Differential phenotype of immune cells in blood and milk following pegylated granulocyte colony-stimulating factor therapy during a chronic Staphylococcus aureus infection in lactating Holsteins.

Neutrophils are principal host innate immune cell responders to mastitis infections. Thus, therapies have been developed that target neutrophil expansion. This includes the neutrophil-stimulating cytokine granulocyte colony-stimulating factor (gCSF). Pegylated gCSF (PEG-gCSF; Imrestor, Elanco Animal Health, Greenfield, IN) has been shown to reduce the natural incidence of mastitis in periparturient cows in commercial settings and reduce severity of disease against experimental mastitis challenge. Pegylated gCSF stimulates neutrophil expansion but also induces changes in monocyte and lymphocyte circulating numbers, surface protein expression changes, or both. We hypothesized that PEG-gCSF modulates surface expression of monocytes and neutrophils and facilitates their migration to the mammary gland. We challenged 8 mid-lactation Holsteins with approximately 150 cfu of Staphylococcus aureus (Newbould 305) in a single quarter via intramammary infusion. All animals developed chronic infections as assessed by bacteria counts and somatic cell counts (SCC). Ten to 16 wk postchallenge, 4 of the animals were treated with 2 subcutaneous injections of PEG-gCSF 7 d apart. Complete blood counts, SCC, bacterial counts, milk yield, feed intake, neutrophils extracellular trap analysis, and flow cytometric analyses of milk and blood samples were performed at indicated time points for 14 d after the first PEG-gCSF injection. The PEG-gCSF-treated cows had significantly increased numbers of blood neutrophils and lymphocytes compared with control cows. Flow cytometric analyses revealed increased surface expression of myeloperoxidase (MPO) on neutrophils and macrophages in milk but not in blood of treated cows. Neutrophils isolated from blood of PEG-gCSF-treated cows had decreased surface expression of CD62L (L-selectin) in blood, consistent with cell activation. Surprisingly, CD62L cell surface expression was increased on neutrophils and macrophages sourced from milk from treated animals compared with cells isolated from controls. The PEG-gCSF-treated cows did not clear the S. aureus infection, nor did they significantly differ in SCC from controls. These findings provide evidence that PEG-gCSF therapy modifies cell surface expression of neutrophils and monocytes. However, although surface MPO+ cells accumulate in the mammary gland, the lack of bacterial control from these milk-derived cells suggests an incomplete role for PEG-gCSF treatment against chronic S. aureus infection and possibly chronic mammary infections in general.

Short communication: Vitamin D status and responses in dairy cows naturally infected with Mycobacterium avium ssp. paratuberculosis.

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 D3 (25OHD3) 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 25OHD3 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 25OHD3 for animals with clinical disease may suggest that these cows have reduced innate immune responses, thereby influencing the ability of animals to fight infection.

Symposium review: Omics in dairy and animal science-Promise, potential, and pitfalls.

Sequencing the first genome took 15 yr and $3 billion to complete. Currently, a genome can be sequenced in a day for a few thousand dollars. Comparing the relative abundance of nearly every mRNA transcript and small RNAs from cells and tissues from different experimental conditions has become so easy that it can take longer to transfer the data between computers than to perform the experiment. Nucleotide sequencing techniques have become so sensitive that the greatest concern is not detecting a gene or transcript but rather, falsely identifying one. Better genome sequencing has led to more complete transcriptomic and proteomic databases and, combined with more sensitive instrumentation and separation techniques, is bringing us closer to detecting complete transcriptomes and proteomes. The promise of these powerful omics techniques is to lead us to new and unexpected connections between molecular processes in the context of animal health. This promise cannot be achieved without hypothesis-driven research that connects omics data with animal health experiments. Any researcher who wishes to invest the time and resources in omics experiments should be aware of the common pitfalls and limitations of these techniques so they can avoid these issues and maximize the use of these research tools. Several important questions must be asked: What is the quality of the databases and how they are annotated? Are the annotations based on experimental results or computational predictions? What assumptions are made by the analysis algorithms, and how will this affect the result? Finally, how can the research community use the vast amount of data being generated by omics experiments in ways to achieve the goals of better animal health and production (which is the promise of omics technologies)? Until the observations shown in omics data sets are used to achieve the goals of better animal health and production, the potential of omics technology will not be fully realized.

The effect of pegylated granulocyte colony-stimulating factor treatment prior to experimental mastitis in lactating Holsteins.

Neutrophils are the first-acting and most prominent cellular defense against mastitis-causing pathogens. This makes neutrophil activation and expansion obvious candidates for targeted therapeutics. The granulocyte colony-stimulating factor (G-CSF) cytokine stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream, which results in neutrophilia as well as increasing the presence of other progenitor cells in the bloodstream. A pegylated form of G-CSF (PEG-gCSF) has been shown to significantly decrease naturally occurring mastitis rates in cows postpartum. The use of PEG-gCSF had not been evaluated in response to an experimental mastitis challenge. In an effort to examine the effect and mechanism of PEG-gCSF treatment, we challenged 11 mid-lactation Holsteins with ∼400 cfu Escherichia coli P4 by intramammary infusion. Five cows received 2 PEG-gCSF injections, one at 14 d and the other at 7 d before disease challenge, and 6 cows remained untreated. To evaluate the response of cows to the PEG-gCSF treatment, we measured complete blood counts, somatic cell counts, bacterial counts, milk yield, and feed intake data. The PEG-gCSF-treated cows had significantly increased circulating levels of neutrophils and lymphocytes after each PEG-gCSF injection, as well as following mastitis challenge. The PEG-gCSF-treated cows had significantly lower bacterial counts and lower milk BSA levels at the peak of infection. In addition, control cows had significant decreases in milk yield postinfection and significantly reduced feed intake postinfection compared with PEG-gCSF-treated cows. Collectively, PEG-gCSF treatment resulted in reduced disease severity when administered before a bacterial challenge. Mechanistically, we show that G-CSF treatment increases cell surface expression of an E-selectin ligand before infection on neutrophils and monocytes found in the blood. These cells quickly disappear from the blood shortly after infection, suggesting a mechanism for the reduced mastitis severity by priming immune cells for quick targeting to the site of infection.

Novel role for IL-22 in protection during chronic Mycobacterium tuberculosis HN878 infection.

Approximately 2 billion people are infected with Mycobacterium tuberculosis (Mtb), resulting in 1.4 million deaths every year. Among Mtb-infected individuals, clinical isolates belonging to the W-Beijing lineage are increasingly prevalent, associated with drug resistance, and cause severe disease immunopathology in animal models. Therefore, it is exceedingly important to identify the immune mechanisms that mediate protection against rapidly emerging Mtb strains, such as W-Beijing lineage. IL-22 is a member of the IL-10 family of cytokines with both protective and pathological functions at mucosal surfaces. Thus far, collective data show that IL-22 deficient mice are not more susceptible to aerosolized infection with less virulent Mtb strains. Thus, in this study we addressed the functional role for the IL-22 pathway in immunity to emerging Mtb isolates, using W-Beijing lineage member, Mtb HN878 as a prototype. We show that Mtb HN878 stimulates IL-22 production in TLR2 dependent manner and IL-22 mediates protective immunity during chronic stages of Mtb HN878 infection in mice. Interestingly, IL-22-dependent pathways in both epithelial cells and macrophages mediate protective mechanisms for Mtb HN878 control. Thus, our results project a new protective role for IL-22 in emerging Mtb infections.

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.

Effect of feeding 25-hydroxyvitamin D3 with a negative cation-anion difference diet on calcium and vitamin D status of periparturient cows and their calves.

Holstein cows (>1 gestation) were fed 1 of 3 diets during the last 13 d of gestation (ranged from 22 to 7 d). The control diet (16 cows) was formulated to provide 18,000 IU/d of vitamin D3 and had a dietary cation-anion difference (DCAD) of 165mEq/kg (DCAD=Na + K - Cl - S). The second diet (DCAD + D) provided the same amount of vitamin D3 but had a DCAD of -139mEq/kg (17 cows). The third diet (DCAD + 25D) had no supplemental vitamin D3 but provided 6mg/d of 25-(OH) vitamin D3 [25-(OH)D3] with a DCAD of -138mEq/kg (20 cows). Diets were fed until parturition and then all cows were fed a common lactation diet that contained vitamin D3. Negative DCAD diets reduced urine pH, with the greatest decrease occurring with the DCAD + D treatment. Urinary Ca excretion was greatest for cows fed DCAD + 25D followed by cows fed DCAD + D. Urinary pH was negatively correlated with urinary excretion of Ca for cows fed DCAD + D. No such correlation was observed with the DCAD + 25D treatment because substantial excretion of urinary Ca occurred at moderate urinary pH values for that treatment. Cows fed DCAD + 25D had greater serum concentrations of 25-(OH)D3 than other treatments from 5 d after supplementation started through 7 d in milk. Concentrations of 1,25-(OH)2D3 in serum were greatest in DCAD + 25D cows starting at 2 d before calving and continued through 7 d in milk. Serum Ca concentrations 5 d before calving were greatest for cows fed DCAD + 25D, but at other time points before and after parturition treatment did not affect serum Ca. Incidence of clinical hypocalcemia was not statistically different between treatments, but cows fed DCAD + 25 had the highest incidence rate (12.5, 0, and 20% for control, DCAD + D, and DCAD + 25D). Calves born from cows fed DCAD + 25D had greater concentrations of 25-(OH)D3 in serum at birth than calves from other treatments (before colostrum consumption), but concentrations were similar by 3 d of age. Concentrations of 25-(OH)D3 in colostrum and transition milk were increased by feeding DCAD + 25D, but by 28 d in milk treatment effects no longer existed. Overall, feeding 25-OH vitamin D with a negative DCAD diet increased vitamin D status of the cow and her newborn calf but had minimal effects on calcium status and did not have positive effects on the incidence of hypocalcemia.

Seasonal variation in vitamin D status of beef cattle reared in the central United States.

The objective was to retrospectively measure seasonal sunlight-associated variation in serum concentrations of 25-hydroxyvitamin D (25OHD) in beef cattle. The concentration of 25OHD was measured in crossbred animals born from March to May in 2011 and 2012. Vitamin D status 2 to 3 mo after birth (period 1) was only available for 2012 calves and was measured in June 2012. Period 1 animals had serum 25OHD concentrations of 26.3 ± 1.5 ng/mL. The 25OHD concentrations for late summer (period 2) were 46.6 ± 1.4 and 51.0 ± 1.5 ng/mL for 2011 and 2012, respectively. Serum concentration of 25OHD in early fall (period 3) were 63.8 ± 1.4 and 55.2 ± 1.5 ng/mL for calves in 2011 and 2012, respectively. Values observed for both late summer and early fall indicated vitamin D sufficiency (P < 0.001) compared with period 1. With diminishing exposure to ultraviolet B and consuming ∼800 IU or 1800 IU (2011 and 2012, respectively) of supplemental vitamin D, the calves' midwinter (period 4) 25OHD concentrations fell to 15.2 ± 1.6 and 16.7 ± 1.5 ng/mL for 2011 and 2012, respectively, after 4 to 5 mo on a finishing diet (P < 0.0001). This is considered vitamin D insufficiency in most species. Results indicate that calves are marginally sufficient to insufficient for vitamin D based on serum 25OHD concentrations soon after birth and during winter. Some individual animals would be classified vitamin D deficient. In the absence of sufficient UVB exposure, the dietary vitamin D requirements for rapidly growing beef cattle may need to be increased.

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.

A genomewide association study identified CYP2J2 as a gene controlling serum vitamin D status in beef cattle.

Vitamin D is an important modulator of calcium homeostasis and has several effects on the immune system. The objective of the study was to estimate its heritability and to identify genomic regions associated with concentration of circulating 25-hydroxyvitamin D (25OHD) in beef cattle. Status of vitamin D was measured in crossbred animals from Cycle VII of the United States Meat Animal Research Center (USMARC) Germplasm Evaluation Project. Progeny were born from March through May in 2008 and in 2010. Heritability was estimated and a genomewide association study was conducted on the concentration of 25OHD measured in 1,432 animals at preconditioning and 1,333 animals at weaning. Genotyping of the population was done by imputing from the parental generation genotyped with a high density array (777,000 SNP) to a target population genotyped with a medium density SNP array (50,000 SNP). After imputation, 675,018 SNP were used in the genomewide association study. Heritability of concentration of circulating 25OHD in cattle at preconditioning and at weaning was 0.41 ± 0.08 and 0.32 ± 0.11, respectively. A region on chromosome 3 was associated with circulating 25OHD. The region on BTA3 had 7 SNP significantly (P < 7.4 × 10(-8)) associated at the genomewide level with serum concentrations of serum 25OHD. Genomewide significant SNP spanned the region between 84.93 and 86.65 megabases (Mb); however, 6 SNP reside between 86.64 and 86.65 Mb. The gene CYP2J2 was identified as a candidate gene associated with concentrations of serum 25OHD in cattle. This is 1 of 6 enzymes involved in metabolizing vitamin D to 25OHD. Results from the present study suggest that CYP2J2 is a gene controlling serum 25OHD concentrations in cattle. CYP2J2 should be considered a prime candidate for understanding both genetic and physiological factors affecting serum 25OHD concentrations in cattle and, therefore, vitamin D status.

Modulation of the bovine innate immune response by production of 1alpha,25-dihydroxyvitamin D(3) in bovine monocytes.

In cattle, the kidney has been the only known site for production of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] from 25-hydroxyvitamin D(3) [25(OH)D(3)] by 1alpha-hydroxylase (1alpha-OHase). Based on human studies, it was hypothesized that bovine monocytes could produce 1,25(OH)(2)D(3) upon activation and 1,25(OH)(2)D(3) would regulate expression of vitamin D-responsive genes in monocytes. First, the effects of 1,25(OH)(2)D(3) on bovine monocytes isolated from peripheral blood were tested. Treatment of nonstimulated monocytes with 1,25(OH)(2)D(3) increased expression of the gene for the vitamin D 24-hydroxylase (24-OHase) enzyme by 51+/-13 fold, but 1,25(OH)(2)D(3) induction of 24-OHase expression was blocked by lipopolysaccharide (LPS) stimulation. In addition, 1,25(OH)(2)D(3) increased the gene expression of inducible nitric oxide synthase and the chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted) in LPS-stimulated monocytes 69+/-13 and 40+/-12 fold, respectively. Next, the ability of bovine monocytes to express 1alpha-OHase and produce 1,25(OH)(2)D(3) was tested. Activation of monocytes with LPS, tripalmitoylated lipopeptide (Pam3CSK4), or peptidoglycan caused 43+/-9, 17+/-3, and 19+/-3 fold increases in 1alpha-OHase gene expression, respectively. Addition of 25(OH)D(3) to LPS-stimulated monocytes enhanced expression of inducible nitric oxide synthase and RANTES and nitric oxide production in a dose-dependent manner, giving evidence that activated monocytes convert 25(OH)D(3) to 1,25(OH)(2)D(3). In conclusion, bovine monocytes produce 1,25(OH)(2)D(3) in response to toll-like receptor signaling, and 1,25(OH)(2)D(3) production in monocytes increased the expression of genes involved in the innate immune system. Vitamin D status of cattle might be important for optimal innate immune function because 1,25(OH)(2)D(3) production in activated monocytes and subsequent upregulation of inducible nitric oxide synthase and RANTES expression was dependent on 25(OH)D(3) availability.

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.

Centennial paper: Proteomics in animal science.

Proteomics holds significant promise as a method for advancing animal science research. The use of this technology in animal science is still in its infancy. The ability of proteomics to simultaneously identify and quantify potentially thousands of proteins is unparalleled. In this review, we will discuss basic principles of doing a proteomic experiment. In addition, challenges and limitations of proteomics will be considered, stressing those that are unique to animal sciences. The current proteomic research in animal sciences will be discussed, and the potential uses for this technology will be highlighted.

Developmental changes in the milk fat globule membrane proteome during the transition from colostrum to milk.

Shotgun proteomics, using amine-reactive isobaric tags (iTRAQ), was used to quantify protein changes in milk fat globule membranes (MFGM) that were isolated from d 1 colostrum and compared with MFGM from d 7 milk. Eight Holstein cows were randomly assigned to 2 groups of 4 cow sample pools for a simple replication of this proteomic analysis using iTRAQ. The iTRAQ labeled peptides from the experiment sample pools were fractionated by strong cation exchange chromatography followed by further fractionation on a microcapillary high performance liquid chromatograph connected to a nanospray-tandem mass spectrometer. Data analysis identified 138 bovine proteins in the MFGM with 26 proteins upregulated and 19 proteins downregulated in d 7 MFGM compared with colostrum MFGM. Mucin 1 and 15 were upregulated greater than 7-fold in MFGM from d 7 milk compared with colostrum MFGM. The tripartite complex of proteins of adipophilin, butyrophilin, and xanthine dehydrogenase were individually upregulated in d 7 MFGM 3.4-, 3.2-, and 2.6-fold, respectively, compared with colostrum MFGM. Additional proteins associated with various aspects of lipid transport synthesis and secretion such as acyl-CoA synthetase, lanosterol synthase, lysophosphatidic acid acyltransferase, and fatty acid binding protein were upregulated 2.6- to 5.1-fold in d 7 MFGM compared with colostrum MFGM. In contrast, apolipoproteins A1, C-III, E, and A-IV were downregulated 2.6- to 4.3-fold in d 7 MFGM compared with colostrum MFGM. These data demonstrate that quantitative shotgun proteomics has great potential to provide new insights into mammary development.

Parturition and hypocalcemia blunts calcium signals in immune cells of dairy cattle.

The stress of parturition in the dairy cow is associated with increased susceptibility to infectious disease. During the periparturient period the demands for calcium are increased; these increased demands for calcium can result in subclinical or clinical hypocalcemia. Periparturient cows also experience significant immune suppression. Because intracellular calcium signaling is a key early feature in immune cell activation, we have hypothesized that the increased demand for calcium in periparturient cows may adversely affect intracellular calcium stores of immune cells. This reduction in intracellular calcium stores in immune cells could blunt intracellular calcium release following an activating stimulus, contributing to the immune suppression seen in these animals. To test this hypothesis, peripheral mononuclear cells were obtained from 27 multiparous dairy cows spanning a period of 2 wk before and 2 wk after parturition. Following activation of these cells by anti-CD3 antibodies plus secondary antibodies, intracellular calcium release from intracellular stores was measured. The intracellular calcium released in response to the activation signal declined as calcium demand for lactation became more intense and recovered as plasma calcium normalized. Intracellular calcium stores in peripheral mononuclear cells, estimated by pretreating cells with pervanadate and ionomycin, significantly decreased at parturition and returned to normal levels as the cows' blood calcium returned to normal levels. Hypocalcemia, which is common in periparturient dairy cows, is associated with decreased intracellular calcium stores in peripheral mononuclear cells. Our data suggest that this is the cause of a blunted intracellular calcium release response to an immune cell activation signal. It is concluded that intracellular Ca stores decrease in peripheral blood mononuclear cells (PBMC) before parturition and development of hypocalcemia. This suggests that systemic calcium stress precedes measurable hypocalcemia, particularly in cows that will develop milk fever. Therefore, PBMC intracellular Ca stores are a more sensitive measure of calcium stresses in transition cow. This decrease in PBMC intracellular Ca stores before parturition and the development of hypocalcemia contributes to periparturient immune suppression.

Expression library immunization confers protection against Mycobacterium avium subsp. paratuberculosis infection.

Currently, paratuberculosis vaccines are comprised of crude whole-cell preparations of Mycobacterium avium subsp. paratuberculosis. Although effective in reducing clinical disease and fecal shedding, these vaccines have severe disadvantages as well, including seroconversion of vaccinated animals and granulomatous lesions at the site of vaccination. DNA vaccines can offer an alternative approach that may be safer and elicit more protective responses. In an effort to identify protective M. avium subsp. paratuberculosis sequences, a genomic DNA expression library was generated and subdivided into pools of clones (approximately 1,500 clones/pool). The clone pools were evaluated to determine DNA vaccine efficacy by immunizing mice via gene gun delivery and challenging them with live, virulent M. avium subsp. paratuberculosis. Four clone pools resulted in a significant reduction in the amount of M. avium subsp. paratuberculosis recovered from mouse tissues compared to mice immunized with other clone pools and nonvaccinated, infected control mice. One of the protective clone pools was further partitioned into 10 clone arrays of 108 clones each, and four clone arrays provided significant protection from both spleen and mesenteric lymph node colonization by M. avium subsp. paratuberculosis. The nucleotide sequence of each clone present in the protective pools was determined, and coding region functions were predicted by computer analysis. Comparison of the protective clone array sequences implicated 26 antigens that may be responsible for protection in mice. This study is the first study to demonstrate protection against M. avium subsp. paratuberculosis infection with expression library immunization.

Short communication: Ca2+-adenosine triphosphatase protein expression in the mammary gland of periparturient cows.

The objectives of this study were to measure the changes in protein expression of the mammary Ca2+-ATPases during the periparturient period and to determine whether Ca2+-ATPase protein expression in the mammary gland is related to milk fever (MF) development. Abundance of Ca2+-ATPase in mammary tissue and milk fat globule membranes was determined by Western blotting. The secretory pathway Ca2+-ATPase was elevated prepartum in mammary tissue from cows that developed MF compared with non-MF cows.

Role of vitamin D in calcium homeostasis and its use in prevention of bovine periparturient paresis.

Calcium (Ca) is essential for life in higher animals. It is involved in the normal functioning of a wide variety of tissues and physiologic processes which include bone formation, muscle contraction, nerve transmission, blood clotting and as a second messenger regulating the actions of many hormones. In order for these functions to be carried out properly, blood Ca concentrations must be monitored and regulated within strict limitations. The discovery of the vitamin D endocrine system has resulted in the realization that Ca regulation in mammals and birds involves a coordinated effort between the hormones parathyroid hormone (PTH), calcitonin and the hormonally-active form of vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Failure of this system to maintain normal blood Ca concentrations at parturition is a common occurrence in ruminants leading to clinical (periparturient paresis, milk fever) and subclinical hypocalcemia. Vitamin D sterols have played a significant role in efforts to avoid parturient hypocalcemia and this report will summarize advantages and disadvantages associated with their use.

Calcitroic acid is a major catabolic metabolite in the metabolism of 1 alpha-dihydroxyvitamin D(2).

Calcitroic acid (1 alpha-hydroxy-23 carboxy-24,25,26,27-tetranorvitamin D(3)) is known to be the major water-soluble metabolite produced during the deactivation of 1 alpha,25-dihydroxyvitamin D(3). This deactivation process involves a series of oxidation reactions at C(24) and C(23) leading to side-chain cleavage and, ultimately, formation of the calcitroic acid. Like 1 alpha,25-dihydroxyvitamin D(3), 1 alpha,25-dihydroxyvitamin D(2) is also known to undergo side-chain oxidation; however, to date there has been no evidence suggesting that 1 alpha,25-dihydroxyvitamin D(2) undergoes side-chain cleavage. To investigate this possibility, we studied 1 alpha,25-dihydroxyvitamin D(2) metabolism in HPK1A-ras cells as well as the well characterized perfused rat kidney system. Lipid and aqueous-soluble metabolites were prepared for characterization. Aqueous-soluble metabolites were subjected to reverse-phase HPLC analysis. The major aqueous-soluble metabolite from both the kidney and cell incubations comigrated with authentic calcitroic acid on two reverse-phase HPLC columns of different chemistry. The putative calcitroic acid from the cell and kidney incubations was methylated and found to comigrate with methylated authentic standard on straight-phase and reverse-phase HPLC columns. The identity of the methylated metabolite from cell incubations was also confirmed by mass spectral analysis. These data show, for the first time, that calcitroic acid is a major terminal product for the deactivation of 1 alpha,25-dihydroxyvitamin D(2). Intermediates leading to the formation of the calcitroic acid in the 1 alpha,25-dihydroxyvitamin D(2) metabolism pathway are currently being studied.