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<OL><LI>Characterization of host mechanisms associated with mastitis susceptibility and resistance <LI>Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses<LI>Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety </ol>Methods: <BR>Objective 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance. <BR>(i) Environment, Nutrition, and Management Related Host Factors Associated with IMI In order to cause an IMI, bacteria must traverse the teat canal. Under normal circumstances this is relatively difficult. However, cold weather and extreme changes in weather in a short time frame, negatively impact teat end condition and increase the chances of mastitis occurring. Clinical trials will be conducted in IA, LA, and WA to evaluate therapeutic approaches to improve teat end condition and prevent the negative teat end changes associated with cold weather. In conjunction with this, new technologies will be assessed to more objectively evaluate teat skin condition. Several stations (MN, IL, WI, IN, PA) will collaborate on adherence and efficacy studies on teat sealants and persistent barrier dips. The risk of mastitis is greater after calving and during early lactation. This is related to a variety of reasons, the stress of calving, the general immune-suppression that occurs during that time, as well as the occurrence of negative energy balance - where cows cannot take in sufficient nutrients to support milk production. To better identify host factors associated with negative energy balance that contribute to impaired host immunity and increased mastitis susceptibility, a series of experiments will evaluate gene expression of mammary tissue and blood neutrophils when cows are exposed to different feeding strategies during early lactation (IL). <BR>(ii) Host-Pathogen Interactions at the Cellular Level Mastitis is caused by a variety of organisms, however several of the most common species are E. coli, K. pneumoniae, and S. aureus. Understanding how these organisms interact with host cells is critical to developing therapies that reduce the incidence and/or severity of disease. Two particular cell types will be evaluated by members of this project - mammary epithelial cells and neutrophils. Mammary epithelial cells are one of the first cells to interact with the invading organisms and initiate host responses, which include the rapid influx of neutrophils to help kill pathogens. The ability of bacteria to subvert either of these responses leads to more severe and/or chronic infections.
NON-TECHNICAL SUMMARY: Mastitis continues to be a major economic risk, capable of devastating the small or large dairy operation. Prevention and control have relied on hygiene during and between milkings, antibiotic treatment or teat sealants during the dry period, antibiotic treatment of clinically detectable mastitis, and culling of seriously affected cattle. Due to human health concerns, dairy farmers follow strict regulations, and are encouraged to avoid exogenous chemicals or drugs. To reduce the need for antibiotics, both innate and adaptive immune responses can be activated in the mammary gland. In innate immunity, the role of "normal flora" on the skin and mucosal surfaces can be vital: "good" bacteria can kill, or simply outcompete, pathogens. That normal flora, if augmented following each disruption by milking, might help form a defense against pathogens. This project will evaluate normal flora of the teat skin, and attempt to augment innate defenses by enhancing that flora between milkings. As well, we will question whether that flora can increase innate host cellular defenses against pathogens. In general, candidates for use as probiotics should be capable of colonizing the site, must have antimicrobial properties, and not be potentially pathogenic. Lactobacilli produce bacteriocidal substances (bacteriocins) and acidify the local microenvironment, suggesting their use to enhance innate defenses. The somatic cell count (SCC) of milk samples reflects a variety of cells that are shed into the lumen of the mammary gland during lactation: epithelial cells, neutrophils, and other cells involved with immune defenses, such as macrophages. High SCC values are associated with infection, but a lower average SCC may actually be associated with a higher incidence of mastitis due to coliforms, or environmental organisms. An optimal number or composition of SCC may exist in the healthy cow's milk, and the activation status of these cells may determine the outcome of infection with pathogens. Innate defenses may be triggered by pathogen-associated molecular patterns (PAMPS) which are perceived by cellular receptors called PRR; pattern recognition receptors. The toll-like receptors (TLR) form a family of PRR. Between the 13 known vertebrate TLR, it is thought that essentially all pathogens can be recognized. Work with intestinal epithelial cells suggests that expression of TLRs are modulated by intestinal microbial flora. Modulation of TLR on the mammary epithelial cell component of the SCC may be a way of detecting up- or down-regulation of the innate immune system of the udder. Expression of TLR or of other innate mechanisms may be modulated by the presence of a probiotic, as occurs in the gut. If true, then a probiotic teat dip will be an active way of protecting the teat end, versus the passive protection via disinfectants such as iodine. Enhancing innate immunity will reduce mastitis, reducing losses from an estimated 11% of total US milk production. <P>APPROACH: We will study host mechanisms associated with mastitis susceptibility and resistance by 1) characterizing teat end endogenous microflora, 2) developing and using a probiotic teat dip to enhance resistance to mastitis, and 3) studying whether a probiotic teat dip enhances innate immunity in the distal teat. We will utilize a novel bacterial phenotypic identification system, Biolog, which will allow rapid and accurate characterization of the endogenous flora. We will investigate the efficacy of endogenous or augmented (probiotic) flora in prevention of mastitis under normal conditions for lactating cattle. We will also investigate the cellular composition of milk in the distal canal, and the changes induced in those cells when non-pathogenic flora are enhanced between milkings, rather than impeded by ordinary (disinfection) teat dip methods. Teat end and streak canal cultures will be collected from dry and lactating cows. Flora will be characterized using the Biolog system. Endogenous lactobacilli or other probiotic bacteria collected above will be cultured and preserved (frozen) for passaging studies. We will use both endogenous and ATCC lactobacilli (already established as capable of colonizing mammalian mucosal surfaces). Bovine mammary epithelial cells will be cultured with lactobacilli to evaluate cell adherence. Biofilm formation and in vitro antimicrobial activity will be evaluated for lactobacillus candidates. Selected probiotic cultures will be tested in vivo as a teat dip between milkings. In a preliminary trial, method and frequency of application will be established using glycerin or other nontoxic vehicle for application of the culture. Milk cows (4 per treatment) from the university dairy will be assigned to treatment (lactobacillus culture teat dip) versus control (iodine-based teat dip only) groups; after 14 days of data collection (28 samples per cow), each cow will be dried off. Teat end skin, strip milk and bulk milk will be cultured for the presence of lactobacilli or other probiotic bacteria; suspect mastitis pathogens will be further identified. "Stripped" milk cells will be harvested and assessed (see below). Milk from treated cows will be collected individually and tested for SCC and microbial growth. In a second set of experiments, we will pursue the effect of selecting effective lactobacilli subcultures and repeatedly inoculating teat ends with passaged lactobacilli as above. Finally, we will evaluate the effects of probiotic use on mammary innate defenses by assessing SCC of milk in treated and control cows (above); modulation of innate immune markers, such as TLR expression, will be investigated by use of available sequence information and RT-PCR of somatic cells. These studies will be immediately applicable in the organic dairy industry, and should lead to further studies utilizing other dairy species such as goats and sheep. Results will be disseminated by the investigators as publications and workshops in collaboration with Extension personnel. If successful, the project may be offered as a clinical study using local small dairies to assess efficacy.