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The proposed experiments will generate data to evaluate the hypothesis that polymorphisms in genes associated with pathogen detection and intracellular signaling pathways contribute to differences between cows in their innate resistance to mastitis. The proposed research stems from anecdotal evidence that some cows appear to have superior endogenous resistance to mastitis than do other cows. Our preliminary data reveals substantial variation between cows in the ability of their dermal fibroblasts to produce cytokines in response to E. coli endotoxin (LPS). Subsequently, of 37 cows tested, four high and four low responders were selected based on their fibroblasts' responses, and were challenged with an intramammary infusion of E. coli. Both groups cleared the infection after five days, yet the high responders had more BSA in milk and a slower decline in milk SCC. The proposed research seeks to extend these findings with additional animals to be screened and then challenged with E. coli, and a second group of animals screened and selected for challenge with S. aureus. The final aim seeks to identify genes that are causative for the extreme variation in dermal fibroblast response to LPS. A combination of microarray, RT-PCR, ELISA and western blot strategies will narrow a list of candidate genes whose coding regions will be sequenced from DNA obtained from select high and low responder animals.
NON-TECHNICAL SUMMARY: The long-term goal of the Kerr lab research is to identify genetic differences between animals in their ability to resist mastitis (infection of the mammary gland). This disease is the most costly infectious disease of dairy cattle, affecting both the quality and quantity of milk produced. Infectious disease must also be controlled to ensure the welfare and longevity of dairy animals. Our recent award from AFRI is to continue this research into natural disease prevention mechanisms. Much anecdotal evidence exists suggesting that some cows are naturally more resistant to infectious disease than are others. Our lab has been exploring the use of cultured skin cells to predict an animal's innate disease resistance potential. Small skin samples were obtained from the shoulder region of 37 cows. The samples were then cultured in laboratory dishes and challenged with a toxin produced by mastitis-causing E. coli. The cells respond to the toxin by producing interleukin-8, which in the animal would summon white blood cells (neutrophils) to combat the infection. Interestingly, cells from different cows produce vastly different quantities of the interlukin-8. The 37 cows were then ranked high to low based on the skin cell IL-8 production. The top and bottom ranked cows were then challenged with intramammary infusion of E. coli bacteria. Both groups cleared he infection within a few days, but the high responding cows had evidence of a greater immune response (increases in milk neutrophils and serum proteins). Considering that both groups cleared the infection in similar fashion then the more pronounced immune response, which actually lowers milk quality, may not be needed. The optimal immune response is one that is sufficient to clear the infection, but does so in a contained fashion such that damage to the cow is minimized.
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APPROACH: Dermal fibroblasts will be obtained from two groups (n=100) of lactating dairy cattle in early lactation. The cells will be challenged in vitro in replicate dishes with E. coli endotoxin and interlukin-1B. Secretion of interlukin-8 into the media over the subsequent 24 hours will then be determined by ELISA. The cows in each group will then be ranked based on the IL-8 secretion and the eight highest and eight lowest ranked animals will be challenged in late lactation with E. coli (group 1) or S. aureus (group 2). Animal response variables to be measured include milk somatic cell count, milk serum albumin concentration, and milk bacteria (colony forming units). The final objective will explore the genetic basis for differences between high and low IL-8 secreting dermal fibroblasts. This will be accomplished by microarray analysis to identify candidate genes, and followed by DNA sequencing to locate candidate polymorphisms.