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The overall objective of this Phase I SBIR project is to jump-start the development of an effective non-antibiotic dairy cow dry-off agent that will ultimately be widely (perhaps, universally) adopted as a standard-of-practice in the US dairy industry. <P> We will accomplish this overall objective by intensive screening of pharmaceutical and natural-product agents. The agents will be screened first with in vitro (i.e., cell culture) assays that we have shown to be highly reliable. <P>The assays are performed using primary bovine mammary epithelial cells (pBMEC). They include an assay for tight junction integrity (trans-epithelial electrical resistance, TEER), and an assay for milk protein gene expression. These assays will reliably predict which compounds are better suited as dry-off agents (involution accelerants).<P> Based on the outcome of these "medium-throughput" in vitro experiments we will field-test a selected set of compounds in dairy cows. <P>Scientific Goal #1: Screen compounds in vitro to determine which are the better candidates for rapid dry-off agents. These assays will use tight junction integrity and milk protein gene expression endpoints, and will compare conventional and tryptophan-sparing medium formulations. <P>Scientific Goal #2: Test three preferred compounds in vivo by infusions into the udders of dairy cows at dry-off. <P>Predictions: We predict that the treatment groups will show larger declines in milk yield at 24h compared with controls. We also predict that plasma lactose concentrations will rise earlier and be higher in treated cows, compared with controls. These findings would be consistent with accelerated dry-off (involution) of the glands. Differences in responses among the treatment groups will inform our choice of the optimum dryoff agent(s).
NON-TECHNICAL SUMMARY: <BR> This project addresses priority areas of 1) Animal Productivity (in both conventional and alternative production systems), 2) Animal Health and Well-Being, and 3) Food Safety and Environmental Mitigation, as described in the NIFA Program Announcement for Phase I SBIR applications. 1) Productivity: Intramammary infection (a.k.a., mastitis) is the largest attributable cause of lost productivity, discarded milk, chronic morbidity, and prophylactic antibiotic use in the dairy industry (Gill et al. 1990, Bar et al. 2008). The average cost of mastitis to the U.S. Dairy industry is at least $184 per cow. This is approximately $1.8 billion or 10% of the total value of farm milk sales, and the majority of new infections are associated with the dry period, during which the udders are temporarily engorged with milk that often leaks from the teatends. Methods that accelerate dry-off that could be applied in both conventional and alternative systems would reduce this major source of lost dairy productivity. 2) Animal Health/Well-being: The engorgement of the udders at dry-off is a major cause of infections that decrease productivity. Udder engorgement also is a cause of discomfort, poor udder health, and negative behaviors (stress vocalization, reduced lying, (Tucker, Lacy-Hulbert & Webster 2009). Our development of a rapid dry-off agent is expected to have two salutary effects on animal health: enhance the effectiveness of therapeutic antibiotics, used in cows with subclinical mastitis at dry-off, and prevent new infections in cows that enter dry-off without mastitis. 3) Food and Environmental Safety: The three major agencies of the U.S. government with responsibilities associated with food safety (USDA, FDA, CDC) have clearly articulated the concern that antibiotic use in agriculture poses threats to food safety, and to the health of agricultural workers (Anonymous, Anonymous). In addition, the EPA has articulated concerns about antibiotics entering the environment from agricultural practices (Anonymous). These concerns are leading in the direction of restricting antibiotics in agriculture to those uses that are legitimately "therapeutic", rather than merely prophylactic. There are, undeniably, larger sources of antibiotic use in agriculture (particularly in the production of poultry and swine). However, our technology provides a scientifically-based opportunity for the dairy industry to contribute to the overall goal of reducing the routine use of non-therapeutic antibiotics.
<P> APPROACH: <BR> Scientific Goal #1: Perform cell culture experiments in which we screen compounds in vitro to determine the better candidates for rapid dry-off agents. These assays will use tight junction integrity and milk protein gene expression endpoints, and will compare conventional and tryptophan-sparing medium formulations. To elucidate the effects of FLX (Sigma, St. Louis, MO) on TJ and milk protein mRNA expression, experiments were designed using primary bovine mammary epithelial cells (pBMEC). Tissue dissociation and pBMEC cell isolation were performed as previously described (Hernandez et al. 2008, McGrath 1987). pBMEC are seeded on 0.4uM Transwell polyester membrane supports in 12-well plates (Corning, Corning NY) at 105 cells/cm2. Cells are fed daily in both the basolateral and apical chambers until plateau resistance (approximately 2500 ohmscm2) is achieved (approximately 8 d). TEER (ohmscm2) is measured daily using the "Epithelial Volt-Ohm Meter, EVOM" device (WPI, Sarasota, FL). At this time, cells are administered their respective treatments in the apical chamber only. Effects are monitored over the subsequent 72h, and results are expressed as a percent change in TEER relative to the control, vehicle-treated cultures. Scientific Goal #2: Perform in vivo lactation experiments in which we test three preferred compounds by infusions into the udders of dairy cows at dry-off. The study will be conducted on a 10,000 cow commercial dairy in Stanfield, Arizona. Prior to the start of the study all cows will be milked 3x daily. A total of 60 multiparous cows will be identified and randomly assigned to one of 4 treatment groups (vehicle, agents 1-3) and transferred to the hospital barn using DHIA (Dairy Head Information Association) test equipment meters. The control group will receive an intramammary infusion of the carrier, followed by Today (prophylactic antibiotic, Boehringer Ingelheim, Saint Joseph, MO) in each quarter on the test day. The treatment groups will be administered the drug agent in carrier (sterile distilled water:mineral oil, 9:1) , followed by Today. The following day (24h after agent infusion) cows will be milked out to determine differences in yield. Blood samples will be obtained by tail venipuncture at time 0 (treatment), and 12h, 24h and 48h after treatment to measure plasma lactose concentrations.