Gamma delta T cells are early responders to Mycobacterium avium ssp. paratuberculosis in colostrum-replete Holstein calves.

Peripheral blood mononuclear cells (PBMC) and mesenteric node lymphocytes (MNL) were obtained from 30 calves that were assigned randomly at birth to 1 of 6 treatment groups with 5 calves per treatment in a 14-d study: (1) colostrum-deprived (CD), no vitamins; (2) colostrum-replacer (CR), no vitamins; (3) CR, vitamin A; (4) CR, vitamin D3; (5) CR, vitamin E; (6) CR, vitamins A, D3, E. Calves were injected with appropriate vitamin supplements and fed pasteurized whole milk (CD calves) or fractionated colostrum replacer (CR calves) at birth. Thereafter, all calves were fed pasteurized whole milk fortified with vitamins according to treatment group. Calves were orally inoculated with 108 cfu of Mycobacterium avium ssp. paratuberculosis (MAP) on d 1 and 3. The PBMC and MNL harvested on d 13 were analyzed by flow cytometry as fresh cells, after 3-d culture with phytohemagglutinin (PHA), and after 6-d culture with a whole-cell sonicate of MAP (MPS). Peripheral γδ T cells were a predominant lymphocyte subset in neonatal calves, with a decreased percentage noted in CD calves compared with CR calves. As well, CD25 expression was higher in γδ T cells compared with other cell subsets, regardless of treatment group. Stimulation of PBMC with PHA resulted in increased CD4+ and CD8+ subsets, whereas MNL response was dominated by expansion of B-cell subpopulations. Stimulation with PHA and MPS decreased the relative abundance of PBMC γδ T cells, but MNL γδ T cells increased upon stimulation with MPS. These results identify γδ T cells as key early responders to intracellular infection in neonatal calves and suggest that colostrum may be an important mediator of this response.

Effects of fractionated colostrum replacer and vitamins A, D, and E on haptoglobin and clinical health in neonatal Holstein calves challenged with Mycobacterium avium ssp. paratuberculosis.

Thirty Holstein calves were obtained from 2 dairy farms in central Iowa at birth and randomly assigned to 1 of 6 treatment groups: (1) colostrum deprived (CD), no vitamins; (2) colostrum replacer (CR), no vitamins; (3) CR, vitamin A; (4) CR, vitamin D3; (5) CR, vitamin E; and (6) CR, vitamins A, D3, E, with 5 calves per treatment in a 14-d study. Calves were fed pasteurized whole milk (CD) or fractionated colostrum replacer (CR) at birth (d 0) and injected with vitamins according to treatment group. From d 1 through d 14 of the study, all calves were fed pasteurized whole milk (PWM) supplemented with vitamins as assigned. All calves were inoculated with Mycobacterium avium ssp. paratuberculosis on d 1 and 3 of age. Calves fed CR acquired IgG1 and haptoglobin in serum within 24 h of birth, whereas CD calves did not. The CR-fed calves were 2.5 times less likely to develop scours, and CR calves supplemented with vitamins D3 and E also demonstrated a decreased incidence of scours. Serum vitamin levels of A, D, and E increased within treatment group by d 7 and 14 of the study. Interestingly, synergistic effects of supplemental vitamins A, D3, and E on serum 25-(OH)-vitamin D were observed at d 7, resulting in higher levels than in calves administered vitamin D only. Further, vitamin D3 deficiency was observed in CD and CR calves fed a basal diet of pasteurized whole milk and no supplemental vitamins. Colonization of tissues with Mycobacterium avium ssp. paratuberculosis was negligible and was not affected by colostrum feeding or vitamin supplementation. Results demonstrated passive transfer of haptoglobin to neonatal calves, and potential health benefits of supplemental vitamins D3 and E to calves fed pasteurized whole milk.

Clinical disease and stage of lactation influence shedding of Mycobacterium avium subspecies paratuberculosis into milk and colostrum of naturally infected dairy cows.

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD). One mode of transmission of MAP is through ingestion of contaminated milk and colostrum by susceptible calves. The objective of this study was to determine if the amount of MAP shed into the milk and colostrum of infected cows was affected by severity of infection as well as the number of days in milk (DIM). Milk was collected over the 305-d lactation period from naturally infected cows in the asymptomatic subclinical (n=39) and symptomatic clinical (n=29) stages of disease, as well as 8 noninfected control cows. All milk samples were assayed for MAP by culture on Herrold's egg yolk medium and either BACTEC 12B (Becton Dickinson, Franklin Lakes, NJ) or para-JEM (Thermo Fisher Scientific, Trek Diagnostic Systems Inc., Cleveland, OH) liquid medium, and by direct PCR for the IS900 target gene. Mycobacterium avium ssp. paratuberculosis was detected in 3.8, 4.1, and 12.6% of milk samples collected from cows with subclinical JD after culture in Herrold's egg yolk medium, liquid medium, and direct PCR, respectively. The frequency of MAP positivity increased to 12.9, 18.4, and 49.2% of milk samples collected from cows with clinical JD by these same methods, respectively. None of the milk samples collected from control cows was positive for MAP by any detection method. Viable MAP was primarily isolated from milk and colostrum of subclinically and clinically infected cows collected in early lactation (DIM 0-60), with negligible positive samples observed in mid (DIM 60-240) and late (DIM 240-305) lactation. This study demonstrates that shedding of MAP into milk is affected by infection status of the cow as well as stage of lactation, providing useful information to producers to help break the cycle of infection within a herd.

Optimization of hexadecylpyridinium chloride decontamination for culture of Mycobacterium avium subsp. paratuberculosis from milk.

A protocol was optimized for the isolation of Mycobacterium avium subsp. paratuberculosis (MAP) from milk and colostrum, with parameters including chemical decontamination, antibiotics, and different culture media. This study demonstrates that the efficiency of MAP recovery from milk is highly dependent upon the culturing protocol, and such protocols should be optimized to ensure that low concentrations of MAP in milk can be detected.

Pasteurization of colostrum reduces the incidence of paratuberculosis in neonatal dairy calves.

In the present study, the potential benefits of feeding pasteurized colostrum were demonstrated in calves born to dams naturally infected with Mycobacterium avium ssp. paratuberculosis. Calves were separated at birth from their dams and randomly allocated into a group fed either the colostrum of their dam (DC; n = 6), followed by feeding the milk of the dam for 3 wk and then milk replacer, or into a group fed pooled pasteurized colostrum (PC; n = 5) from healthy noninfected dams, followed by milk replacer. At 6 wk of age, calves were weaned onto calf starter, housed together, and fed in a similar manner throughout the rest of the 12-mo study. Calves were necropsied at the end of the study, and 25 tissue sites were sampled from each animal and cultured for M. avium ssp. paratuberculosis. Sixteen of the 25 tissue sites were positive for calves across both treatment groups, with 14 of the 16 tissue sites positive for DC calves and 9 of the 16 tissue sites positive for PC calves. The degree of colonization within a tissue was low and variable for calves within treatment groups, and fecal shedding of M. avium ssp. paratuberculosis was minimal during the 12-mo study. As a measure of the early immune response to infection, blood obtained from calves was stimulated in vitro with M. avium ssp. paratuberculosis antigen preparations, and IFN-Upsilon secretion was measured. Antigen-specific IFN-Upsilon was consistently greater throughout the study in DC calves (0.95 +/- 0.19) compared with PC calves (0.43 +/- 0.10). Although long-term benefits are unknown, these results indicate that feeding a source of colostrum from paratuberculosis-free dams may decrease the initial exposure of neonates to M. avium ssp. paratuberculosis, perhaps decreasing dissemination of infection over time.

Destruction of Mycobacterium paratuberculosis, Salmonella spp., and Mycoplasma spp. in raw milk by a commercial on-farm high-temperature, short-time pasteurizer.

The 2002 NAHM's Dairy Survey indicated that 87.2% of dairy farms in the United States feed waste milk to their neonatal calves. Although cost-effective, this practice can lead to increased calf morbidity and mortality due to ingestion of pathogenic agents. In an effort to reduce the risk of infection, dairy producers are implementing on-farm pasteurization of the waste milk as a control procedure before feeding the milk to calves. In the present study, the efficacy of a commercial high-temperature, short-time (HTST) on-farm pasteurizer unit to destroy Mycobacterium paratuberculosis, Salmonella enterica spp., and Mycoplasma spp. in raw milk was evaluated. Replicate experiments were run for 3 isolates of M. paratuberculosis, 3 serovars of Salmonella (derby, dublin, typhimurium); and 4 species of Mycoplasma (bovis, californicum, canadense, serogroup 7) at 2 different levels of experimental inoculation. In addition, HTST pasteurization experiments were performed on colostrum experimentally inoculated with M. paratuberculosis. After culture of the pasteurized milk samples, no viable M. paratuberculosis, Salmonella, or Mycoplasma were recovered, regardless of species, strain, or isolate. Pasteurization of colostrum was also effective in the destruction of M. paratuberculosis but resulted in an average 25% reduction in colostral immunoglobulin. These results suggest that HTST pasteurization is effective in generating a safer product to feed to young calves.

Effects of supplemental energy on metabolic and immune measurements in periparturient dairy cows with Johne's disease.

The present study was designed to evaluate whether feeding supplemental energy would improve the metabolic profile and alleviate some of the immunosuppression typically noted during the periparturient period in dairy cows with Johne's disease. Twelve dairy cows naturally infected with Mycobacterium paratuberculosis were fitted with rumen cannulas in late gestation and assigned to treatment groups: control, n = 6; or stuffed, n = 6. Cows in the control group were allowed to consume feed ad libitum. Cows assigned to the stuffed treatment group were also fed ad libitum but received additional total mixed rations by manually stuffing their rumens with refused feed to maintain dry matter intake of 2% body weight per day before calving and 2.5% body weight per day after calving. Serum nonesterified fatty acid levels were significantly decreased in stuffed cows compared with control cows, indicating that stuffing to maintain dry matter intake improved the energy balance in the cows. In addition, periparturient serum calcium and magnesium concentrations were significantly higher in stuffed cows. Stuffing modulated cell-mediated immunity by reducing lymphocyte proliferative responses to T-cell mitogens during early lactation. Stuffing resulted in an increase in the secretion of in vitro immunoglobulin by peripheral blood mononuclear cells after parturition when compared to control cows. These data demonstrate that energy balance is improved by providing additional energy in this manner and suggest energy supplementation can improve some aspects of immune function during the periparturient period.

1,25-dihydroxyvitamin D3 enhances milk antibody titers to Escherichia coli J5 vaccine.

Recent evidence in cattle and mice has suggested that 1,25-dihydroxyvitamin D3 may stimulate T-lymphocyte differentiation pathways responsible for humoral immunity. The use of 1,25-dihydroxyvitamin D3 as an adjuvant for an Escherichia coli J5 vaccine was tested. Ten midlactation cows received J5 vaccine and were revaccinated 6 wk after the first vaccine administration. Five of these cows were given 200 micrograms of 1,25-dihydroxyvitamin D3, in addition to the primary vaccination, and they received an additional 200 micrograms of 1,25-dihydroxyvitamin D3 1 wk after the primary vaccination. No 1,25-dihydroxyvitamin D3 was administered with the wk 6 J5 vaccine booster. Milk immunoglobin (Ig)M, IgG, and IgA antibodies to E. coli J5 were significantly increased in cows receiving 1,25-dihydroxyvitamin D3 + E. coli J5 vaccine compared with cows that received only E. coli J5 vaccine. Serum IgG and IgG1 antibodies to E. coli J5 were also significantly enhanced by 1,25-dihydroxyvitamin D3 treatment compared with cows receiving vaccine alone. In contrast, serum IgG2 titers tended to decline in cows receiving 1,25-dihydroxyvitamin D3. Taken together, these data suggest that 1,25-dihydroxyvitamin D3 has potential usefulness in boosting humoral responses to vaccines such as J5 and may further enhance the protective qualities of vaccines.

Effect of copper deficiency on tissue, blood characteristics, and immune function of calves challenged with infectious bovine rhinotracheitis virus and Pasteurella hemolytica.

Fourteen Holstein steers, averaging 30 d of age, were fed a semipurified diet (1.5 mg of Cu/kg) supplemented with 0 (-Cu) or 10 mg of Cu/kg of diet (+Cu) for 5 mo. Calves were then challenged by consecutive exposure to aerosol preparations of infectious bovine rhinotracheitis virus (IBRV) and Pasteurella hemolytica on d 0 and 7, respectively, of the 30-d study. Serum ceruloplasmin and plasma copper were higher in +Cu calves throughout the challenge period and increased in +Cu calves after microbial challenge. Heart weights were higher in -Cu calves, although weights of liver, spleen, and thymus were not different between treatments. Copper concentrations in all tissues as well as thymus zinc were higher in +Cu calves. Serum immunoglobulin M tended to be higher in +Cu calves and increased in both treatments after IBRV challenge. Serum IBRV antibody titers were higher in -Cu calves with detectable seroconversion by d 10 postinfection. In contrast, antigen-specific antibodies to P. hemolytica tended to be higher in +Cu calves on d 21. Copper status did not affect blastogenic response, but phytohemagglutinin (PHA)-stimulated blastogenesis was higher in both treatments after IBRV challenge. Repletion of lymphocyte cultures with copper chloride increased proliferative responses to PHA in both +Cu and -Cu calves, and greater responses at all levels of copper (1 to 16 micrograms/mL) were noted in -Cu calves. These results indicate that copper deficiency affects various physiological characteristics that may be important in immunological defense to pathogenic challenge.

Vitamin E effects on in vitro immunoglobulin M and interleukin-1 beta production and transcription in dairy cattle.

Experiments were conducted to determine the effect of dietary vitamin E on in vitro IgM and interleukin-1 production and its transcription by bovine peripheral blood mononuclear cells. Cells were isolated from Jersey cows and cultured with pokeweed mitogen, a T-cell-dependent, B-lymphocyte mitogen, to stimulate polyclonal IgM production. Addition of 55 and 110 ng/ml of alpha-tocopherol at time 0 to cell cultures containing pokeweed mitogen significantly enhanced IgM production compared with control cultures containing pokeweed mitogen alone. Cultures supplemented with 55 ng/ml of alpha-tocopherol at 0, 24, or 48 h after incubation with mitogen had enhanced IgM production compared with control cultures incubated for the same duration. However, addition of alpha-tocopherol to cultures at 72 and 96 h did not affect IgM production. Production of interleukin-1 in culture supernatants obtained 24 h after stimulation with pokeweed mitogen was similar between control cultures and cultures supplemented with alpha-tocopherol. At 48 h, secretion of interleukin-1 was maintained in the supplemented cultures but declined in control cultures. Mononuclear cells obtained from steers receiving vitamin E supplement or control steers were used to examine the effects of in vivo vitamin E status on interleukin-1 mRNA expression. Concanavalin A-stimulated cells from Jersey steers fed diets supplemented with vitamin E expressed 55% higher interleukin-1 mRNA than cells from control steers.

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.

Selenium effects on glutathione peroxidase and the immune response of stressed calves challenged with Pasteurella hemolytica.

The present study was conducted to determine whether a marginal Se deficiency affects health, blood characteristics and the immune response of calves subjected to stresses associated with weaning, shipping (332 km) and Pasteurella hemolytica inoculation. Treatments were 1) -Se, 2) -Se/P. hemolytica, 3) +Se (.1 mg Se/kg feed) and 4) +Se/P. hemolytica. Previous Se intake was controlled; dams of -Se calves were fed diets marginally deficient in Se (.03 to .05 mg/kg), whereas dams of +Se calves received a s.c. injection of 30 mg Se (as sodium selenite) every 60 d. Calves were inoculated with P. hemolytica intratracheally on d 3 following weaning and transport. Inoculation with P. hemolytica increased (P less than .05) body temperatures, platelet counts, serum IgM concentrations and serum antibody titers and decreased serum albumin concentrations at 4 to 7 d postinoculation. Weight gains for the 21-d study were not affected by Se status, although whole blood and plasma glutathione peroxidase (GSH-Px) were higher (P less than .05) for +Se calves. Plasma GSH-Px increased (P less than .01) in calves showing signs of morbidity. Increases in plasma GSH-Px were correlated positively with body temperature. Serum IgM concentrations were higher (P less than .05) in +Se calves on d 17, but Se-supplemented calves had lower (P less than .05) anti-P. hemolytica titers on d 17 than -Se calves. Selenium status did not affect body temperatures, plasma creatine phosphokinase or serum IgG and albumin concentrations. These results indicate that Se status can affect IgM concentrations following stress.