INVESTIGATING MECHANISMS OF INNATE AND RESIDENT IMMUNITY IN THE BOVINE MAMMARY GLAND

Mastitis in dairy cattle continues to be a challenge. Dairy cows are constantly exposed to mastitis pathogens, thus it is critical to develop strategies that enable them to resist and quickly eliminate infections. A number of strategies have been developed and improved through continuous innovation - this includes teat disinfection, milking equipment, nutrition, environmental management, bedding management, genetic improvement, and vaccine development. We've made considerable progress over the last 30-40 years. Despite this, mastitis remains present on all dairies, the overwhelmingly dominant reason dairy cows are treated with antibiotics, and explains why approximately 1 in 4 cows are culled from the herd. Mastitis impairs more than cow health and welfare, as it also lowers milk production, increases costs, and negatively impacts herd efficiency; inflammation and milk component changes limit product yields, shelf-life, and taste; and increases risk of food borne illness. In this proposal, we will focus on two potential opportunities to boost innate immunity - preventing entry into the gland and rapid reaction to invading pathogens to promote killing.Prevent bacteria from entering the mammary gland. The teat end opening is the doorway for bacteria to gain entry to the mammary gland and potentially cause infection. The teat end has a sphincter of 6-10 mm (0.25 - 0.4 in) which prevents entry further into the canal. After milking the muscle of this sphincter becomes fatigued and does not close quickly. This has served as the basis for the recommendation to keep cows standing after milking and allow time for the teat end to close prior to lying down and directly exposing the teat end to bedding material containing infectious organisms (McDonald, 1975). Relatively recently, DeVries and colleagues (2010) evaluated new intramammary infections in relation to standing time and determined a reduction in new infections when cows remained standing 30-60 min after milking, but increased thereafter until approximately 3 hr after milking. Thus the recommendation to keep cows standing for 30-60 min after milking holds. However, the secondary rise in new intramammary infections (DeVries, 2010) corresponds to increases in teat end diameter that occurs with increasing pressure from milk on the teat end (McDonald, 1975). In addition to changes in teat diameter, the teat sphincter also has rhythmic contractions which has been proposed to occur as a result of holding increasing milk volumes in the cistern and helps to sweep organisms and debris out of the teat can. These contractions are dramatically reduced following milking and did not start to return until approximately 5 hr after milking (Lefcourt, 1981). Clinical and subclinical which has reductions in blood calcium availability, also have been proposed to increase the time required for the teat end to close (Goff and Kimura, 2002). However, this has not been evaluated. Teat end closure also may be impaired post-milking in the southeast, where cows are heat stressed routinely in the summer months. We hypothesize the ability of the teat sphincter to close during periods of heat stress and hypocalcemia may be compromised. Identifying strategies to rapidly close the teat end after milking would serve as a barrier to prevent bacteria from entering the gland after milking. By testing our hypothesis and completing our objectives, we will be able to develop novel methods to promote closure of the teat end between milkings.Mammary immune development. We've made considerable progress in understanding how the mammary gland responds to different types of infection - the primary immune cell types present, the cytokines and oxylipids that communicate responses, the generation of antibodies and antimicrobial factors, cellular communication, and the shifts in immune responsiveness that occur with parturition and early lactation (Bannerman, 2009; Schukken et al., 2011; Sordillo and Streicher, 2002; Sordillo, 2018). However, we lack knowledge regarding the immunological development of the gland as research to date has focused on heifers greater than 14 months of age and mature dairy cows. This is a significant gap in our knowledge when we consider resident immune cell populations in the gastrointestinal tract can be seeded during fetal and post-natal development, and their maturation shaped by local environments (Agace and McCoy, 2017). In the gastrointestinal tract, resident dendritic cells and intraepithelial lymphocytes help maintain epithelial integrity and shape responses to resident and pathogenic bacteria. In the mammary gland of non-infected pubertal heifers and mature dairy cows, intraepithelial T lymphocytes, iba1+ monocytes, and antibody-producing plasmocytes have been observed, indicating the mammary gland has resident immune cell populations (Magro et al., 2017; Quiroga and Nickerson, 1993). However, our lack of knowledge regarding when resident immune cell populations seed the mammary gland, their functional role in homeostasis, and how it changes through growth and development prevents us from targeting these cells for promoting mastitis resistance. We hypothesize intraepithelial lymphocytes seed the mammary gland during post-natal development to help maintain the integrity of the mammary gland and shape responses to host microbiomes and pathogens. By testing our hypothesis and completing our objectives, we will obtain critical knowledge needed regarding targeting the mammary immune gland early in life to protect against mastitis.To accomplish our goal of developing strategies to prevent pathogen entry and to rapidly activate resident lymphoid populations, we propose the two following objectives:Objective 1. Evaluate the conditions which influence the strength and closure of the teat sphincter. Objective 2. Determine development and functional role of resident immune cells in the bovine mammary gland.