Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
Animal & Veterinary Sciences
Non Technical Summary
This seed grant application describes a preliminary investigative activity into a new aspect of mammary gland physiology. We will test whether a specialized cell type, the M-cell, exists in the mammary gland. M-cells are found in many organs and help the immune system to promote a strong response to disease causing organisms. Unfortunately, there is no current data about M-cells in the mammary gland. The present dearth of information about mammary M-cells is quite surprising, but the experiments outlined in this proposal will provide a focused determination of whether M-cells form in response to bacterial invasion of the udder though extensive microscope examination of tissues from animals challenged with bacterial infections. If our hypothesis is borne out and M-cells are identified, there will be many possibilities for future research projects. As an example, understanding more about the formation of M-cells, and the process of antigen sampling, and immune cell movements could aid in targeted vaccine development and delivery strategies. Engineered nanoparticles could be developed and optimized to generate a more pronounced mammary mucosal immune response that would confer greater resistance or recovery from mastitis. If M-cells are not identified, the study will still provide novel insights into the timing and progression of bacterial infections in the mammary gland.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Mastitis caused by Staphylococcus aureus (S. aureus) is a major management challenge and source of economic loss for dairy producers. Current vaccine and antibiotic therapies have only limited success and chronic infections are common. There is a well-populated literature detailing the mechanisms through which S. aureus establish infection and resist treatment, but new knowledge of host-pathogen interactions is required to improve prevention and control measures. One promising research area for improving resistance to mastitis causing organisms is in mucosal immunity. Classically, the mucosal immune system is represented by specialized structures in the gut called Peyer's patches. In regions where the lymphoid accumulations directly contact the mucosal epithelium, the morphology of the epithelial cells changes so that microvilli become less prominent or absent. The follicle associated epithelium (FAE) contains many "M-cells," named for the characteristic microfolds on the lumenal surface of the cell and attenuated membranous cytoplasm. The M-cells completely envelop and surround one or more leukocytes but maintain intact junctional complexes. Even more surprisingly, the M-cells then begin a process whereby antigen components from the lumenal space are endocytosed, transported across the cell, and delivered to the waiting leukocytes, essentially without any further processing. The M-cell arrangement thus allows for a physically sequestered environment for antigen sampling and presentation of live pathogens to leukocytes. A number of studies have demonstrated that M-cells are found in mucosal locations outside the gut, including the tonsils, conjunctiva, and respiratory tract. Curiously, there are no available reports that specifically describe an M-cell phenotype in the mammary gland of any species. The purpose of this project is test the hypothesis that M-cells form in the mammary gland in response to bacterial challenge. The pilot study will consist of an in vivo challenge with live S. aureus, followed by extensive histologic analyses.
Project Methods
Our hypothesis is that M-cells form in the mammary mucosa after exposure to bacteria. To investigate the possibility of M-cell formation during the host-response to S. aureus, we will conduct an in vivo challenge experiment and perform extensive histologic analysis. In light of the observations from the published literature and our preliminary data, the histologic analyses will focus first on the mucosa of Furstenberg's rosette. Multiparous Holstein cattle will be selected from the Clemson University herd after veterinary inspection. Infusions of live S. aureus will be delivered via the streak canal to lactating cows at 3 separate time points (one per teat) so that the duration of staph exposure is varied and the number of treated animals is minimized. Infusions will begin after the evening milking, 12h before sacrifice, and will be repeated at 2h and 1h prior to tissue collection. The fourth gland quarter in each animal will serve as an un-inoculated control. At the last two time points, we will also introduce S. aureus into the conjunctival space in an effort to secure an additional within-animal positive control. Each animal will be sacrificed for tissue collection. At sacrifice, teats, conjunctiva, and sections of ileal tissue containing Peyer's patches will be collected. Additional samples of mammary parenchyma will be collected from the annular ring at the base of the teat, from the gland cistern, and from more distal secretory tissue. Transmission electron microscopy will be performed to inspect the mucosal epithelia in samples collected from the challenged animals. Standard lead citrate/uranyl acetate staining will be used for all the initial studies. Particular attention will be paid to search for signs of invading leukocytes, endocytic vesicles, altered epithelial morphology, disruptions to the basal lamina, and similar ultrastructural features of M-cells. As much as possible, serial sections will be captured onto each grid so that if M-cells are observed, multiple profiles through the same grouping of cells can be visualized. Our assumption is that exposure to live bacteria will induce a pronounced physiologic response. If M-cell formation is part of that response, we will have an exquisitely sensitive analysis to evaluate the development of M-cell morphology on a cell-by-cell basis. In this pilot trial, we are not expressly seeking to quantify the development or change in distribution of M-cells, just to determine whether M-cells even exist in the bovine mammary gland. Clear demonstration of the M-cell phenotype at any time point would be a very important outcome from the study and would provide important validation for future studies of bovine mammary mucosal immunity.