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An HPLC to Strengthen Analytical Opportunities in Bovine Mastitis

Investigators
Rezamand, Pedram
Institutions
University of Idaho
Start date
2009
End date
2010
Objective
Our overall goal is to test the hypothesis that 1) hyporetinemia (low vitamin A) associated with an intra-mammary infection (IMI) is due to a decrease in retinol-binding protein (RBP) complex, that 2) innate immune response to occurrence of an IMI is impaired by altered retinoid metabolism, and that 3) milk lipids, particularly fatty acids, reduce the risk of mastitis in cows.

Specific Objectives:

  1. To determine the effect of dietary supplementation of vitamin A and protein on retinoid metabolism and circulating RBP during the periparturient period in vivo
  2. To investigate whether dietary supplementation of vitamin A and protein modifies innate immune response to an experimentally (Escherichia (E.) coli) -induced IMI during early lactation
  3. To determine the fatty acid composition of different lipid fractions in cows infected with S. aureus
  4. To investigate the relationship between fatty acid composition of free fatty acids and infectivity (duration of infection and extent of SCC changes) of S. aureus.
More information
NON-TECHNICAL SUMMARY: The risk of mastitis is greatest during the transition period. The cost of mastitis is greater than $1.7 billion each year and represents a significant loss for the dairy industry. During the last 25 years, there has been improvement in mastitis prevention and control as well as a greater understanding of the effects of dietary nutrients on immune function. However, research on nutritional factors affecting immune function in particular, bovine retinoid metabolites and RBP, the carrier protein for retinol, are limited for dairy cows and in response to intra-mammary infection. Our laboratories have been investigating the metabolic challenges during the transition period in dairy cows and their effects on specific immune measures and, in particular, on mastitis. This proposed project will create new knowledge of the effects of the retinoids and RBP status on immune system enhancement in dairy cows. Mastitis affects the composition of fluid milk and the extent to which composition is altered depends on the causative agent and host inflammatory response to the infection. In response to an infection, major changes occur in the mammary gland. One of the perceived minor changes that occurs is in the lipid composition of milk. Increased concentrations of free fatty acids in milk are common during mastitis in cows. Increases of 50 to 100% in free fatty acid content of milk from cows with mastitis have been detected. The increase in free fatty acids during infection of the mammary gland is not constant among all fatty acids. Preference for fatty acids C4:0, C6:0 and C8:0 is at the sn-3 position, while C12:0, C14:0, and C16:0 are located at the sn-2 position. These "released" fatty acids in milk may act as antimicrobials reducing the growth of the pathogen.

APPROACH: 1-Periparturient study: Forty eight high producing multiparous Holstein cows from ~ 4 weeks prepartum to 3 weeks postpartum will be used in a CRB design in a 2 x 2 factorial arrangement. Rations will be developed containing either 12.0% CP or 15.7% CP. At each ration CP level, diets will be supplemented with vitamin A to provide either no supplement or 110 IU/kg of BW. Twelve cows will be randomly assigned to each dietary treatment blocked based on expected parturition date (EPD) and previous milk yield. The cows will be fed experimental TMR twice a day, starting ~4 weeks prepartum through parturition. A common lactation ration will be fed after parturition for all cows and cows will be milked twice daily. Weekly BW, weekly BCS, and daily DMI will be measured. Cows will be fed individually using Calan gates and will have free access to water at all times. Postpartum, milk yield will be recorded and sampled two times daily and all cows will receive the same TMR. Rations and refusals will be sampled for dry matter and composited monthly for nutrient analysis including retinol and beta-carotene contents. Blood samples will be collected at day -28, -21, -14, -7 EPD, at parturition, and at day +1, +2, +3, +6, +9, +12, +15, +18 and +21 postpartum. Concentrations of plasma albumin, HPT, beta-carotene, retinol, RBP, at-A, 9-cis RA, and 13-cis RA will be determined using HPLC. Blood neutrophil retinol content, and killing of E. coli will be determined at d -28 relative to EPD and immediately before IM challenge (day +7 relative to EPD. Milk will be sampled at 1st, 2nd, 3rd milking and day +3, +6, +9, +12, +15, +18, and +21 and analyzed for retinoids and beta-carotene. Liver biopsy will be performed at days -28, -3, +3, +10, and +17 in order to determine retinol content and RBP mRNA expression in the liver. 2- Mastitis study: Using a subset of cows from Periparturient Study (n = 3/treatment), one quarter of each cow will be infused via the teat canal with E. coli LPS. Quarter milk will be sampled at 0, 6, 12, 18 h after the intra-mammary E. coli infusion and at day +2,+ 3, +4, +7, +14 and +21, and used for bacteriological analysis. Quarter milk samples will be analyzed for â-carotene, retinoids, fat, protein, total solid (TS), lactose, and SCC. Quarter milk samples will be also used to isolate milk leukocytes, and the population of neutrophils, B cells and T cells will be determined. Gene expression of pro-inflammatory cytokines including tumor necrosis factor-á (TNF-alpha), G/M-CSF, IL-1beta, IL-6 and IL-8 will be measured in milk cells via quantitative real time rt-PCR. Blood samples will be collected at day -7 and 0 (relative to LPS infusion), and at the same time point as milk samples via coccygeal venipuncture. Concentrations of plasma albumin, haptoglobin, beta-carotene, retinol, RBP, at-RA, 9-cis RA, 13-cis RA, total reactive oxygen species, thiobarbituric acid-reactive substances and leukocyte populationswill be determined. Real-time reverse transcribed PCR will be carried out in a 7500 Fast real time PCR system using custom designed TaqMan MGB probes.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
View this project
Project number
IDA00907-CG
Accession number
220755
Categories
Escherichia coli
Risk Assessment, Management, and Communication
Bacterial Pathogens