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Serum samples were obtained from Holstein dairy control cows and cows naturally infected with Mycobacterium avium ssp. paratuberculosis (MAP) to evaluate the effects of disease status on serum 25-hydroxyvitamin D 3 (25OHD 3 ) levels. Disease status was stratified for infected cows into asymptomatic, subclinical infection (n = 25), and cows demonstrating clinical signs (n = 20), along with noninfected control (n = 12) cows for comparison. In addition, portions of the ileocecal valve were taken from a subsample of cows (n = 5 per treatment group) at necropsy and processed for RNA sequencing gene transcription studies. Genes associated with vitamin D metabolism were queried to determine any association between infection and gene expression. Serum 25OHD 3 levels were significantly lower in cows in the clinical stage of disease compared with either cows in the subclinical stage and noninfected control cows. Differential expression for genes associated with the vitamin D pathway such as CYP27A1, CYP27B1, vitamin D-binding protein (DBP), and IFNG was dependent upon infection status. An upregulation of CYP27A1 was noted for cows in subclinical status, whereas CYP27B1 expression was enhanced for clinical cows. Increased expression of vitamin D-binding protein was observed for infected cattle, regardless of infection status. In summary, decreases in circulating 25OHD 3 for animals with clinical disease may suggest that these cows have reduced innate immune responses, thereby influencing the ability of animals to fight infection., (Copyright © 2019 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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 D 3 ; (5) CR, vitamin E; (6) CR, vitamins A, D 3 , 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 10 8 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., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Mycobacterium avium ssp. paratuberculosis (MAP) is shed into the milk of cattle affected by Johne's disease and, therefore, is a route of transmission for infection in young stock in dairy herds. The objective of this study was to validate a decontamination and culture protocol for the recovery of MAP from individual bovine milk samples from known infected herds. Decontamination of milk samples (n = 17) with either 0.75% hexadecylpyridinium chloride for 5h or N-acetyl-L-cysteine-1.5% sodium hydroxide (NALC-1.5% NaOH) for 15 min before culture in BACTEC 12 B (Becton Dickinson, Franklin, NJ), para-JEM [Thermo Fisher Scientific (TREK Diagnostic Systems, Inc.), Cleveland, OH], and Herrold's egg yolk (HEY; Becton Dickinson) media was compared. Treatment with NALC-NaOH resulted in a lower percentage (6%) of contaminated samples than did treatment with hexadecylpyridinium chloride (47%), regardless of culture medium. The decontamination protocol (NALC-1.5% NaOH) was then applied to milk samples (n = 144) collected from cows at 7 US dairies. Recovery of viable MAP from the milk samples was low, regardless of culture medium, with recovery from 2 samples cultured in BACTEC 12 B medium, 1 sample cultured in para-JEM medium, and no viable MAP recovered on HEY medium. However, 32 cows were fecal culture positive and 13 milk samples were positive by direct PCR, suggesting that several cows were actively shedding MAP at the time of milk collection. Contamination rates were similar across media, with 39.6, 34.7, and 41.7% of samples contaminated after culture in BACTEC 12 B, para-JEM, and HEY media, respectively. Herd-to-herd variation had a major effect on sample contamination, with the percentage of contaminated samples ranging from 4 to 83%. It was concluded that decontamination of milk with NALC-1.5% NaOH before culture in BACTEC 12 B medium was the most efficacious method for the recovery of viable MAP from milk, although the ability to suppress the growth of contaminating microorganisms varied greatly between herds., (Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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.

Osteopontin (Opn), an important mediator of the cell-mediated immune response, enhances the host immune response against mycobacterial infections. Infections caused by Mycobacterium avium ssp. paratuberculosis (MAP) have a devastating effect on the dairy industry. We sought to characterize Opn at the level of gene and protein expression in periparturient dairy cows naturally infected with MAP. Peripheral blood mononuclear cells (PBMC) were isolated from control, subclinical, and clinical periparturient dairy cows naturally infected with MAP beginning 3 wk precalving to 5 wk postcalving and incubated with medium alone (non-stimulated: NS), concanavalin A (ConA), or a whole-cell sonicate of MAP (MPS). Real-time PCR was performed to evaluate expression of Opn and classical Th1 and Th2 cytokines. Results demonstrated greater Opn expression in nonstimulated PBMC isolated from subclinical cows compared with control and clinical cows. For clinical cows, there was a strong correlation between Opn expression and expression of the Th1 cytokines IFN-gamma and IL-1 alpha for nonstimulated PBMC and IFN-gamma and IL-12 for PBMC stimulated with MPS. Expression of tumor necrosis factor-alpha was greater in clinical cows than the other groups. Nonstimulated, ConA, and MPS-stimulated PBMC from subclinical cows secreted more IFN-gamma, and MPS-stimulated PBMC from clinical cows secreted more IL-4 compared with the other groups. Immunoblot analysis of PBMC detected 4 Opn proteins at 60, 52, 34, and 27 kDa. This is the first study to evaluate the role of Opn on the immune response of dairy cows naturally infected with MAP, and results suggest Opn may be a key regulator against MAP infection.

Mastitis, caused by bacterial infection of the mammary gland, is a major disease of dairy cattle. The greatest risks of intramammary infection occur at the end of lactation and at the initiation of the next lactation when the cow calves. Treating serum with zymosan (yeast cell wall preparation) causes the complement to cleave, allowing this serum to serve as a source of complement fragment 5a (C5a), a potent chemoattractant and activator of the immune system. Our hypothesis was that intramammary infusion of zymosan-treated serum (ZTS) would recruit polymorphonuclear neutrophils (PMN) and generate prolonged activity in lymphocytes within the mammary gland. Ultimately this could help prevent bacterial infections in cows at dry-off and at the initiation of lactation. Two ipsilateral quarters of the mammary gland of each cow were infused with ZTS (12.5 mL/quarter), and 2 contralateral quarters were infused with saline in 8 cows shortly after lactation ended. Mammary secretions were collected periodically throughout the dry period and the first 2 wk of the next lactation. Activation status of lymphocytes and PMN in those secretions was assessed based on the intracellular presence or absence of IFN-gamma and IL-8 as determined by flow cytometry. The ZTS infusion greatly increased PMN numbers in mammary secretions for the first week only. The percentage of IFN-gamma positive lymphocytes and PMN, and the percentage of IL-8 positive PMN, exhibited a sustained increase in secretions from ZTS-treated quarters through the first 2 wk of lactation. The ZTS can stimulate PMN and lymphocyte-mediated immune defense mechanisms in the mammary gland, which may provide a useful means of preventing new intramammary infections during the dry period as well as at the initiation of lactation.

Bovine spongiform encephalopathy (BSE) is a neurodegenerative disease of cattle caused by abnormally folded prion proteins. Two regulatory region polymorphisms in the bovine prion gene are associated with resistance to classical BSE disease: a 23-bp region in the promoter that contains a binding site for the repressor protein RP58, and a 12-bp region in intron 1 that has a binding site for the transcription factor SP1. The presence of these binding sites enhances BSE resistance in cattle, whereas cattle that lack these regions are more susceptible to the disease. The present study examined the allele, genotype, and haplotype frequencies for the 23-bp and 12-bp polymorphisms in Holstein cattle from 9 different US states, and these frequencies were compared with data previously established for Holstein cattle from the United Kingdom, Germany, and Japan. Additionally, the coding region of the prion gene was sequenced from the US samples. Finally, archival samples from US Holstein sires born between 1953 and 1957 were analyzed. We found that the resistant allele and genotype frequencies for the US Holstein cattle were as high, or higher, relative to that observed in other countries. Furthermore, the current US frequencies were comparable to those determined in the archival samples from the 1950s. Based on the frequencies of these regulatory region polymorphisms, the US Holstein population is not at a greater risk for BSE than Holsteins worldwide.

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.

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.

Objectives were to estimate percentages of seropositive herds with cows shedding Mycobacterium paratuberculosis in feces and milk, and to estimate sensitivity, specificity, and predictive value of an ELISA relative to fecal culture. Dairy cows (n = 712) were randomly selected from 61 herds previously identified by ELISA as positive for Johne's disease. Fecal and bulk tank milk samples (n = 52 of 61 herds) were obtained from 10 states in the United States. Fecal samples were processed by a double centrifugation, double decontamination culture procedure. Milk samples were processed for both culture and DNA analysis by using polymerase chain reaction (PCR). Of 24 herds with at least three cows that had tested ELISA-positive, 79% were also culture-positive, compared with 18 of 37 herds with one or two ELISA-positive cows. Both fecal-culture and ELISA results were available on 651 cows; only 25% of cows that were fecal-culture positive also tested positive by ELISA and over 6% of cows that were fecal-culture negative tested ELISA-positive. Milk samples all cultured negative, but analysis of milk samples by PCR resulted in 68% of herds positive for M. paratuberculosis DNA including 24 of 31 herds with positive fecal cultures and 11 of 21 herds with negative fecal cultures. Sensitivity and specificity of the ELISA compared with fecal culture is lower than previously reported and perhaps best used in screening herds because of limited efficacy to predict infection in individual cows. In addition, contamination of bulk tank milk samples with M. paratuberculosis does occur in seropositive herds, even in some with negative fecal cultures.

A recent dairy survey conducted in 1996 by the National Animal Health Monitoring System suggests between 20 and 40% of dairy herds in the United States have some level of Johne's disease. This figure will continue to increase unless producers implement management regimes that will help control the spread of this disease within their herds. The neonatal calf is the target for infection with Mycobacterium paratuberculosis, the causative agent of Johne's disease. Calves become infected via exposure to the bacterium through contaminated feces, bedding, colostrum, and milk. Shedding of viable M. paratuberculosis has been documented in the colostrum and milk of infected dams. This study evaluated the efficacy of on-farm pasteurization to destroy M. paratuberculosis in waste milk fed to calves to circumvent this mode of transmission. In three replicate experiments, waste milk was experimentally inoculated with M. paratuberculosis and heated at 65.5 degrees C for 30 min. No viable bacteria were recovered after 28 wk of incubation. These results suggest that batch pasteurization of waste milk contaminated with M. paratuberculosis was effective at generating a clean product to feed to young calves.

Mycobacterium paratuberculosis, an acid-fast bacillus that causes enteritis in ruminants, has been suggested as an etiological agent of Crohn's disease in humans. The mode of transmission is unclear; however, some evidence suggests that humans may become infected via contaminated milk. Currently, it is not known whether commercial pasteurization effectively kills M. paratuberculosis in contaminated raw milk. Using a laboratory-scale pasteurizer unit designed to simulate the high-temperature, short-time method (72 degrees C, 15 sec) currently used by commercial dairies, we previously demonstrated that treatment of raw milk inoculated with 10(4) to 10(6) cfu of M. paratuberculosis/ml reduced numbers to an undetectable level. However, M. paratuberculosis is an intracellular pathogen that resides within the macrophages of the host and evades destruction. We subsequently performed further experiments examining heat treatment of milk inoculated with mammary gland macrophages containing ingested M. paratuberculosis. Heat treatment of these samples under high-temperature, short-time conditions demonstrated that the macrophage does not protect the organism because we were unable to recover any viable M. paratuberculosis from the samples. Conversely, other researchers have demonstrated that a residual population of M. paratuberculosis may survive heat treatment of milk. In addition, a recent news report stated that viable M. paratuberculosis organisms have been cultured from retail-ready milk in Ireland. A summary of past and current studies concerning this issue along with a discussion of methodologies used to recover M. paratuberculosis from experimentally inoculated milk will be presented in this paper.

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.

Paratuberculosis, which is also known as Johne's disease, is a chronic, progressive enteric disease of ruminants caused by infection with Mycobacterium paratuberculosis. Cattle become infected with M. paratuberculosis as calves but often do not develop clinical signs until 2 to 5 yr of age. The clinical disease is characterized by chronic or intermittent diarrhea, emaciation, and death. Although animals with clinical disease are often culled from the herd, animals with subclinical paratuberculosis may cause economic losses because of reduced milk production and poor reproductive performance. Although the economic impact of paratuberculosis on the national cattle industry has not been determined, it is estimated to exceed $1.5 billion/yr. The diagnosis of subclinical paratuberculosis is difficult. Bacteriologic culture is the most definitive method of diagnosis, but culture is time consuming and labor intensive. Serological assays are not very useful because animals do not develop an antibody response until the clinical stages of disease. Development of assays to measure cell-mediated immunity is critical to accurate detection of paratuberculosis in subclinically infected animals. Although not considered a zoonotic agent, M. paratuberculosis has been identified in intestinal biopsy tissue from patients with Crohn's disease, an inflammatory enteritis in humans. Currently, the potential human health risk is being addressed by research evaluating pasteurization of dairy products in the US.

The associations between alleles at the BoLA (bovine lymphocyte antigen) DRB3 locus and 20 indicator traits of innate and adaptive immunity were investigated. Periparturient Holsteins (n = 127) were genotyped at the BoLA DRB3 locus using polymerase chain reaction and restriction fragment length polymorphism. Twenty-two alleles were observed in the study population, and frequencies ranged from 21 to < 1%. The same cattle were tested for a total of 20 innate and adaptive immunity traits, including lymphocyte response to mitogens (proliferative responses and Ig secretion), serum Ig, complement and conglutinin concentrations, total leukocyte count, and selected assays for neutrophil function. Models with gene substitution effects were used to investigate associations between BoLA DRB3 alleles and each of the immunological variables. Significant associations were found with 13 of the leukocyte functions tested. The number of immune parameters with significant associations with any allele ranged from 0 (with alleles DRB3.2*23 and DRB3.2*27) to 7 (with DRB3.2*8). The immunological parameter that had the most associations with alleles was serum IgG2 concentration with 6 alleles. One group of 4 alleles (representing 46% of the total allele frequency) was uniformly associated with increased IgM and complement and decreased mononuclear cell numbers. Thus, we demonstrated that the BoLA DRB3 genotype can influence measures of innate and adaptive immunity.

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.

Immunomodulatory effects of recombinant bovine granulocyte colony-stimulating factor were evaluated in periparturient dairy cows. Eleven of 21 cows were experimentally infected with Staphylococcus aureus in one mammary quarter prior to the study. Cows were assigned to four groups in a randomized complete block design to evaluate the effects of recombinant bovine granulocyte colony-stimulating factor (5 micrograms/kg of body weight or placebo injected subcutaneously once daily beginning 14 d prepartum through 10 d postpartum) on infected and uninfected cows during the periparturient period. Blood lymphocytes were isolated and evaluated from 5 wk before expected parturition through 7 wk postpartum. Lymphocyte function was evaluated using a blastogenesis assay, a mitochondrial methylthiazoltetrazolium cleavage activity assay, and an in vitro assay of IgM production. Serum concentrations of IgM, IgG1, conglutinin, and hemolytic complement were also determined. Injections of cows with recombinant bovine granulocyte colony-stimulating factor resulted in enhanced lymphocyte blastogenesis and mitochondrial methylthiazoltetrazolium cleavage activity in unstimulated cultures, higher serum IgM, and increased in vitro IgM production by B lymphocytes. These data provide support for the use of recombinant bovine granulocyte colony-stimulating factor to alleviate immunosuppression in periparturient cows.

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.

Retinol (vitamin A), alpha-tocopherol (vitamin E), and Zn are micronutrients essential for health and performance. We determined the effects of parturition, lactation, and periparturient Ca status on plasma retinol, alpha-tocopherol, and Zn in 18 Jersey cows during the 2 wk before and after parturition. Six cows developed clinical milk fever. Prepartum plasma concentrations of retinol, alpha-tocopherol, and Zn decreased progressively in all animals. A nadir was reached at 1 d postpartum when concentrations declined to 38, 47, and 67%, respectively, of prepartal baseline concentrations. Plasma Zn returned to baseline concentrations within 3 d of calving, and plasma alpha-tocopherol returned toward baseline about 10 d after calving. Plasma retinol remained below baseline concentrations throughout the first 2 wk of lactation. The decline in plasma Zn observed at calving was more severe in cows with milk fever (parturient paresis) than in cows without milk fever. The decrease in plasma retinol and alpha-tocopherol observed at parturition was similar in cows with or without milk fever. These data document an acute decline in plasma retinol, alpha-tocopherol, and Zn in the immediate periparturient period and indicate that the decline in plasma Zn is more severe in cows with milk fever.