Role of glucuronidated 25-hydroxyvitamin D on colon gene expression in mice.

25-Hydroxyvitamin D3-3ß-glucuronic acid (25OHD-Gluc) is produced in the liver and is a constituent of human blood and bile. Bacterial glucuronidases (GUS) in mammalian digestive microbiota cleave glucuronide conjugates, such as 25OHD-Gluc, and release the free aglycone (i.e., 25OHD) inside the intestinal lumen. We hypothesized that 25OHD-Gluc would elicit a VDR-dependent mRNA response in the colon after cleavage by gut microbiota. The activity of 25OHD-Gluc was investigated by measuring expression of cytochrome P450 24A1 (Cyp24) mRNA both in vitro and in vivo. In cell culture, Caco2 cells responded to 25OHD-Gluc, whereas HT29 cells did not. When coincubated with GUS, both cell lines elicited a robust response as indicated by a 5 Ct (32-fold) increase in Cyp24 mRNA. In vitamin D-sufficient mice, we found that both oral and subcutaneous administration of 1 nmol 25OHD-Gluc induced expression of Cyp24 mRNA in the colon whereas 25OHD did not. In contrast, 25OHD, but not 25OHD-Gluc, was active in the duodenum. When the jejunum was surgically ligated to block flow of digesta to the colon, neither oral nor subcutaneous administration of 2 nmol 25OHD-Gluc was able to induce expression of Cyp24 in the colon. Our findings suggest that 25OHD-Gluc, a vitamin D metabolite found in bile, induces VDR-mediated responses in the colon by crossing the apical membrane of the colon epithelium.NEW & NOTEWORTHY We found that 25OHD-Gluc, an endogenously produced metabolite, is delivered to the colon via bile to induce vitamin D-mediated responses in the colon.

Oral 25-Hydroxycholecalciferol Acts as an Agonist in the Duodenum of Mice and as Modeled in Cultured Human HT-29 and Caco2 Cells.

BACKGROUND: 25-Hydroxycholecalciferol [25(OH)D] is the predominant circulating metabolite of vitamin D and serves as the precursor for 1α,25-dihydroxycholecalciferol [1,25(OH)2D], the hormonally active form. The presence of 1α-hydroxylase (1α-OHase) in the intestine suggests that 1,25(OH)2D can be produced from 25(OH)D, but the effects of oral 25(OH)D on the intestine have not been determined. OBJECTIVES: We investigated the acute intestinal response to orally consumed 25(OH)D in mice by assessing mRNA induction of cytochrome p450 family 24 subfamily A member 1 (Cyp24), a vitamin D-dependent gene. The mechanism of action then was determined through in vitro analyses with Caco2 and HT-29 cells. METHODS: Adult male C57BL6 mice were given a single oral dose of 40, 80, 200, or 400 ng 25(OH)D (n = 4 per dose) or vehicle (n = 3), and then killed 4 h later to evaluate the duodenal expression of Cyp24 mRNA by qPCR and RNA in situ hybridization. The 25(OH)D-mediated response was also evaluated with Caco2 and HT-29 cells by inhibition assay and dose-response analysis. A cytochrome p450 family 27 subfamily B member 1 (CYP27B1) knockdown of HT-29 was created to compare the dose-response parameters with wild-type HT-29 cells. RESULTS: Oral 25(OH)D induced expression of Cyp24 mRNA in the duodenum of mice with 80 ng 25(OH)D by 3.3 ± 0.8 ΔΔCt compared with controls (P < 0.05). In vitro, both Caco2 and HT-29 cells responded to 25(OH)D treatment with 200-fold and 175-fold greater effective concentration at 50% maximal response than 1,25(OH)2D, yet inhibition of 1α-OHase and knockdown of CYP27B1 had no effect on the responses. CONCLUSIONS: In mice, orally consumed 25(OH)D elicits a vitamin D-mediated response in the duodenum. In vitro assessments suggest that the response from 25(OH)D does not require activation by 1α-OHase and that 25(OH)D within the intestinal lumen acts as a vitamin D receptor agonist.

Localization of the 1,25-dihydroxyvitamin d-mediated response in the intestines of mice.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D) elicits a transcriptional response in the intestines. Assessments of this response are often derived from crude tissue homogenates and eliminate the ability to discriminate among different cell types. Here, we used an RNA in situ hybridization assay, RNAScope (Advanced Cell Diagnostics, Newark, CA), to identify the cells in the intestine that respond to 1,25(OH)2D with expression of cytochrome P450 family 24 subfamily A member 1 (Cyp24a1) mRNA. Mice were gavaged with a single bolus dose of 1,25(OH)2D to target the duodenum or a glucuronic acid conjugate of 1,25(OH)2D, ß-G-1,25(OH)2D, to target the colon. QRT-PCR analysis of Cyp24a1 mRNA verified that the 1,25(OH)2D-induced responses were present. RNAScope revealed that the mRNA response present after six hours is limited to mature enterocytes exposed to the intestinal lumen in both the duodenum and colon. No detectable expression was observed in goblet cells, lamina propria, muscularis mucosa muscle, submucosa and submucosal lymphoid follicles, or tunica muscularis. Our findings have identified epithelial enterocytes to be the intestinal targets for 1,25(OH)2D in both the duodenum and colon.

Comparison of 0.46% calcium diets with and without added anions with a 0.7% calcium anionic diet as a means to reduce periparturient hypocalcemia.

Most studies demonstrating that diets with low dietary cation-anion difference (DCAD) reduce hypocalcemia in cows add enough anions to the diet to reduce urine pH below 7.0. One objective of these experiments was to determine whether there is any benefit to periparturient plasma Ca concentration if diet anion addition results in a lesser degree of acidification of the cow and urine pH does not go below 7.0. Another method for reducing hypocalcemia involves feeding a prepartal diet that is Ca deficient. This places the cow in negative Ca balance before calving, stimulating parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D secretion before calving and thus promoting Ca homeostasis at calving. As practiced in the field, low-Ca diets are often about 0.5% Ca. Our second objective was to determine whether a 0.46% Ca diet would be sufficiently low in Ca to stimulate PTH secretion before calving. A meta-analysis of the literature suggests that a 0.5% Ca, low-DCAD diet will reduce hypocalcemia better than a 0.7% Ca diet. A third objective was to compare periparturient plasma Ca in cows fed 0.46 or 0.72% Ca diets with similar DCAD. In experiment 1, anions (primarily chloride) or anions plus Ca were added to a 1.4% K basal diet to create the following diets: 0.46% Ca and +167 mEq/kg of DCAD, 0.46% Ca and -13 mEq/kg of DCAD, and 0.72% Ca and -17 mEq/kg of DCAD. In experiment 2, the same amounts of anion were added to a 2.05% K basal diet to create the following diets: 0.46% Ca and +327 mEq/kg of DCAD, 0.46% Ca and +146 mEq/kg of DCAD, and 0.72% Ca and +140 mEq/kg of DCAD. In experiment 1, cows fed the diet with 0.46% Ca and +167 mEq/kg of DCAD had significantly lower plasma Ca concentration after calving than cows fed the 0.46 or 0.72% Ca diets with anions. Periparturient plasma Ca concentrations did not differ in cows fed the low-DCAD diets with 0.46 or 0.72% Ca. Urine pH was reduced from 8.27 in the diet with 0.46% Ca and +167 mEq/kg of DCAD to 7.07 and 7.41 in the 0.46 and 0.72% Ca anion diets, respectively. Precalving plasma PTH and 1,25-dihydroxyvitamin D concentrations were similar in cows fed the 0.46% Ca diets and the 0.72% Ca diets, suggesting that the 0.46% Ca diets were not low enough in Ca to place the cow in negative Ca balance before calving. In experiment 2, adding the anion supplements to a 2.05% K diet did not reduce urine pH below 8.0. Periparturient plasma Ca concentrations did not differ in cows in any group in experiment 2. Precalving diets that are 0.46% Ca fed ad libitum are too high in Ca to stimulate Ca homeostasis before calving. Adding anions to a diet can benefit periparturient cow plasma Ca concentration, but only if it alters acid-base status enough to reduce urine pH below 7.5.

Invited review: Mineral absorption mechanisms, mineral interactions that affect acid-base and antioxidant status, and diet considerations to improve mineral status.

Several minerals are required for life to exist. In animals, 7 elements (Ca, P, Mg, Na, K, Cl, and S) are required to be present in the diet in fairly large amounts (grams to tens of grams each day for the dairy cow) and are termed macrominerals. Several other elements are termed microminerals or trace minerals because they are required in much smaller amounts (milligrams to micrograms each day). In most cases the mineral in the diet must be absorbed across the gastrointestinal mucosa and enter the blood if it is to be of value to the animal. The bulk of this review discusses the paracellular and transcellular mechanisms used by the gastrointestinal tract to absorb each of the various minerals needed. Unfortunately, particularly in ruminants, interactions between minerals and other substances within the diet can occur within the digestive tract that impair mineral absorption. The attributes of organic or chelated minerals that might permit diet minerals to circumvent factors that inhibit absorption of more traditional inorganic forms of these minerals are discussed. Once absorbed, minerals are used in many ways. One focus of this review is the effect macrominerals have on the acid-base status of the animal. Manipulation of dietary cation and anion content is commonly used as a tool in the dry period and during lactation to improve performance. A section on how the strong ion theory can be used to understand these effects is included. Many microminerals play a role in the body as cofactors of enzymes involved in controlling free radicals within the body and are vital to antioxidant capabilities. Those same minerals, when consumed in excess, can become pro-oxidants in the body, generating destructive free radicals. Complex interactions between minerals can compromise the effectiveness of a diet in promoting health and productivity of the cow. The objective of this review is to provide insight into some of these mechanisms.

Targeted delivery of 1,25-dihydroxyvitamin D3 to colon tissue and identification of a major 1,25-dihydroxyvitamin D3 glycoside from Solanumglaucophyllum plant leaves.

Leaves of the Solanum glaucophyllum (Sg) plant, indigenous to South America, have long been known for their calcinogenic toxicity in ruminant animals. It was determined the leaves contained glycosidic derivatives of 1,25-dihydroxyvitamin D3 (1,25D3) and liberation of the free hormone by rumen bacterial populations elicited a hypercalcemic response. Our interest in the leaves is predicated on the concept that the glycoside forms of 1,25D3 would target release of the active hormone in the lower gut of non-ruminant mammals. This would provide a means of delivering 1,25D3 directly to the colon, where the hormone has been shown to have beneficial effects in models of inflammatory bowel disease (IBD) and colon cancer. We fed mice for 10 days with variable amounts of Sg leaf. Feeding 7-333µg leaf/day produced no changes in plasma Ca(2+) and 1,25D3 concentrations, and only at ≥1000µg leaf/day did these values become significantly elevated compared to controls. Gene expression studies from colon tissue indicated a linear relationship between the amount of leaf consumed and expression of the Cyp24a1 gene. In contrast, Cyp24a1 gene expression in the duodenums and ileums of these mice was unchanged compared to controls. One of the major 1,25D3-glycosides was isolated from leaves following extraction and purification by Sep-Pak cartridges and HPLC fractionation. Ultraviolet absorbance was consistent with modification of the 1-hydroxyl group, and positive ion ESI mass spectrometry indicated a diglycoside of 1,25D3. 2-Dimensional NMR analyses were carried out and established the C1 proton of the A-ring was interacting with a C1' sugar proton, while the C3 proton of the A-ring was linked with a second C1' sugar proton. The structure of the isolated compound is therefore consistent with a ß-linked 1,3-diglycoside of 1,25D3. Thus, Sg leaf administered to mice at up to 333 ug/day can elicit colon-specific enhancement of Cyp24a1 gene expression without inducing hypercalcemia, and the 1,3-diglycoside is one of the major forms of 1,25D3 found in the leaf. This article is part of a Special Issue entitled '17th Vitamin D Workshop'.

Calcium and magnesium disorders.

Hypocalcemia is a clinical disorder that can be life threatening to the cow (milk fever) and predisposes the animal to various other metabolic and infectious disorders. Calcium homeostasis is mediated primarily by parathyroid hormone, which stimulates bone calcium resorption and renal calcium reabsorption. Parathyroid hormone stimulates the production of 1,25-dihydroxyvitamin D to enhance diet calcium absorption. High dietary cation-anion difference interferes with tissue sensitivity to parathyroid hormone. Hypomagnesemia reduces tissue response to parathyroid hormone.

Effect of recombinant bovine granulocyte colony-stimulating factor covalently bound to polyethylene glycol injection on neutrophil number and function in periparturient dairy cows.

Dairy cows often experience decreased immune function around the time of calving, typified by impaired polymorphonuclear neutrophil (PMN) function and a transient neutropenia. This is associated with increased disease incidence, including mastitis, retained placenta, and metritis. In an attempt to improve PMN functional capacity during the periparturient period, we injected cows with recombinant bovine granulocyte colony-stimulating factor covalently bound to polyethylene glycol (PEG rbG-CSF) twice subcutaneously, about 6d before calving and within 24h after calving. Twenty-one cows in their second pregnancy were enrolled in this study and divided into 2 groups: PEG rbG-CSF treated (n=11) and saline-treated controls (n=10). The PMN numbers quickly and dramatically increased after PEG rbG-CSF administration and remained elevated through the end of the experiment (13d after calving). Exocytosis of myeloperoxidase by stimulated PMN, which is generally decreased in periparturient cows, was markedly increased by PEG rbG-CSF after injection. Higher myeloperoxidase exocytosis persisted for at least 10d after calving. The PMN superoxide anion release and phagocytosis activity did not differ between groups. Injection of PEG rbG-CSF was safe for cows, with no significant negative effects observed. The greatest single effect of PEG rbG-CSF administration was a dramatic increase in circulating numbers of PMN. The increased numbers of PMN ready to move to a site of infection early in the course of an infection may improve the ability of the cow to ward off clinical disease in the periparturient period.

Human UGT1A4 and UGT1A3 conjugate 25-hydroxyvitamin D3: metabolite structure, kinetics, inducibility, and interindividual variability.

25-Hydroxyvitamin D3 (25OHD3) is used as a clinical biomarker for assessment of vitamin D status. Blood levels of 25OHD3 represent a balance between its formation rate and clearance by several oxidative and conjugative processes. In the present study, the identity of human uridine 5'-diphosphoglucuronyltransferases (UGTs) capable of catalyzing the 25OHD3 glucuronidation reaction was investigated. Two isozymes, UGT1A4 and UGT1A3, were identified as the principal catalysts of 25OHD3 glucuronidation in human liver. Three 25OHD3 monoglucuronides (25OHD3-25-glucuronide, 25OHD3-3-glucuronide, and 5,6-trans-25OHD3-25-glucuronide) were generated by recombinant UGT1A4/UGT1A3, human liver microsomes, and human hepatocytes. The kinetics of 25OHD3 glucuronide formation in all systems tested conformed to the Michaelis-Menten model. An association between the UGT1A4*3 (Leu48Val) gene polymorphism with the rates of glucuronide formation was also investigated using human liver microsomes isolated from 80 genotyped livers. A variant allele dose effect was observed: the homozygous UGT1A4*3 livers (GG) had the highest glucuronidation activity, whereas the wild type (TT) had the lowest activity. Induction of UGT1A4 and UGT1A3 gene expression was also determined in human hepatocytes treated with pregnane X receptor/constitutive androstane receptor agonists, such as rifampin, carbamazepine, and phenobarbital. Although UGT mRNA levels were increased significantly by all of the known pregnane X receptor/constitutive androstane receptor agonists tested, rifampin, the most potent of the inducers, significantly induced total 25OHD3 glucuronide formation activity in human hepatocytes measured after 2, but not 4 and 24 hours, of incubation. Finally, the presence of 25OHD3-3-glucuronide in both human plasma and bile was confirmed, suggesting that the glucuronidation pathway might be physiologically relevant and contribute to vitamin D homeostasis in humans.

Rickets: case series and diagnostic review of hypovitaminosis D in swine.

Rickets can be attributed to nutritional, genetic, hormonal, or toxic disturbances and is classified as a metabolic bone disease. Rickets is most often associated with inappropriate dietary levels of calcium, phosphorus, and/or vitamin D. During a 27-month period (January 2010 through March 2012), the Iowa State University Veterinary Diagnostic Laboratory investigated causes of sudden, unexpected death and lameness in growing pigs throughout the Midwestern United States. Clinical observations from 17 growing pig cases included weakness, lameness, reluctance to move, muscle fasciculations and/or tremors, tetany, and death. Ribs were weak, soft, and bent prior to breaking; rachitic lesions were apparent at costochondral junctions in multiple cases. Acute and/or chronic bone fractures were also noted in multiple bones. Failure of endochondral ossification, expanded physes, infractions, thin trabeculae, and increased osteoclasts were noted microscopically. Decreased bone ash and serum 25(OH)D(3), combined with clinical and microscopic evaluation, confirmed a diagnosis of vitamin D-dependent rickets in all cases. In 3 cases, disease was linked to a specific nutrient supplier that ultimately resulted in a voluntary feed recall; however, most cases in the current investigation were not associated with a particular feed company. The present report describes vitamin D-associated rickets and its importance as a potential cause of weakness, lameness, muscle fasciculations, recumbency or sudden unexpected death in swine, and describes appropriate samples and tests for disease diagnosis.

Importance of apical membrane delivery of 1,25-dihydroxyvitamin D3 to vitamin D-responsive gene expression in the colon.

Synthetic conjugation of a glucuronide to 1,25-dihydroxyvitamin D3 (1,25D3) to produce ß-25-monoglucuronide-1,25D3 (ßGluc-1,25D3) renders the hormone biologically inactive and resistant to mammalian digestive enzymes. However, ß-glucuronidase produced by bacteria in the lower intestinal tract can cleave off the glucuronide, releasing the active hormone. In mice given a single oral dose of 1,25D3, 24-hydroxylase (Cyp24a1) gene expression was strongly enhanced in the duodenum, but not in the colon, despite circulating concentrations of 1,25D3 that peaked at ∼3.0 nmol/l. In contrast, in mice treated with an equimolar dose of ßGluc-1,25D3, Cyp24a1 gene expression increased 700-fold in the colon but was significantly weaker in the duodenum compared with mice treated with 1,25D3. Similar results were observed with another vitamin D-dependent gene. When administered subcutaneously, 1,25D3 weakly stimulated colon Cyp24a1 gene expression while ßGluc-1,25D3 again resulted in strong enhancement. Surgical ligation to block passage of ingesta beyond the upper intestinal tract abolished upregulation of colon Cyp24a1 gene expression by orally and subcutaneously administered ßGluc-1,25D3. Feeding ßGluc-1,25D3 for 5 days revealed a linear, dose-dependent increase in colon Cyp24a1 gene expression but did not significantly increase plasma 1,25D3 or calcium concentrations. This study indicates that the colon is relatively insensitive to circulating concentrations of 1,25D3 and that the strongest gene enhancement occurs when the hormone reaches the colon via the lumen of the intestinal tract. These findings have broad implications for the use of vitamin D compounds in colon disorders and set the stage for future therapeutic studies utilizing ßGluc-1,25D3 in their treatment.

Targeted delivery of vitamin D to the colon using ß-glucuronides of vitamin D: therapeutic effects in a murine model of inflammatory bowel disease.

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D] has been shown to inhibit development of dextran sodium sulfate (DSS)-induced colitis in mice but can also cause hypercalcemia. The aim of this study was to evaluate whether ß-glucuronides of vitamin D could deliver 1,25(OH)(2)D to the colon to ameliorate colitis while reducing the risk of hypercalcemia. Initial studies demonstrated that bacteria residing in the lower intestinal tract were capable of liberating 1,25(OH)(2)D from 1,25-dihydroxyvitamin D(3)-25-ß-glucuronide [ß-gluc-1,25(OH)(2)D]. We also determined that a much greater upregulation of the vitamin D-dependent 24-hydroxylase gene (Cyp24) was induced in the colon by treatment of mice with an oral dose of ß-gluc-1,25(OH)(2)D than 1,25(OH)(2)D, demonstrating targeted delivery of 1,25(OH)(2)D to the colon. We then tested ß-glucuronides of vitamin D in the mouse DSS colitis model in two studies. In mice receiving DSS dissolved in distilled water and treated with 1,25(OH)(2)D or ß-gluc-1,25(OH)(2)D, severity of colitis was reduced. Combination of ß-gluc-1,25(OH)(2)D with 25-hydroxyvitamin D(3)-25-ß-glucuronide [ß-gluc-25(OH)D] resulted in the greatest reduction of colitis lesions and symptoms in DSS-treated mice. Plasma calcium concentrations were lower in mice treated with ß-gluc-1,25(OH)(2)D alone or in combination with ß-gluc-25(OH)D than in mice treated with 1,25(OH)(2)D, which were hypercalcemic at the time of death. ß-Glucuronides of vitamin D compounds can deliver 1,25(OH)(2)D to the lower intestine and can reduce symptoms and lesions of acute colitis in this model.

Prevalence of subclinical hypocalcemia in dairy herds.

The prevalence of subclinical hypocalcemia in the transition cow is unknown. Cows with subclinical hypocalcemia have no clinical signs of hypocalcemia but may be more susceptible to other diseases. The objective of this study was to determine the prevalence of subclinical hypocalcemia in the US dairy herds. As a part of the United States Department of Agriculture's National Animal Health Monitoring System 2002 Dairy study, serum samples were collected from 1462 cows within 48 h of parturition. The samples were sorted by lactation number: 1st (n=454), 2nd (n=447), 3rd (n=291), 4th (n=166), 5th (n=72), and 6th (n=32). Subclinical hypocalcemia (<2.0 mM) increased with age and was present in 25%, 41%, 49%, 51%, 54%, and 42% of 1st-6th lactation cows, respectively. Cows with serum calcium concentrations >2.0 mM had significantly lower serum non-esterified fatty acids indicating better energy balance than those with subclinical hypocalcemia. Subclinical hypocalcemia may make cows more susceptible to secondary diseases but more research will be required to determine if this is true.

Evaluation of breed-dependent differences in the innate immune responses of Holstein and Jersey cows to Staphylococcus aureus intramammary infection.

Mastitis is one of the most prevalent diseases of cattle. Various studies have reported breed-dependent differences in the risk for developing this disease. Among two major breeds, Jersey cows have been identified as having a lower prevalence of mastitis than Holstein cows. It is well established that the nature of the initial innate immune response to infection influences the ability of the host to clear harmful bacterial pathogens. Whether differences in the innate immune response to intramammary infections explain, in part, the differential prevalence of mastitis in Holstein and Jersey cows remains unknown. The objective of the current study was to evaluate several parameters of the innate immune response of Holstein and Jersey cows to intramammary infection with Staphylococcus aureus, a common mastitis-inducing pathogen. To control for non-breed related factors that could influence these parameters, all cows were of the same parity, in similar stages of milk production, housed and managed under identical conditions, and experimentally infected and sampled in parallel. The following parameters of the innate immune response were evaluated: acute phase protein synthesis of serum amyloid A and lipopolysaccharide-binding protein; total and differential circulating white blood cell counts; milk somatic cell counts; mammary vascular permeability; milk N-acetyl-beta-d-glucosaminidase (NAGase) activity; and production of the cytokines, interferon (IFN)-gamma, interleukin (IL)-12, tumour growth factor(TGF)-alpha, and TGF-beta1. The temporal response of all of these parameters following infection was similar between Holstein and Jersey cows. Further, with the exception of changes in circulating neutrophils and NAGase activity, the overall magnitude of these parameters were also comparable. Together, these data demonstrate that the innate immune response of Holstein and Jersey cows to Staph. aureus intramammary infection remains highly conserved despite previously reported differences in mastitis prevalence, as well as genotypic and phenotypic traits, that exist between the two breeds.

The monitoring, prevention, and treatment of milk fever and subclinical hypocalcemia in dairy cows.

The periparturient cow undergoes a transition from non-lactating to lactating at calving. The animal is tremendously challenged to maintain calcium homeostasis. Those that fail can develop milk fever, a clinical disorder that is life threatening to the cow and predisposes the animal to a variety of other disorders. Guidelines for monitoring the incidence of hypocalcemia and methods for treating milk fever are reviewed. The physiological factors that cause milk fever and strategies for prevention of milk fever are discussed, focusing on the effects diet cation-anion difference can have on tissue sensitivity to parathyroid hormone. Another major risk factor for milk fever is hypomagnesemia, which is observed when animals are fed inadequate amounts of magnesium, or some factor is present in the diet that prevents adequate absorption of magnesium. Moderate hypomagnesemia impairs the ability of the cow to maintain calcium homeostasis and hypocalcemia occurs.

Neutrophil extracellular trap formation by bovine neutrophils is not inhibited by milk.

Neutrophils are the first line of defense in a mammary gland infection. However, the process of neutrophil transmigration across a membrane and ingestion of fat and/or casein when incubated in milk have been shown to inhibit bacterial phagocytosis and oxidative burst functions. Recently, a killing mechanism has been described whereby stimulated neutrophils release nuclear and granule material in fibrous webs that physically trap and kill bacteria. We demonstrate that these neutrophil extracellular traps are also produced by bovine blood neutrophils stimulated with PMA/ionomycin. Importantly, neutrophil extracellular traps can be formed when neutrophils have been incubated for up to 6h in milk prior to stimulation. This contrasts milk's rapid inhibition of bacterial phagocytosis and oxidative burst functions in the neutrophil. Furthermore, stimulation of neutrophils with bacteria common to mammary gland infections leads to neutrophil extracellular traps being formed in milk. Some bacteria tested stimulated enhanced formation of neutrophil extracellular traps in milk compared to culture media. Therefore, being unaffected by incubation in milk may indicate an important role for neutrophil extracellular traps in defense against mastitis.

Effect of administration of a controlled-release monensin capsule on incidence of calving-related disorders, fertility, and milk yield in dairy cows.

OBJECTIVE: To determine the effect of a controlled-release monensin capsule administered at cessation of lactation on incidence of calving-related disorders, fertility, and milk yield in dairy cows. ANIMALS: 290 dairy cows treated with monensin and 290 untreated control cows. PROCEDURE: Treated cows received a capsule that released monensin at 335 mg/d for 95 days. Incidence of calving-related disorders; daily milk yield up to 20 days postpartum; test-day milk yield, fat, protein, and mature-equivalent 305-day milk production; and body condition score at calving were determined. Reproductive variables were conception rate at first service, pregnancy rate, and calving-to-conception interval. RESULTS: Cows treated with monensin were 2.1 times as likely to develop dystocia and 0.8 times as likely to develop metritis as control cows. For milk yield, there was an interaction of treatment X time X parity. In multiparous cows, monensin significantly improved milk yield at test days 4 and 7. In addition, monensin increased body condition score at calving. CONCLUSIONS AND CLINICAL RELEVANCE: Despite increasing the likelihood of developing dystocia and metritis, administration of monensin improved the lactational performance of multiparous cows and may be a promising additive for use at the time of cessation of lactation.

Expression of lymphocyte homing and adhesion molecules during intramammary infection of cows with Serratiamarcescens or Streptococcusuberis: correlation with bacterial colonization and clinical signs.

We wished to determine the expression of trafficking/adhesion molecules on the surface of lymphocytes isolated from infected mammary glands of cows challenged with either Serratia marcescens or Staphylococcus uberis. Healthy Holstein cows in mid lactation were infected by intramammary infusion with S. marcescens or S. uberis. Following infection, milk samples were collected at various time points. Body temperatures of the cows were taken, and milk was analyzed for colony forming units (CFU) of bacteria and somatic cell counts (SCC). Leukocytes were isolated from the milk and analyzed by flow cytometry. Percentages and types of lymphocytes were determined as well as expression of CD62L, CD11a, LPAM-1 and CD44 on these cells. We found that the percentage of lymphocytes expressing either CD62L or CD11a showed a marked increase 12 h post infection (PI) with S. marcescens that was not seen in cows infected with S. uberis. Conversely, the percentage of lymphocytes expressing CD44 increased in cows infected with S. uberis at 12 h PI, but the increase was not seen in cows infected with S. marcescens. Expression of LPAM-1 was low at all time points in both groups of cows. Body temperatures became elevated in both groups of cows, peaking at 24 h PI in S. marcescens-infected cows and dropping thereafter. In contrast, temperatures of S. uberis-infected cows continued to rise and were still elevated 96 h PI. CFU of bacteria isolated from mammary glands of S. marcescens-infected cows dropped precipitously 24 h PI but continued at high levels in S. uberis-infected cows. SCC began falling in S. marcescens-infected cows 48 h PI but continued to increase in S. uberis-infected cows. Thus, a greater percentage of lymphocytes in milk had a phenotype consistent with recruitment from the peripheral pool following infection with S. marcescens than was seen following infection with S. uberis. Concurrent with the increases seen in percentages of this lymphocyte phenotype, clinical signs lessened in the S. marcescens-infected cows.

Lymphocytes from one side of the bovine mammary gland migrate to the contra lateral gland and lymph node tissue.

The four quarters of bovine mammary glands are completely separated and two quarters on each side (right or left) are connected to ipsi lateral supra mammary lymph nodes. It is not clear whether cells infused into the cistern of the mammary gland are capable of migrating to lymph nodes or the general circulation. To examine cell migration, a prescapular lymph node was removed from each of two lactating and three non-lactating dairy cows, and isolated lymphocytes were stained with Hoechst 33342. Autologous stained cells were infused into the mammary gland and then activated by intramammary infusion of zymosan-stimulated serum (source of C5a). After 17 h, Escherichia coli J5 bacterin was infused into the contra lateral mammary gland to mimic infection. After 43 h cows were euthanized and tissue samples (mammary quarters, right and left supra mammary, mesenteric, ileocecal and prescapular lymph nodes, liver and spleen) were collected for microscopic examination as well as flow cytometric analysis. Hoechst stained cells were detected not only in infused quarters, but also in contra lateral quarters as well as in both supra mammary lymph nodes. This indicates that cells infused into the mammary gland migrate to contra lateral tissues and supra mammary lymph nodes.

Adapting to the transition between gestation and lactation: differences between rat, human and dairy cow.

Adequate blood calcium concentrations are vital for the normal function of mammals. Mechanisms for maintaining normal blood calcium function adequately most of the time; however, occasionally they fail and calcium homeostasis is compromised. Milk fever or periparturient hypocalcemia in dairy cattle is a well-documented example of a breakdown in the mechanisms of calcium homeostasis. This disease occurs at the time of parturition and is unique to adult dairy animals. The disease results from the inability of animals to cope with the sudden demand for calcium in support of colostrum formation. Animals developing the disease become hypocalcemic and require intravenous calcium to survive. The precise metabolic disorder(s) responsible for the onset of milk fever is still being debated. This report will highlight some of the current concepts related to the causes and prevention of milk fever in dairy cattle, as well as contrasting differences in calcium demands that exist between dairy cattle, humans and rats at the onset of lactation.