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 d-α-tocopherol and dietary energy on growth and health of preruminant dairy calves.

To observe the effects of supplemental dietary d-α-tocopherol in relation to dietary energy on growth and immune status in dairy calves, 32 newborn Holstein bull calves were assigned to 1 of 4 treatments for 5 wk in a 2 × 2 factorial, randomized complete block, split-plot design. Calves received moderate growth (MG) or low growth (LG) all-milk dietary treatments, formulated to support daily gains of 0.5 or 0.25 kg/d, respectively, per the dietary energy recommendation for milk-fed calves according to the National Research Council's Nutrient Requirements of Dairy Cattle. Calves in both groups were either injected i.m. with Vital E-A+D (injectable solution of vitamins E, A, and D) on d 1 and supplemented with Emcelle Tocopherol (micellized vitamin E) via milk daily (MG-S and LG-S), or were not supplemented (MG-C and LG-C) during the study period. Total weight gain of MG calves was greater than that of LG calves and tended to be greater in MG-S calves than in MG-C calves. Calves receiving vitamin supplementation demonstrated greater concentrations of plasma α-tocopherol, retinol, and 25-(OH)-vitamin D than did control calves, whereas MG calves demonstrated a lower concentration of plasma α-tocopherol than did LG calves. The apparent increased utilization of α-tocopherol by MG calves was accompanied by a rise in serum haptoglobin, a positive acute-phase protein and indicator of inflammation, especially in MG-C calves. Serum amyloid A, also a positive acute-phase protein, was not different among groups, but was elevated from baseline in all groups during wk 1 through 3. Plasma IgG1 concentrations were higher in MG-S and LG-S calves than in their nonsupplemented dietary counterparts, whereas plasma IgG2, IgA, and IgM concentrations were not different among groups. In summary, dietary supplementation of d-α-tocopherol improved plasma α-tocopherol status and tended to increase growth in calves fed for 0.5 kg of average daily gain. Vitamin supplementation ameliorated the rise of serum haptoglobin associated with acute inflammation in MG calves, and may have improved passive transfer of maternal antibody. These results indicate a role for α-tocopherol in prevention of proinflammatory state associated with greater dietary energy and onset of infectious disease.

Adaptive immunity in the colostrum-deprived calf: response to early vaccination with Mycobacterium bovis strain bacille Calmette Guerin and ovalbumin.

Responses of the newborn calf to vaccination are frequently characterized by marginal antibody (Ab) responses. The present study evaluated effects of colostrum ingestion on the adaptive immune response of the preruminant calf to early vaccination. Colostrum-fed (CF) and colostrum-deprived (CD) calves were vaccinated at 2 d of age with Mycobacterium bovis, Pasteur strain of bacille Calmette Guerin (BCG), and ovalbumin (OVA) to track development of the adaptive immune response during the first 8 wk of life. Dams were also vaccinated with BCG prepartum. At wk 0, serum IgG(1), IgG(2), IgA, and IgM were elevated in CF calves, with IgG(1) predominating. In these calves, IgG(2), IgA, and IgM concentrations decreased with age. The CD calves, in contrast, had very low or undetectable serum immunoglobulin concentrations at wk 0 followed by an age-related increase in IgG(1), IgG(2), and IgM concentrations, suggesting endogenous production of these immunoglobulin classes. Immunoblot and ELISA analyses of Ab response to BCG vaccination indicated that colostrum ingestion was associated with measurable serum anti-mycobacterial Ab in CF calves during the first month postpartum, with substantially lower levels at 7 wk of age. Although mycobacteria-specific Ab was undetectable in CD calves at wk 0, it was present at 4 and 7 wk of age, suggesting that these calves, unlike CF calves, were capable of generating an Ab response to BCG vaccination. Antibody responses of CF and CD calves to vaccination with OVA, an antigen not present in the natural environment of dairy cattle, were of comparable magnitude and characterized by a progressive increase in Ab levels from birth (wk 0) to 7 wk of age. The disparate Ab responses of CF calves to BCG and OVA suggest that maternal antigenic experience or exposure influences Ab responses of the colostrum-fed preruminant calf to early vaccination. Ex vivo, antigen [OVA and M. bovis-derived purified protein derivative (PPDb)]-induced IFN-γ and nitric oxide responses of blood mononuclear cells (PBMC) from CF and CD calves were comparable at wk 0 and wk 7. As expected, responses were very low or nonexistent at wk 0. Responses for all calves were greater at wk 7 than at wk 0, suggesting a colostrum-independent maturation of the cell-mediated immune response capacity of the preruminant calf. The consistently greater proliferative responses of antigen-stimulated T-cell subsets at wk 7 versus wk 0 indicate the development of antigen-specific lymphocyte responses to early vaccination. Total numbers of blood leukocytes as well as numbers of lymphocytes and monocytes were unaffected by colostrum feeding; however, granulocyte numbers were higher in CD than in CF calves at wk 0. Granulocyte numbers decreased and monocyte numbers increased with age in all calves. Within the lymphocyte population, only natural killer (NK(+)) cell percentages were affected by colostrum ingestion, with higher percentages of NK(+) cells in CD calves at wk 0 and wk 7. Antigen-induced proliferation of lymphocyte subsets including IgM(+) cells was unaffected by colostrum ingestion. In conclusion, ingestion of colostrum within hours after birth inhibited the capacity of the calf to produce antigen-specific immunoglobulin (i.e., antibody) in response to vaccination, with little or no effect on cell-mediated immune responses. Although colostrum appeared to block endogenous antibody production, certain B-cell functions were retained. These findings will aid in development of new vaccination strategies for improving health of the preruminant calf.

Short communication: Fat-soluble vitamin and mineral status of milk replacer-fed dairy calves: effect of growth rate during the preruminant period.

Effects of growth rate on fat-soluble vitamin and macro- and micromineral concentrations in the circulation of preruminant dairy calves were evaluated. Dietary treatments were designed to achieve 3 targeted rates of gain [no growth (NG)=0.0 kg/d; low growth (LG)=0.55 kg/d; or high growth (HG)=1.2 kg/d] over a 7-wk period. Milk replacer (MR) intakes necessary to achieve these growth rates were estimated using the National Research Council's Nutrient Requirements of Dairy Cattle calf model computer program. All of the calves were fed a 30% crude protein, 20% fat MR reconstituted to 14% dry matter. The diets were formulated to ensure that protein was not a limiting nutrient. No-growth and LG calves were supplemented additionally with vitamins A, D, and E to compensate for treatment differences in dry matter intake relative to the HG calves; however, no attempt was made to adjust mineral intake based on MR consumption. Growth rates for NG (0.11 kg/d), LG (0.58 kg/d), and HG (1.16 kg/d) calves differed during the study. Health was minimally affected by growth rate and this was reflected by comparable and relatively low serum haptoglobin concentrations in all calves during the 7-wk period. Concentrations of serum retinol, 25-(OH)-vitamin D(3), and zinc were unaffected by growth rate. The HG calves had lower RRR-alpha-tocopherol concentrations than NG and LG calves at wk 7, suggesting that the increased growth rate of HG calves was associated with increased utilization of vitamin E. Serum concentrations of all vitamins increased with age. Copper, calcium, and phosphorous concentrations in HG calves exceeded those in LG and NG calves during the latter weeks of the study, likely because of increased MR intake by HG calves. Fat-soluble vitamin and mineral concentrations for all treatment groups remained within ranges considered normal for preruminant calves.

Short communication: the preruminant calf as a model for characterizing the effects of vitamin D status in the neonate.

The objective of this study was to evaluate the feasibility of using the preruminant dairy calf as a model for evaluating effects of vitamin D status in the neonate. Because the newborn calf can be sustained during the first weeks of life solely on a fluid diet having a defined composition, has documented nutritional requirements, and is minimally affected by repeated samplings of peripheral blood, it has the potential to serve as a model for characterizing nutrient-specific effects on the growth and health of the neonate. Colostrum-fed Holstein bull calves (n = 13) entered the trial at approximately 4 d of age. All calves were fed a custom-formulated milk replacer devoid of vitamin D. Plasma 25-hydroxyvitamin D(3) concentrations in all calves were determined on a regular basis beginning at d 0. Using this information, low- and high-status groups of calves were established by subcutaneous administration of 25-hydroxyvitamin D(3). To maintain targeted plasma 25-hydroxyvitamin D(3) concentrations in low (<30 ng/mL) and high (>60 ng/mL) vitamin D-status calves, low-status calves (n = 6) received a total of 8,600 IU (2,225 IU/wk) of vitamin D during the experimental period and high-status calves (n = 7) received 54,000 IU (13,500 IU/wk). Concentrations of 25-hydroxyvitamin D(3) in low-status calves averaged 27 ng/mL, compared with 78 ng/mL in high-status calves, and were less at all sampling times from d 7 to d 28. Concentrations of 1,25-dihydroxyvitamin D(3) and 25-hydroxyvitamin D(3) were not correlated. Calcium, magnesium, and phosphorous concentrations were unaffected by 25-hydroxyvitamin D(3) administration; however, plasma calcium and 1,25-dihydroxyvitamin D(3) concentrations were correlated. Calcium and magnesium concentrations decreased with age but remained within normal ranges for dairy cattle. These results indicate that it is possible to predictably control vitamin D status over a 28-d period and suggest that the preruminant calf might be useful as a model for studying effects of vitamin D on growth, development, and immune function in the neonate.

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.

Blood culture and stimulation conditions for the diagnosis of tuberculosis in cervids by the Cervigam assay.

Mitogen- and antigen-induced interferon-gamma (IFN-gamma) responses of peripheral blood leucocytes from cervids were evaluated by a commercial whole-blood assay. The assay was applied to Mycobacterium bovis-infected white-tailed deer and reindeer, M bovis BCG-vaccinated white-tailed deer and elk, and unvaccinated, uninfected white-tailed deer, fallow deer, elk and reindeer. The responses of the M bovis-infected white-tailed deer to pokeweed mitogen (PWM) varied with time and between individuals. The responses of the M bovis-infected reindeer to PWM and M bovis purified protein derivative (PPD) were positively associated. Samples from tuberculosis-free captive herds in various parts of the USA were also evaluated. Four per cent of fallow deer, 20 per cent of elk, 44 per cent of white-tailed deer, and 91 per cent of reindeer had responses to PWM exceeding 0.25 Delta optical density, that is, PWM stimulation minus no stimulation. The specificity of the responses to M bovis PPD and a Mycobacterium tuberculosis complex-specific antigen rESAT-6:CFP-10, excluding animals not responding to PWM, ranged from 78 per cent to 100 per cent and was dependent upon the species and the positive response cut-off value. The results show that the commercial assay is valid for the detection of TB in reindeer; however, further development of the assay will be required before it is used in surveillance programmes for white-tailed deer, fallow deer, and elk.

Effects of pre-culture holding time and temperature on interferon-gamma responses in whole blood cultures from Mycobacterium bovis-infected cattle.

The Bovigam assay is approved for use within the United States as a complementary tuberculosis test. Prior to whole blood culture and the ensuing ELISA to detect interferon-(IFN)-gamma, samples are subjected to various holding time/temperature combinations due, in part, to practical constraints associated with shipment of samples to approved laboratories. To evaluate these effects, 5-month-old Holstein calves (n = 7) received 10(3) cfu Mycobacterium bovis by aerosol. Heparinized blood was collected 2 months after challenge and held at 4 or 22 degrees C for 0, 8 or 24 h prior to culture with mycobacterial antigens or pokeweed mitogen (PWM). Responses of samples held for 8 or 24 h were comparable and lower than responses of cultures prepared immediately after collection, regardless of holding temperature. Differences in responses of samples held at 4 degrees C versus 22 degrees C were also minimal. A subset of samples was held for 2 h at 37 degrees C at the beginning of the holding period. This subset of samples had diminished responses to all stimulants and increased holding times (i.e., 24 h versus 8 h) negatively impacted the response. Pre-processing conditions, particularly delays in set-up and initial high sample temperatures, reduces IFN-gamma responses of cells from infected cattle increasing the risk of false negatives in this assay of regulatory importance.

High growth rate fails to enhance adaptive immune responses of neonatal calves and is associated with reduced lymphocyte viability.

The objective of the study was to evaluate the effects of 3 targeted growth rates on adaptive (i.e., antigen-specific) immune responses of preruminant, milk replacer-fed calves. Calves (9.1 +/- 2.4 d of age) were assigned randomly to one of 3 dietary treatments to achieve 3 targeted daily rates of gain [no growth (maintenance) = 0.0 kg/d, low growth = 0.55 kg/d, or high growth = 1.2 kg/d] over an 8-wk period. The NRC Nutrient Requirements of Dairy Cattle calf model computer program was used to estimate the milk replacer intakes needed to achieve target growth rates. All calves were fed a 30% crude protein, 20% fat, all-milk protein milk replacer reconstituted to 14% dry matter. Diets were formulated to ensure that protein would not be limiting. All calves were vaccinated 3 wk after initiation of dietary treatments with Mycobacterium bovis, strain bacillus Calmette-Guerin and ovalbumin. Growth rates for no-growth (0.11 kg/d), low-growth (0.58 kg/d), and high-growth (1.16 kg/d) calves differed throughout the experimental period. Blood glucose concentrations in high-growth calves increased with time and were higher than in low- and no-growth calves. Mononuclear and polymorphonuclear leukocyte percentages in peripheral blood were unaffected by growth rate but did change with advancing age. Percentages of CD4(+) T cells increased with age in no-growth and low-growth calves, a characteristic of maturation, but failed to increase in high-growth calves. Growth rate did not affect the percentages of CD45RO(+) (memory) CD4(+) and CD8(+) T cells, antigen (i.e., ovalbumin)-specific serum IgG concentrations, or antigen (i.e., purified protein derivative)-induced IFN-gamma and nitric oxide secretion by mononuclear cell cultures. Antigen-elicited cutaneous delayed-type hypersensitivity responses of no-growth calves exceeded responses of low-growth, but not high-growth, calves. In resting- and antigen-stimulated cell cultures, viabilities of CD4(+), CD8(+), and gammadeltaTCR(+) T cells from high-growth calves were lower than those of the same T cell subsets from no-growth and low-growth calves. Alternatively, resting cultures of mononuclear leukocytes from high-growth calves produced more nitric oxide than those from no-growth and low-growth calves. In conclusion, adaptive immune responses were affected minimally by growth rate. The results suggest that protein-energy malnutrition in the absence of weight loss is not detrimental to antigen-specific responses of neonatal vaccinated calves and that a high growth rate does not enhance these responses. The negative effect of a high growth rate on the viability of circulating T cell populations may influence infectious disease resistance of the calf.

Antigen-specific B-cell responses by neonatal calves after early vaccination.

The objective of this research was to evaluate the effects of early vaccination on the phenotype (i.e., activation marker expression) and functional capacity of B cell populations in neonatal calves. In the first of 2 experiments, 6 calves were vaccinated with ovalbumin at 3 and 5 wk of age. Three of the 6 calves also were vaccinated with Mycobacterium bovis, strain bacillus Calmette-Guerin (BCG) at 3 wk of age. Mycobacterium bovis lipoarabinomannan-reactive IgG1 and IgG2 were detected in calf sera prior to vaccination, indicative of colostral transfer of maternal Ig cross-specific to BCG. Ovalbumin-specific IgG1 and IgG2 were not detected before vaccination. Vaccination of 3-wk-old calves with ovalbumin elicited antigen-specific IgG1 and IgG2 anti-body responses that were amplified by secondary vaccination. Vaccination with BCG did not elicit a measurable antibody response. In the second experiment, 6 calves were vaccinated with ovalbumin at 3 and 5 wk of age in addition to BCG at 3 wk of age. Lymph node cell populations stimulated with ovalbumin had decreased CD5, CD21, and CD40 expression and increased B-B2, CD25, and CD80 expression on IgM+ cells. Stimulation of the same population with purified-protein derivative increased CD25 and CD80 expression on IgM+ cells. Expression of activation molecules on ovalbumin- and purified protein derivative-stimulated CD5+ IgM+ cells was similar to expression on the larger IgM+ cell population. An increased expression of major histocompatibility class II on CD5+ IgM+ cells after stimulation was the only exception. Interestingly, IgM+ cells isolated from the superficial cervical lymph node draining the vaccination site, but not from the opposing cervical lymph node, responded to antigen stimulation in vitro. In conclusion, calves generated B cell responses to ovalbumin and BCG after vaccination. Additional studies are necessary to determine whether maternal immunologic experience transferred via colostral immunoglobulin inhibits production of mycobacteria-specific immunoglobulin production in the calf.

Cumulative physiological events influence the inflammatory response of the bovine udder to Escherichia coli infections during the transition period.

A high proportion of intramammary coliform infections present at parturition develop disease characterized by severe inflammatory signs and sepsis during the first 60 to 70 d of lactation. In the lactating bovine mammary gland, the innate immune system plays a critical role in determining the outcome of these infections. Since the beginning of the 1990s, research has increased significantly on bovine mammary innate defense mechanisms in connection with the pathogenesis of coliform mastitis. Neutrophils are key effector cells of the innate immune response to intramammary infection, and their function is influenced by many physiological events that occur during the transition period. Opportunistic infections occur when the integrity of the host immune system is compromised by physical and physiological conditions that make the host more susceptible. The innate immune system of many periparturient cows is immunocompromised. It is unlikely that periparturient immunosuppression is the result of a single physiological factor; more likely, several entities act in concert, with profound effects on the function of many organ systems of the periparturient dairy cow. Their defense system is unable to modulate the complex network of innate immune responses, leading to incomplete resolution of the pathogen and the inflammatory reaction. During the last 30 yr, most efforts have been focused on neutrophil diapedesis, phagocytosis, and bacterial killing. How these functions modulate the clinical outcome of coliform mastitis, and how they can be influenced by hormones and metabolism has been the subject of intensive research and is the focus of this review. The afferent (sensing) arm of innate immunity, which enables host recognition of a diverse array of pathogens, is the subject of intense research interest and may contribute to the variable inflammatory response to intramammary infections during different stages of lactation. The development of novel interventions that modulate the inflammatory response or contribute to the elimination of the pathogen or both may offer therapeutic promise in the treatment of mastitis in periparturient cows.

Effects of dexamethasone treatment on IL-1 receptor mRNA levels in vivo.

Using semiquantitative reverse transcriptasepolymerase chain reaction and Northern analysis, we observed in vivo up-regulation of interleukin-1 (IL-1) RI and IL-1RII mRNA levels in peripheral blood mononuclear cells (PBMCs) and neutrophils (PMNs) from Holstein cattle injected with dexamethasone (0.04 mg/kg). Baseline levels of IL-1RI mRNA were greater than IL-1RII mRNA levels in PBMCs and PMNs before dexamethasone treatment. This is in contrast with the previously reported predominance of IL-1RII in unstimulated human PMNs. IL-1RII mRNA was strongly induced in both bovine PBMCs and PMNs at 24 h and returned to baseline levels by 72 h, after dexamethasone injection. Conversely, the greatest increase in IL-1RI mRNA in PBMCs and PMNs was not detected until 72 h after dexamethasone injection. These data provide evidence for sequential in vivo up-regulation of first IL-1RII mRNA and later IL-1RI mRNA by dexamethasone that is consistent with the anti-inflammatory activity of glucocorticoids.

Effects of age and nutrition on expression of CD25, CD44, and L-selectin (CD62L) on T-cells from neonatal calves.

Effects of the plane of nutrition and age on the proliferation and activation of lymphocyte subsets from milk replacer-fed calves were investigated in vitro. Holstein calves were fed a standard (0.45 kg/d of a 20% crude protein, 20% fat milk replacer, n = 4) or intensified (1.14 kg/d of a 28% crude protein, 20% fat milk replacer, n = 4) diet from 1 to 8 wk of age. Average daily weight gain of intensified-diet (0.66 kg/d) calves was greater than that of standard-diet (0.27 kg/d) calves. Relative to the pokeweed mitogen-induced responses of CD4(+) cells from steers (5 to 6 mo of age), CD4(+) cells from 1-wk-old calves showed decreased proliferative activity, delayed increase in CD25 expression, and no demonstrable increase in CD44 expression or decrease in CD62L expression. Calf CD8(+) and gammadeltaT-cell receptor(+) cells, unlike T-cells from the older animals, did not demonstrate decreased expression of CD62L after stimulation with mitogen. The increased expression of CD44 by mitogen-stimulated gammadeltaT-cell receptor(+) cells from older animals was not seen in gammadeltaT-cell receptor(+) cells from 1-wk-old calves. At wk 8 of age, mitogen-induced proliferation and expression of activation antigens by T-cells from standard-fed calves were similar to responses of T-cells from steers indicating rapid maturation of T-cell function during the neonatal period. Feeding calves an intensified milk replacer was associated with decreased proliferation of mitogen-stimulated CD4(+), CD8(+), and gammadeltaT-cell receptor(+) cells; decreased CD25 expression by mitogen-stimulated CD4(+) and CD8(+) cells; and decreased CD44 expression by mitogen-stimulated CD8(+) cells. These results indicate that the functional capacity of the calf's T-cell population becomes more adult-like during the first weeks of life and suggest that nutrition modulates T-cell function during this period of immune maturation.

Mycobacterium bovis bacille Calmette-Guerin vaccination of cattle: activation of bovine CD4+ and gamma delta TCR+ cells and modulation by 1,25-dihydroxyvitamin D3.

SETTING: 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) is a potent modulator of immune responses and may be beneficial in the treatment of tuberculosis. Recent evidence suggest that 1,25(OH)(2)D(3) may affect T-dependent responses in cattle; however, mechanisms by which this vitamin modulates activation of bovine T cells are unclear. OBJECTIVE: Determine the effects of 1,25(OH)(2)D(3) on the expression of CD25, CD44, and CD62L by bovine T cell subsets proliferating in response to antigen stimulation. DESIGN: Antigen-specific recall responses of Mycobacterium bovis bacille Calmette-Guerin (BCG) vaccinated cattle were used as a model system to evaluate effects of 1,25(OH)(2)D(3) on the proliferation and activation of bovine T cell subsets. RESULTS: CD4(+) and gamma delta TCR(+) cells were the predominant T cell subsets responding to soluble crude M. bovis-derived antigens (i.e., purified protein derivative and a BCG whole cell sonicate) by proliferation and activation-induced alterations in phenotype. These subsets exhibited increased CD25 and CD44 mean fluorescence intensity (mfi) and decreased CD62L mfi upon antigen stimulation. Addition of 1,25(OH)(2)D(3) inhibited proliferation of CD4(+) cells and decreased the expression of CD44 on responding (i.e., proliferating) CD4(+) and gamma delta TCR(+) cells. CONCLUSION: These findings suggest that the production of 1,25(OH)(2)D(3) by macrophages within tuberculous lesions would inhibit proliferation and CD44 expression by co-localized CD4(+) and gamma delta TCR(+) cells.

The effect of local and parenteral vaccination on the response of the guinea pig mammary gland to staphylococcal challenge.

Preparturient guinea pigs vaccinated locally (orally and intramammarily) or parenterally (intradermally) with killed Staphylococcus aureus (KS), were challenged intramammarily (IMM) with KS or viable S. aureus during the next lactation. The number and types of leukocytes emigrating into the milk were determined before and after IMM challenge. The milk leukocytosis after challenge with KS was the greatest in the intradermally (ID) vaccinated animals, moderate in the IMM vaccinated animals, and insignificant in the unvaccinated or orally vaccinated animals. The polymorphonuclear (PMN) leukocyte predominated in the milk of the IMM and ID vaccinated animals during the initial 30 h after challenge with KS. Later (30-102 h postchallenge), the mononuclear leukocyte (macrophage and lymphocytes) was the major cell-type. No significant changes in the number or types of leukocytes occurred in the milk of the unvaccinated or orally vaccinated animals after challenge. Intramammary challenge with viable S. aureus caused a large increase in the number of leukocytes in the milk of all animals. The milk leukocytosis occurred more rapidly in locally vaccinated guinea pigs than unvaccinated or ID vaccinated guinea pigs. The PMN leukocyte predominated in the milk of all animals during the period of maximum response. The incidence and severity of staphylococcal mastitis were less in guinea pigs vaccinated locally than ID vaccinated or unvaccinated animals.

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.

Regulation of mitogenic responses by bovine milk leukocytes.

Bovine milk lymphocytes are less responsive to in vitro mitogen stimulation than peripheral blood lymphocytes (PBL). In this study, milk leukocytes (ML) or their soluble products, were co-cultured with mitogen stimulated PBL to determine if suppression could be transferred to normally responsive cells. Addition of either ML (treated with mitomycin C to prevent cell division), or supernatant from ML cultures to cultures of autologous PBL resulted in a reduction of mitogenesis by the PBL, but no suppression was seen with addition of treated PBL or PBL supernatant. Suppression was greater when the ML were from animals with chronic staphylococcal infection. Suppression by ML supernatant was not due to toxicity to the responders, since addition at the latter stages of culture had no effect on the response. These results indicate that reduced mitogenesis by milk lymphocytes may be due to the presence of suppressor cells or molecules.

Local and systemic immune response in the cow after intramammary vaccination during lactation.

The local and systemic immune response of the lactating cow during the 10 week period after intramammary (IMM) vaccination with killed Mycobacterium bovis (M. bovis) was evaluated. Antigen (tuberculin)-reactive lymphocytes were present in the milk as early as 2 weeks post-vaccination, and in the blood at 6 weeks after vaccination. The milk lymphocytes, compared to the blood lymphocytes were consistently more responsive to tuberculin. Both blood and milk lymphocytes responded in vitro to the lectins, phytohemagglutinin-A (PHA-P) and concanavalin A (Con A), although the milk lymphocytes were consistently less responsive than the blood lymphocytes during the period. Anti-tuberculin antibody was significantly elevated in the milk and blood of the vaccinated animals at 10 weeks post-vaccination. Infusion of tuberculin into the mammary glands of the cows 10 weeks after vaccination resulted in a marked increase in the number of milk leukocytes. The influx of leukocytes initially consisted of polymorphonuclear leukocytes (PMNL), and later, mononuclear leukocytes. Intramammary vaccination also resulted in antigen recognition at sites distant from the mammary gland.

Phenotypic analysis of peripheral blood lymphocytes and intestinal intra-epithelial lymphocytes in calves.

The phenotype of intestinal intraepithelial lymphocytes (IEL) of young calves has not been described. In order to determine the potential role of IEL in protection against enteric infection, it is important to characterize these cells in normal calves. Therefore, IEL of calves were analyzed phenotypically and compared with peripheral blood lymphocytes (PBL) via flow cytometry using monoclonal antibodies to cell surface markers. Approximately 25% of PBL and IEL expressed the gamma/delta T-cell receptor (TCR-1+). TCR-1+ PBL co-expressed WC1 (antigen expressed on a subset of lymphocytes expressing the gamma/delta T-cell receptor), whereas only a small percent of TCR-1+ IEL co-expressed WC1. TCR-1+ cells in the PBL co-expressed the interleukin-2 receptor (IL-2r), whereas TCR1+ cells in the IEL co-expressed ACT-2 (null cell and CD-8+ T-cell activation marker). There were approximately twice as many CD-4+ cells as CD-8+ cells in the PBL, whereas there were three to five times as many CD-8+ cells as CD-4+ cells in the IEL. Thus, IEL of calves are antigenically distinct from PBL.

Effects of Cryptosporidium parvum infection on lymphocyte phenotype and reactivity in calves.

Cryptosporidium parvum is a protozoan parasite now recognized as a significant cause of neonatal diarrhea in calves, and infection is also widespread in both immunocompetent and immunocompromised humans. No effective treatment or preventive measures against C. parvum infection are available, owing largely to the lack of understanding of immunologic mechanisms of resistance to and recovery from this parasite. In the present study, we compared phenotypes of lymphocytes from peripheral blood, spleen, mesenteric, and prescapular lymph nodes of calves infected or not infected with C. parvum. We also compared reactivity of these lymphocytes to mitogens and C. parvum antigen in vitro. There were more non-T, non-B (null) lymphocytes in all tissues of infected compared with control calves. The percent of CD8+ lymphocytes was significantly increased in spleens of infected compared with control calves, and there were markedly less CD4+ than CD8+ cells in spleens of both groups (i.e. low CD4/CD8 ratios). Splenic lymphocytes showed significantly decreased in vitro proliferation to pokeweed mitogen and C. parvum antigen stimulation compared with lymphocytes from other tissues. These findings suggest that null lymphocytes and CD8+ lymphocytes may be important in the expression and regulation of bovine immune responses to C. parvum in vivo.