CD44 and decreased expression of CD62L compared to cells that had not proliferated. In contrast to proliferation, activation-induced apoptosis of CD4(+) cells resulted in a significant down regulation of CD44 expression. Lymphocytes obtained from lungs of M. bovis-infected cattle also had reduced expression of CD44 compared to lymphocytes from lungs of noninfected cattle. These alterations in surface molecule expression upon activation likely impact trafficking to sites of inflammation and the functional capacity of these cells within tuberculous granulomas.

Use of the relative dose response (RDR) assay to determine vitamin A status of calves at birth and four weeks of age.

An accurate assessment of vitamin A status can be determined by analysis of liver biopsy samples; however, liver biopsies are not always feasible. Plasma concentrations of vitamin A do not provide an accurate indication of vitamin A status. The objective of this study, therefore, was to determine the ability of the relative dose response assay to indicate the vitamin A status of Holstein calves. Calves were obtained at birth and assigned to vitamin A treatments (0, 1700, 34,000, or 68,000 IU/d) added to milk replacer. Liver biopsies and relative dose response assays were performed at birth and 4 wk. Calves supplemented with 1700, 34,000, or 68,000 IU of vitamin A/d had adequate (greater than 20 microg/g) liver concentrations of vitamin A at 4 wk of age. The relative dose response assay at 4 wk was correlated with liver concentrations of vitamin A. Both the relative dose response assay and liver concentrations of vitamin A indicated that calves not supplemented with vitamin A had low vitamin A status, whereas other treatment groups had adequate vitamin A status. Plasma concentrations of retinol increased by 4 wk of age in calves receiving supplemental vitamin A at 34,000 IU and 68,000 IU/d and decreased in unsupplemented calves; however, all calves had concentrations of <20 microg of retinol/dl of plasma. The relative dose response assay agreed with liver biopsies as an indication of vitamin A status, whereas plasma concentrations of retinol incorrectly indicated all treatment groups were deficient in vitamin A.

Effects of replacement of native fat in colostrum and milk with coconut oil on fat-soluble vitamins in serum and immune function in calves.

Fat-soluble vitamins and their metabolites modulate immune function in a variety of animal species. The objective of the present study was to determine the role of fat-soluble vitamins in colostrum and milk in the development of specific aspects of immune function in the calf during the 1st wk postpartum. During this period, control calves (n = 6) were fed normal colostrum and milk, and calves in the treatment group (n = 6) were fed skimmed colostrum and skimmed milk supplemented with coconut oil. Treated calves did not experience the progressive increase in concentrations of retinol, beta-carotene, alpha-tocopherol, 1,25-dihydroxyvitamin D, or retinoic acids in serum that was observed in control calves. Acquisition of passive immunity, which is indicated by concentrations of immunoglobulin G1 in serum, was unaffected by treatment. Composition and functional capacities of populations of blood mononuclear leukocytes that were collected from birth to 7 d postpartum were also unaffected by treatment. Major changes in the function and composition of mononuclear leukocyte populations from all calves occurred during the experimental period and were unrelated to the concentrations of fat-soluble vitamins in serum. Populations of blood mononuclear leukocytes from calves were functionally hyporesponsive and compositionally different from populations of blood mononuclear leukocytes from adult nongravid cows. These differences likely reflected the immaturity of the immune system of the neonatal calf and may contribute to the increased susceptibility of the calf to infectious disease.

Effects of supplemental chromium on production of cytokines by mitogen-stimulated bovine peripheral blood mononuclear cells.

This study determined whether supplementing the diets of dairy cows during the peripartum period with organic trivalent Cr influenced the capacity of their peripheral blood mononuclear cells to produce activation cytokines in response to stimulation with mitogens in vitro. Nine cows were fed 0.5 ppm of Cr/d per cow from 6 wk prepartum to 16 wk postpartum; 10 other periparturient cows served as unsupplemented controls. Mononuclear leukocytes, enriched from peripheral blood during wk 0, 2, 4, and 6 of lactation, were cultured with or without the T-lymphocyte mitogen, concanavalin A. Culture supernatants, harvested at 24, 48, or 72 h, were assayed for interleukin-2, interferon-gamma, and tumor necrosis factor-alpha. The cytokines were barely detectable in the supernatants from the unstimulated cultures, but supernatants from mitogen-stimulated cultures contained higher concentrations of each cytokine. For cows fed Cr, concentrations of all three cytokines in the culture supernatants of the mitogen-stimulated mononuclear cells decreased significantly relative to values for unsupplemented cows, particularly around peak lactation for the 24- and 48-h cultures. Theses results extended our previous observations and supported the hypothesis that organic Cr is immunomodulatory in high producing cows.

Immunomodulatory activity of blood serum from chromium-supplemented periparturient dairy cows.

Our previous research showed enhanced immune responses, including mitogen-induced blastogenesis of peripheral blood mononuclear cells from feedlot calves and periparturient dairy cows supplemented with dietary chromium (Cr). The objective of the present study were to test whether blood sera from Cr-supplemented periparturient cows contained immunomodulatory activity for mitogen-stimulated peripheral blood mononuclear cells and, if so, to determine if this activity was explicable by differences in blood profiles of some glucose-regulating hormones (insulin, cortisol, growth hormone, insulin-like growth factor I, and tumor necrosis factor-alpha) between Cr-supplemented and unsupplemented (control) animals. Blood sera from ten unsupplemented cows and nine Cr-supplemented cows (0.5 ppm day-1) were collected weekly from 2 weeks before to 6 weeks after parturition, and were used to supplement (1, 10, and 20% vol/vol) culture medium supporting concanavalin A (Con A) stimulated mononuclear cells enriched from blood of four nulliparous donor cows. Hormone concentrations were determined using radioimmunoassays. Con A-induced blastogenesis was enhanced when 1, 10, and 20% sera from Cr-supplemented cows was added to the mononuclear cell cultures, and this was particularly evident around parturition. Conversely, peripartum sera from unsupplemented cows depressed Con A-induced blastogenesis. Except for a marginal rise in blood cortisol 2-4 weeks after parturition, no significant effects of Cr supplementation on other hormones (insulin, growth hormone, insulin-like growth factor I, tumor necrosis factor-alpha) were observed. These observations suggest that factors in peripheral blood serum from Cr-supplemented cows, other than absolute concentrations of the glucose-regulating hormones studied, modulate Con A-induced blastogenesis of mononuclear leukocytes.

Effect of selenium and reducing agents on in vitro immunoglobulin M synthesis by bovine lymphocytes.

A series of experiments was conducted to evaluate the effects of inorganic and organic forms of Se with or without reducing agents on in vitro IgM production by bovine lymphocytes. Peripheral mononuclear cells were isolated from nonlactating Jersey cows fed a diet with adequate Se. Cells were stimulated with pokeweed mitogen and, in addition, were cultured with various Se compounds at a concentration of 100 ng Se/ml. Mercaptoethanol (50 microM) and glutathione (1 mM) were included in cultures of cells stimulated by pokeweed mitogen with and without inorganic Se. Sodium selenite was less effective than selenomethionine and selenocystine in augmenting pokeweed mitogen-induced Ig synthesis. The addition of mercaptoethanol to pokeweed mitogen-stimulated control cultures enhanced in vitro IgM production, whereas the addition of glutathione had a negligible effect, but addition of either in combination with sodium selenite dramatically depressed IgM production. These results suggest that Se in inorganic or organic forms enhances B-cell function in vitro.

Phagocytic activity of milk leukocytes during chronic staphylococcal mastitis.

Receptors for IgG on milk leukocytes were detected by rosette formation using sensitized erythrocytes. The percentage (41) of milk leukocytes from uninfected glands forming sensitized erythrocyte rosettes was significantly greater than the percentage (13) of leukocytes from glands with chronic staphylococcal mastitis. A greater percentage of polymorphonuclear leukocytes than macrophages formed sensitized erythrocyte rosettes, regardless of the infection status of the gland from which they were obtained. Both IgG-receptor and nonimmunologic receptor-mediated phagocytosis were greater for milk leukocytes from uninfected glands than for milk leukocytes from chronically infected glands. Preincubation of normal milk leukocytes in whey prepared from mastitic milk resulted in a decrease in their capacity to form sensitized erythrocyte rosettes as well as a reduction in their phagocytic capacity. Immune complexes prepared in vitro also reduced the phagocytic capacity of normal milk leukocytes and inhibited their capacity to form sensitized erythrocyte rosettes. These data indicate a factor, possibly consisting of immune complexes, was present in secretions from glands chronically infected with staphylococci. This factor reduced the phagocytic capacity of milk leukocytes.

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 ammonia on in vivo and in vitro immune responses.

The effects of exposure of animals to ammonia on their delayed type of dermal response, the mitogenic and antigenic responses of their lymphocytes, and the bactericidal and phagocytic activities of their alveolar macrophages were examined. Experimental guinea pigs vaccinated with Mycobacterium bovis BCG were exposed to 3.75 micrograms of ammonia per dl of air (50 ppm) or 6.75 micrograms of ammonia per dl of air (90 ppm), whereas control animals also vaccinated with BCG were maintained in the normal environment. The delayed type of dermal response to tuberculin injected 3 weeks later was significantly (P less than 0.05) less in experimental animals exposed to 6.75 micrograms of ammonia per dl than in control animals. In vitro, the response of blood lymphocytes and bronchial lymphocytes to phytohemagglutinin, concanavalin A, and tuberculin stimulation was significantly (P less than 0.01) less than the response of lymphocytes from control animals. The response of normal blood lymphocytes to phytohemagglutinin incubated in medium containing 1 or 10 mg of ammonia per dl was significantly (P less than 0.01) reduced as compared with the response of lymphocytes incubated without ammonia. The viability of lymphocytes incubated with these concentrations of ammonia was significantly (P less than 0.01) affected. There was no significant difference in the bactericidal or phagocytic activities of alveolar macrophages collected from animals exposed to ammonia and control animals. However, ammonia added to the culture of alveolar macrophages from normal animals significantly inhibited their bactericidal activity.

Cell-mediated immune response of the mammary gland in guniea pigs. I. Effect of antigen injection into the vaccinated and unvaccinated glands.

The leukocytic response of the mammary gland in the locally vaccinated guniea pigs to challenge with specific antigen during lactation was investigated. The response was measured by enumerating cells in milk collected at various times prior to and following antigenic challenge. A significant leukocytosis was observed in milk from vaccinated animals. The maximum cellular response by vaccinated animals was observed at 16 h to 30 h after challenge. The majority of leukocytes collected at that time did not form EA rosettes. Differential cell counts showed that the polymorphonuclear leukocytes were the major cell type until 30 h postchallenge while the mononuclear leukocytes predominated later. The delayed cellular response to challenge and the predominancy of leukocyte type at various times after challenge are discussed. It is proposed that the leukocyte response of the mammary glands in vaccinated animals was a cell-mediated immune reaction.