Cellular proliferation and apoptosis in Staphylococcus aureus-infected heifer mammary glands experiencing rapid mammary gland growth.

Intramammary infections in nonlactating mammary glands are common and can occur during periods of rapid mammary epithelial cell (MEC) accumulation, which may ultimately reduce total MEC numbers. Reduced MEC numbers, resulting from impaired MEC proliferation and increased cellular apoptosis, are expected to reduce future milk yields. The objective of this study was to measure the degree of cellular proliferation and apoptosis in the epithelial and stromal compartment of uninfected and Staphylococcus aureus-infected mammary glands hormonally induced to grow rapidly. Nonpregnant heifers (n = 8) between 11 and 14 mo of age were administered supraphysiological injections of estradiol and progesterone for 14 d. One mammary gland of each heifer was randomly selected and infused with Staph. aureus (CHALL) while another mammary gland was designated as an uninfected control on d 8 of injections. Mammary tissues were collected on the last day of hormonal injections from center and edge parenchymal regions and subject to proliferation assessment via Ki-67 staining and apoptotic assessment via terminal deoxynucleotidyl transferase dUTP nick-end labeling. Differences in cellular proliferation between CHALL and uninfected control quarters were not apparent, but proliferation of MEC was marginally greater in edge parenchyma than in center parenchyma. Coincidently, CHALL quarters experienced a greater percentage of apoptotic MEC and lower percentage of stromal cells undergoing apoptosis than uninfected control quarters. This study also provides the first insight into the mechanisms that allow the mammary fat pad to be replaced by expanding mammary epithelium as edge parenchyma contained a greater percentage of apoptotic stromal cells than center parenchyma. When taken together, these data suggest that Staph. aureus intramammary infection impairs mammary epithelial growth through reductions in MEC number and by preventing its expansion into the mammary fat pad. These factors during periods of rapid mammary growth are expected to impair first lactation milk yield.

Histological tissue structure alterations resulting from Staphylococcus aureus intramammary infection in heifer mammary glands hormonally induced to rapidly grow and develop.

Intramammary infections (IMI) are common in nonlactating dairy cattle and are expected to impair mammary growth and development and reduce future milk production. The objective of this study was to histologically evaluate how IMI alter tissue structure in growing and developing heifer mammary glands. A total of 18 nonpregnant, nonlactating heifers between 11 and 14 mo of age were used in the present study. Heifers received daily supraphysiological injections of estradiol and progesterone for 14 d to stimulate rapid mammary growth and development. One-quarter of each heifer was subsequently infused with Staphylococcus aureus (CHALL) while a second quarter served as an uninfected control (UNINF). Heifers were randomly selected and euthanized either the last day of hormonal injections to observe IMI effects on mammary gland growth (GRO), or 13 d post-injections, to observe IMI effects on mammary development (DEV). Mammary tissues were collected from the center and edge parenchymal regions of each mammary gland for morphometric tissue area evaluation. For GRO tissues, CHALL quarters had less epithelial tissue area and marginally more intralobular stroma tissue area than UNINF quarters. Tissue areas occupied by luminal space, extralobular stroma, adipose, and lobular tissue were similar. For DEV tissues, area occupied by epithelium, luminal space, intralobular stroma, and extralobular stroma did not differ between quarter treatments, but UNINF quarters had more adipose tissue area and marginally less lobular area than CHALL quarters. Results indicate that IMI in growing and developing mammary glands reduces mammary epithelial growth and alters mammary gland development by impairing epithelial branching into the mammary fat pad. Taken together, these tissue changes before calving may have adverse effects on milk production. Therefore, an important focus should be placed on improving udder health in replacement heifers through management strategies that mitigate the deleterious effects of IMI and promote the positive development of the mammary gland.

Bovine Mammary Gland Biopsy Techniques.

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

A 100-Year Review: Mammary development and lactation.

What is old is new again-and with respect to the study of the mammary development and function in dairy animals, the expression resonates. Many of the mammary and milk production questions raised in the early years of the Journal of Dairy Science apply today. To be sure, scientists have filled in many details regarding, for example, identification of hormones and growth factors important in the control of mammary growth, the onset of copious milk production at calving, and maintenance of lactation. Early years focused on identification and subsequent availability of classic mammogenic, lactogenic, and galactopoietic hormones (e.g., steroids, prolactin, and growth hormone). The advent of sensitive assays to measure concentrations of these hormones and, subsequently, myriad growth factors in blood, milk, and tissues, allowed creation of multiple hypotheses to explain mammary cell proliferation and regulation of function. It is also apparent that we understand many of the fundamentals of milk removal, milking frequency, milking management, and milk ejection for successful lactation. However, some questions remain. Are the principles that were identified when cows produced markedly less milk still valid for the high-producing cows of today and the future? What mechanism(s) explain the positive effects of early increased milking frequency on subsequent milk production? Can the persistency of lactation be improved (secretory cell number vs. secretory cell function) or does early management "program" future mammary development or productivity (epigenetics, immune responsiveness, other)? The explosion of tools and techniques (Southern and Northern blots, PCR, and the "-omics" revolution) has driven an almost overwhelming evaluation of cellular and molecular functions in the mammary gland and other tissues. One key may be the discovery of a "Rosetta stone" that will allow understanding of this mass of detailed information on gene expression, cell signaling, and so on. Many scientists can now better appreciate the difficulty of the dairy farmer seeking to process DHIA or Dairy Comp 305 data, milking data, weights, feeding reports, pedometer readings, or genomic evaluations to manage their operations.

The influence of postnatal nutrition on reproductive tract and endometrial gland development in dairy calves.

Uterine gland development occurs after birth in cattle and other mammals. The timeline of gland development has been described in various species, but little is known about how postnatal diet influences uterine gland development. This is especially concerning in dairy heifers, where a variety of milk replacer and whole milk nutrition options exist. Little work also exists in cattle to describe how early exposure to steroids influences reproductive tract and uterine gland development. The objective of this work was to determine the effects of early postnatal plane of nutrition and estrogen supplementation on uterine gland development in calves. In both studies, Holstein heifer calves were assigned to restricted milk replacer (R-MR) or enhanced milk replacer (EH-MR) diets. In study 1, calves (R-MR, n = 6; EH-MR, n = 5) were euthanized at 8 wk. In study 2, calves were weaned at 8 wk and administered estradiol (R-MR, n = 6; EH-MR, n = 6) or placebo (R-MR, n = 6; EH-MR, n = 5) for an additional 14 d before euthanasia. Average daily gain and final body weight was greater in both studies in heifers fed the enhanced diet. At 8 wk, EH-MR calves had a greater number of glands and a smaller average gland size, but total gland area was not different from the R-MR group. At 10 wk, uterine gland number and size were not affected by diet or estrogen. Expression profiles of several paracrine mediators of gland development were examined. Increases in transcript abundance for IGF1 and IGFBP3 and a decrease in abundance of WNT7A were detected in calves fed the enhanced diet at 8 wk of age. Plane of nutrition did not affect transcript profiles at 10 wk of age, but estradiol supplementation decreased MET and WNT7A transcript abundance. To conclude, heifer calves on a restricted diet exhibited a uterine morphology and transcript profile suggestive of delayed uterine gland development. These changes appear to be corrected by wk 10 of life. Also, this work provides evidence supporting the contention that early estradiol exposure has detrimental effects on uterine gene expression.

Circannual changes in progesterone secretion in intact ewes, luteinizing hormone secretion in ovariectomized estradiol-implanted ewes, and prolactin secretion in three sheep breeds anticipated to differ in seasonality of reproduction.

Changes in progesterone secretion in intact ewes (7 or 9 per breed) and luteinizing hormone secretion in ovariectomized, estradiol-implanted ewes (9 or 10 per breed) were monitored for 12 mo in Suffolk, tropically adapted St. Croix, and OOS ewes. The OOS line is a composite population of 50% Dorset, 25% Rambouillet, and 25% Finnish Landrace breeding that was selected for 10 yr for ability to lamb in October and early November. Ewes were isolated from rams, and blood samples were collected twice weekly. Circulating prolactin concentrations were also determined from blood samples collected near the summer and winter solstice and vernal and autumnal equinox. Intact OOS ewes entered anestrus later, began the subsequent breeding season sooner, and had a shorter seasonal anestrus than Suffolk and St. Croix ewes (P ≤ 0.005). St. Croix ewes did not differ from Suffolk ewes in date of onset or cessation of breeding or duration of anestrus (P ≥ 0.06). Breed differences in duration of luteinizing hormone inhibition in ovariectomized ewes were essentially identical to those observed for duration of anestrous. Prolactin concentrations varied during the year: annual changes were larger in relatively seasonal Suffolk ewes than in tropically-derived St. Croix ewes (P<0.01), and OOS ewes were intermediate to, and tended to differ from (P<0.10), the other two breeds. We conclude that OOS ewes developed by selection for fertility in spring matings had an abbreviated seasonal anestrus that is one of the shortest ever reported for temperate breeds, and that tropical St. Croix sheep did not have a shorter seasonal anestrus than Suffolk sheep under temperate conditions and ram isolation.

Mastitis and its impact on structure and function in the ruminant mammary gland.

It is a given in biology that structure and function go hand-in-hand. At the level of the mammary alveoli, copious milk production depends on the proliferation of mammary epithelial cells and the biochemical and structural differentiation of these cells after parturition. For example, data from quantitative structural studies demonstrate that differences in milk production between beef and dairy cows correspond with a relative failure of alveolar cell differentiation in cattle not specifically selected for milk yield. It is likely, but not proven, that production differences within or between dairy breeds are also determined by differences in the capacity of alveolar cells to differentiate or to maintain an adequate state of differentiation. These observations strongly support the belief that insults from mastitis that lead to losses in mammary function are directly related to disruption of alveolar cell integrity, sloughing of cells, induced apoptosis, and increased appearance of poorly-differentiated cells. Ironically, reduced milk production in cases of subclinical mastitis, is also associated with increases in milk somatic cell count. Thus the elevated neutrophil migration evoked to fight inflammation can inadvertently rendered alveolar epithelial cells non-secretory. A challenge to future researchers will be to devise mastitis treatments and therapies that prevent and/or repair damage to alveolar structure and maximize subsequent secretory cell differentiation.

Effect of feeding level on serum IGF1 response to GH injection.

This study was conducted to further understand the mechanism by which nutrition modulates GH-induced changes in serum IGF1 concentration in cattle. Cows were fed hay only or corn-based concentrates in addition to hay for 8 weeks. At week 8, serum concentrations of IGF1, IGF-binding protein 3 (IGFBP3), and acid-labile subunit (ALS) as well as their mRNA levels in liver were determined immediately before and 7 days after an injection of GH formulated for sustained release. GH injection caused greater increases in both serum IGF1 concentration and liver IGF1 mRNA expression in the cows fed concentrates than in those fed hay. In the cows fed concentrates, the magnitude of the GH-induced increase in serum IGF1 concentration was, however, much greater than that in liver IGF1 mRNA expression. This difference was not due to GH-induced IGF1 mRNA being translated more efficiently. GH injection also induced greater increases in serum IGFBP3 and ALS concentrations in the cows fed concentrates than fed hay. The injection, however, did not induce a greater increase in IGFBP3 or ALS mRNA in the cows fed concentrates than fed hay. These data and the fact that the ternary complex of IGF1/IGFBP3/ALS stabilizes each of the components in the blood suggest that in cows, increased nutrition enhances serum IGF1 response to GH not only by increasing IGF1 mRNA response to GH in the liver, but also by elevating IGFBP3 and ALS concentrations in the blood.

The origin and evolution of lactation.

The presence of mammary glands is the defining morphological feature of mammals. The recent assembly of the bovine genome and a report in Genome Biology that links the milk and lactation data of bovine and other mammalian genomes will help biologists investigate this economically and medically important feature.

Gene expression ratio stability evaluation in prepubertal bovine mammary tissue from calves fed different milk replacers reveals novel internal controls for quantitative polymerase chain reaction.

Prepubertal mammary development can be affected by nutrition partly through alterations in gene network expression. Quantitative PCR (qPCR) remains the most accurate method to measure mRNA expression but is subject to analytical errors that introduce variation. Thus, qPCR data normalization through the use of internal control genes (ICG) is required. The objective of this study was to mine microarray data (> 10,000 genes) from prepubertal mammary parenchyma and stroma to identify the most suitable ICG for normalization of qPCR. Tissue for RNA extraction was obtained from calves ( approximately 63 d old; n = 5/diet) fed a control (200 g/kg crude protein, 210 g/kg crude fat, fed at 441 g/d dry matter) or a high-protein milk replacer (280 g/kg crude protein, 200 g/kg crude fat, fed at 951 g/d dry matter). ICG were selected based on both absence of expression variation across treatment and of coregulation (gene network analysis). Genes evaluated were ubiquitously expressed transcript, protein phosphatase 1 regulatory (inhibitor) subunit 11 (PPP1R11), matrix metallopeptidase 14 (MMP14), ClpB caseinolytic peptidase B, SAPS domain family member 1 (SAPS1), mitochondrial GTPase 1 (MTG1), mitochondrial ribosomal protein L39, ribosomal protein S15a (RPS15A), and actin beta (ACTB). Network analysis demonstrated that MMP14 and ACTB are coregulated by v-myc myelocytomatosis viral oncogene, tumor protein p53, and potentially insulin-like growth factor 1. Pairwise comparison of expression ratios showed that ACTB, MMP14, and SAPS1 had the lowest stability and were unsuitable as ICG. PPP1R11, RPS15A, and MTG1 were the most stable among ICG tested. We conclude that the geometric mean of PPP1R11, RPS15A, and MTG1 is ideal for normalization of qPCR data in prepubertal bovine mammary tissue. This study provides a list of candidate ICG that could be used by researchers working in bovine mammary development and allied fields.

Autocrine production of insulin-like growth factor-I (IGF-I) affects paracellular transport across epithelial cells in vitro.

Autocrine production of growth factors can have significant effects on cell activity. We report for the first time that autocrine production of insulin-like growth factor-I (IGF-I) alters paracellular transport across bovine mammary epithelial cells in vitro. Paracellular transport was assessed by measuring phenol red transport across mammary alveolar cells-large T antigen (MAC-T cells) derived from parental mammary epithelial cells, cultured on porous membranes and compared with two different transfected MAC-T cell lines that constitutively secrete IGF-I. Phenol red transport was essentially blocked in parental cell culture after six days, while IGF-I secreting cells provided essentially no barrier. Surprisingly, neither co-culture studies between parental and IGF-I-secreting cells nor addition of exogenous IGF-I or IGF-binding protein-3 reversed the phenol red transport properties. IGF-I-secreting cells did however express lower levels of the junction components occludin and E-cadherin than parental cells, suggesting that localized autocrine IGF-I activity might lead to increased permeability via changes in both the tight and adherens junction protein levels.

Alginate encapsulation impacts the insulin-like growth factor-I system of monolayer-expanded equine articular chondrocytes and cell response to interleukin-1beta.

Alginate hydrogel culture has been shown to reestablish chondrocytic phenotype following monolayer expansion; however, previous studies have not adequately addressed how culture conditions affect the signaling systems responsible for chondrocyte metabolic activity. Here we investigate whether chondrocyte culture history influences the insulin-like growth factor-I (IGF-I) signaling system and its regulation by interleukin-1 (IL-1). Articular chondrocytes (ACs) from equine stifle joints were expanded by serial passage and were either encapsulated in alginate beads or maintained in monolayer culture for 10 days. Alginate-derived cells (ADCs) and monolayer-derived cells (MDCs) were then plated at high density, stimulated with IL-1beta (1 and 10 ng/mL) or IGF-I (50 ng/mL) for 48 h, and assayed for levels of type I IGF receptor (IGF-IR), IGF binding proteins (IGFBPs), and endogenously secreted IGF-I. Intermediate alginate culture yielded relatively low IGF-IR levels that increased in response to IL-1beta, whereas higher receptor levels on MDCs were reduced by cytokine. MDCs also secreted substantially more IGFBP-2, the predominant binding protein in conditioned media (CM), though IL-1beta suppressed levels for both cell populations. Concentrations of autocrine/paracrine IGF-I paralleled IGFBP-2 secretion. Disparate basal levels of IGF-IR and IGFBP-2, but not IGF-I, were attributed to relative transcript expression. Systemic differences coincided with varied effects of IL-1beta and IGF-I on cell growth and type I collagen expression. We conclude that culture strategy impacts the IGF-I signaling system of ACs, potentially altering their capacity to mediate cartilage repair. Consideration of hormonal regulators may be an essential element to improve chondrocyte culture protocols used in tissue engineering applications.

Transcriptional and proteolytic regulation of the insulin-like growth factor-I system of equine articular chondrocytes by recombinant equine interleukin-1beta.

Interleukin-1 (IL-1) and insulin-like growth factor-I (IGF-I), which have opposing effects on matrix metabolism within articular cartilage, are thought to play prominent roles in the pathogenesis of osteoarthritis. To better understand the link between these anabolic (IGF-I) and catabolic (IL-1) stimuli, we examined exogenous IL-1 regulation of the IGF-I signaling system of articular chondrocytes (ACs). Equine ACs from non-arthritic stifle joints were expanded in monolayer culture, encapsulated for 10 days in alginate beads, and stimulated as high-density monolayers with recombinant equine IL-1beta (0, 1, 10 ng/ml) for 48 h. IL-1beta enhanced expression of IGF-IR levels, as determined by both [125I]-IGF-I binding studies and Western blotting, while reducing the concentration of endogenous IGF-I detected in conditioned media by radioimmunoassay. Western ligand blotting revealed that chondrocytes primarily secreted IGF binding proteins (IGFBPs) with molecular weights of 28-30 and 32-34 kDa, which were identified as IGFBPs 5 and 2, respectively, and that IL-1beta treatment diminished IGFBP-2, the prominent homolog in conditioned media. Northern blot analysis suggested IL-1beta regulation of IGF-I and, to some extent, IGF-IR was mediated by transcription; however, the cytokine did not affect IGFBP-2 expression. To test for evidence of proteolysis by matrix metalloproteinases (MMPs), additional cultures were co-incubated with inhibitors for MMPs 2/9, 3, and 8. IGFBP-2 suppression was partially reversed by gelatinase (MMP-2/9) inhibition. In summary, these findings further delineate the role of IL-1 as a key regulator of the IGF-I system within articular cartilage, demonstrating that regulation occurs through both direct (transcriptional) and indirect (proteolytic) mechanisms.

Modulation of protein adsorption and cell adhesion by poly(allylamine hydrochloride) heparin films.

Electrostatic layer-by-layer film assembly is an attractive way to non-covalently incorporate proteins and bioactive moieties into the surface of conventional biomaterials. Selection of polycationic and polyanionic components and deposition conditions can be used to control the interfacial properties, and through them protein adsorption, cell adhesion, and tissue development. In this study the polycation was poly(allylamine hydrochloride) (PAH), which is a weak base and consequently adsorbs at interfaces in a pH-dependent manner, and the polyanion was heparin, which is capable of interacting with many adhesion ligands and growth factors. PAH/heparin multilayer films were formed using PAH solutions of pH 6.4, 7.4, 8.4, and 9.4. Film thickness increased both with the number of PAH/heparin bilayers and the pH of the PAH solution. Films consisting of 10 bilayers with heparin topmost exhibited similar bulk atomic compositions and penetration of PAH into the heparin top layer. Finally, fibronectin adsorption and cell adhesion were maximal at an intermediate pH (pH 8.4>pH 9.4>pH 7.4). These results demonstrate that heparin-containing electrostatic films support cell adhesion and protein adsorption in a manner sensitive to film deposition conditions.

Reduced serum insulin-like growth factor (IGF) I is associated with reduced liver IGF-I mRNA and liver growth hormone receptor mRNA in food-deprived cattle.

Nutritional deprivation decreases blood insulin-like growth factor I (IGF-I) concentrations in a variety of species. In this study, we explored the underlying mechanism by determining the effects of food deprivation on the levels of total IGF-I mRNA and total growth hormone receptor (GHR) mRNA, as well as the levels of individual IGF-I mRNA variants and GHR mRNA variants in the liver of steers. Food deprivation for nearly 3 d decreased the levels of serum IGF-I by 63% (P < 0.01), and this decrease was associated with a 75% decrease (P < 0.01) in total IGF-I mRNA in the liver. The food deprivation-induced decrease in liver total IGF-I mRNA was associated with an equivalent decrease in the levels of both class 1 and class 2 IGF-I mRNA. In addition to IGF-I mRNA, food deprivation also decreased the levels of total GHR mRNA in the liver (P < 0.05), and this decrease was associated with a decrease in the liver expression of GHR mRNA variants 1C3 (P < 0.05) and 1A (P = 0.08). Food deprivation did not affect the levels of two other major GHR mRNA variants, 1B and 1C2, in the liver. These results demonstrate that the food deprivation-induced decrease in circulating IGF-I in steers is associated with a coordinate decrease in the expression of different IGF-I mRNA variants and a specific decrease in the expression of GHR mRNA variants 1C3 and 1A in the liver.

Chronic pulsatile shear stress alters insulin-like growth factor-I (IGF-I) binding protein release in vitro.

Insulin-like growth factor-I (IGF-I) is a potent smooth muscle cell mitogen indicated to have a role in vascular disease. IGF-I stimulates proliferation via receptor activation but its activity is mediated by IGF binding proteins (IGFBPs). Since hemodynamics have been linked to vascular proliferative disorders, we studied how pulsatile low (5 +/- 2 dynes/cm2) and high (23 +/- 8 dynes/cm2) shear stresses impacted IGFBP metabolism in bovine aortic endothelial cells using the Cellmax capillary system. We modeled the pulsatile flow in our system using the Womersley model for flow inside a rigid tube and harmonic analysis revealed that the flow was sinusoidal with a frequency of approximately 0.3 Hz for both shear stress treatments. Laminar flow was confirmed and the phase lag between the pressure and the flow found to be insignificant. Thus, our study provides a necessary characterization of this in vitro system as well as an investigation into how shear impacts the IGF axis. We found a significant difference in IGFBP distribution between treatments and, given that IGFBPs regulate IGF-I activity and that IGF-I-independent activities have been suggested for IGFBP-3, suggest that shear stress may indirectly regulate IGF-I activity, and, by extension, the effect of IGF-I on vascular pathologies.

Evaluation of frequent milkout for treatment of cows with experimentally induced Escherichia coil mastitis.

OBJECTIVE: To evaluate the effect of frequent milkout (FMO) on the outcome of experimentally induced Escherichia coli mastitis in cows. DESIGN: Randomized complete block study. ANIMALS: 16 Holstein dairy cows. PROCEDURE: Cows were randomly assigned to 1 of 4 groups and were either not infected and not treated (NI-NT), experimentally infected with E coli and not treated (EC-NT), not infected and FMO (NI-FMO), or experimentally infected with E coli and FMO (EC-FMO). The infected quarter in cows in FMO groups was milked out every 4 hours from 16 to 36 hours and every 6 hours from 36 to 84 hours after challenge, with the aid of oxytocin administration. Somatic cell counts (SCC); times to bacterial, clinical, and systemic cures; and serum concentrations of a-lactalbumin were determined. RESULTS: Use of FMO did not appear to affect SCC. For EC-NT and EC-FMO groups, mean bacterial cure times were 203 and 159 hours, clinical cure times were 276 and 360 hours, and systemic cure times were 144 and 159 hours, respectively; these times were not significantly different. Concentrations of a-lactalbumin were significantly increased in the EC-NT group at 12 hours and in the NI-FMO group at 36 and 60 hours after challenge, compared with values of cows in other treatment groups. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with results in control cows, FMO does not appear to be an efficacious treatment for experimentally induced moderate to severe E coli mastitis.