Non Technical Summary
Parturition is a risky period when the incidence of disease is high in dairy cows. These diseases include both metabolic (ketosis, displaced abomasum, hypocalcemia, and retained placenta) and infectious diseases (mastitis and metritis). According to the USDA 2007 NAHMS report, 37% of cows experience at least one disorder during the postpartum period. These diseases are quite costly. To provide an example of this, the cost of clinical mastitis during early lactation is estimated at $444 per case, and the annual cost of mastitis for the US dairy industry is estimated at $2 billion. As such, innovative animal health solutions will be required to solve complex problems in an economically strained dairy industry. In this research proposal, we strive to improve our understanding of the nexus between systemic inflammation and mammary gland health in periparturient dairy cattle.The objective of this proposal is to elucidate the direct effects of chronic low-grade systemic inflammation on mammary gland health. Elevated concentrations of inflammatory markers during the peripartum period have been associated with diseases including mastitis during the postpartum period in dairy cattle. Nevertheless, these associations do not necessarily demonstrate a causal relationship. We hypothesize that low-grade systemic inflammation will impair mammary gland immune responses and increase severity during an intramammary challenge.Therefore, in Aim 1, we propose to study the role of chronic, low-grade systemic inflammation in postpartum dairy cattle on mammary gland immune responses during an intramammary Streptococcus uberis challenge. For this objective, we will use recombinant tumor necrosis factor α as a model pro-inflammatory cytokine to induce chronic, low-grade inflammation. For Aim 2, we propose to assess the effects of postpartum administration of meloxicam, a non-steroidal anti-inflammatory drug used to inhibit postpartum systemic inflammation, on mammary gland immune responses during an intramammary Streptococcus uberis challenge. We anticipate that systemic inflammation will impair mammary gland immune responses leading to a more severe intramammary infection, whereas postpartum meloxicam administration will improve mammary gland immune responses leading to an enhanced ability to control and eliminate the Streptococcus uberis.The proposed study will define for the first time the impact of systemic inflammation on mammary gland immune responses. Postpartum dairy cattle experience chronic, low-grade systemic inflammation, which has been associated with disease incidence. Nevertheless, it is unclear if this systemic inflammatory response is protective or pathological. As such, it has been incredibly challenging to develop solutions for postpartum disorders because we do not understand the pathology of many of these disorders and diseases. Therefore, it comes as no surprise that the incidence of many clinical diseases during the postpartum period including clinical mastitis have stubbornly remained unchanged over the past few decades. Future research projects are needed to develop solutions for postpartum health problems; however, these projects must be informed by studies evaluating the causes of these disorders to identify potentially effective treatment strategies. The results from these studies will be used to inform future research projects to develop solutions to reduce the incidence of postpartum disorders including mastitis. The long-term goals of these projects are to improve dairy cattle health and reduce antimicrobial usage.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3410	1020	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
3410 - Dairy cattle, live animal;

Field Of Science
1020 - Physiology;

Goals / Objectives
The major goals of this project are to determine the effects of systemic inflammation on mammary gland immune responses in dairy cattle. Our long-term goals are to improve mammary gland health and reduce antimicrobial usage on dairy farms.Our first objective is to assess the effects of systemic TNFα signaling on mammary gland immune responses during an intramammary challenge in periparturient dairy cattle. We will assess the effects of subcutaneous recombinant TNFα (rbTNF) injection during the postpartum period to determine if systemic inflammation impairs mammary gland immune responses. There will be 2 treatment groups: 1) a control group that receives daily subcutaneous (SC) injections of a placebo (CON, n = 10) and 2) daily SC injections of rbTNF (rbTNF, n = 10). Multiparous Holstein cows will be injected daily for the first 7 d of lactation (7 total injections) starting on the day after calving. An intramammary Streptococcus uberis challenge will be conducted on d 4 after calving.Our second objective is to assess the impact of postpartum meloxicam administration on mammary gland immune responses during an intramammary challenge in periparturient dairy cattle. In Aim 1, we will determine the effects of inducing systemic inflammation on mammary gland immune responses. Conversely, in Aim 2, we will determine the effects of inhibiting inflammation on mammary gland immune responses using meloxicam, a non-steroidal anti-inflammatory drug (NSAID). In our previous work [24], we found that postcalving meloxicam administration increased energy-corrected milk yield by 2.5 kg/d for the first 15 wk of lactation [24]. In other studies, post-calving meloxicam administration increased milk yield by 4 kg/d over the 305-d lactation [25] and reduced SCC by 100,000 cells/mL during the first month after calving in a large field trial [26]. In light of these results, we propose to test the effects of postpartum meloxicam administration on mammary gland immune responses. Multiparous Holstein cows (n = 17 per treatment group) will be randomly assigned to postcalving meloxicam (approximately 24 h after calving) or control treatments. An intramammary Streptococcus uberis challenge will be conducted on d 4 after calving.

Project Methods
Aim 1. Multiparous Holstein cows will be blocked by parity, previous lactation milk yield, and calving month (to control for seasonality) and randomly assigned within block to 1 of 2 treatment groups: 1) control (CON, n = 10) and 2) recombinant bovine TNF (rbTNF, n = 10). The recombinant TNF will be administered subcutaneously (SC) starting on d 1 after calving until d 7 at a dose of 2 µg/kg of body weight in 10 mL of phosphate-buffered saline (PBS); 10 mL of PBS will be administered SC starting on d 1 after calving until d 7 to serve as a placebo in the CON group. Cows will receive daily SC injections on the upper half of the neck. To induce a chronic, low-grade systemic inflammation, rbTNF will be dosed at 2 µg/kg of body weight. Cows will be weighed on the day of calving to determine body weight and appropriate dosages. Recombinant bovine TNFα will be sourced by a vendor that specializes in recombinant protein expression (GenScript, Piscataway, NJ) and has synthesized rbTNF previously for similar research projects on a large-scale [13].Aim 2. Multiparous Holstein cows will be blocked by parity, previous lactation milk yield, and calving month (to control for seasonality), and randomly assigned within block to 1 of 2 treatment groups: 1) a control group (CON, n = 17) that receives a gel capsule as a placebo and 2) a single oral administration of a bolus of meloxicam (MEL, n = 17) in a gel capsule dosed at approximately 1 mg/kg of body weight. Approximately 24 h after calving, cows will be weighed, dosages will be determined using the body weight, and treatments will be administered. Meloxicam (15 mg tablets; Zydus Pharmaceuticals) will be administered by mouth in a gel capsule using a bolus gun on d 1 after calving; an empty gel capsule will be administered orally approximately 24 h after calving to serve as a placebo in the CON group. All cows will receive an intramammary Streptococcus uberis (S. uberis 0140J strain, 2,000 cfu in 2 mL of sterile PBS [1,000 cfu/mL]) challenge on d 4 after calving.Individual quarter milk samples will be collected pre-challenge on d 3 and d 4 using aseptic technique prior to the intramammary challenge from all four quarters using standard microbiologic procedures to identify any cows with pre-existing infections. On d 5, 6, 7, and 8, milk samples will be collected from all four quarters for standard microbiologic procedures to monitor bacteria presence in the gland. Bacterial enumeration of S. uberis from individual quarter milk samples from the challenged quarter will be collected daily on d 5, 6, 7, and 8. For bacterial counts determination, a selective agar for streptococci (Edwards Modified Medium) will be used with serial dilutions. At each milk sample time point, mastitis severity will be recorded using a previously defined scale.Daily milk yield will be recorded from calving until 8 d after calving. Composite milk samples will be collected once daily on d 1 (just prior to treatment), 2, 3, 4, 5, 6, 7, and 8 to determine milk composition and composite somatic cell count (SCC). Individual quarter foremilk samples will be collected from all four quarters including the challenged quarter just prior to milking on d 1 (just prior to treatment), 2, 3, 4, 5, 6, 7, and 8 for quarter-level SCC. Additionally, milk collected from the challenged quarter on d 4 (just prior to challenge), 5, 6, 7, and 8 (just prior to biopsy) will be defatted using centrifugation to acquire milk serum. We will quantify TNFα in milk serum samples using an ELISA (Bovine TNFα DuoSet ELISA, R&D Systems) that we previously validated for milk TNFα.Individual dry matter (feed) intake will be recorded starting 1 wk prior to calving until the end of the trial (8 d post-calving). Prepartum dry matter intake will be used as a covariate for postpartum intake during the trial. Rectal temperatures will be recorded once daily (0900 h) from d 1 through d 8. Body weights will be recorded daily starting on the day of calving until d 8. Body condition score at calving (1 to 5 scale) will be recorded prior to treatment application to test as a covariate in the statistical analyses.Mammary tissue will be collected from the challenged rear quarter on d 8 (4 d following the S. uberis challenge) to assess tissue-specific inflammatory responses to the S. uberis challenge. This time point was selected as peak clinical signs from S. uberis mastitis occur between 3 to 4 d following intramammary challenge. Cows will undergo standard biopsy procedure using analgesia, as previously described. Samples (approximately 0.75 to 1 gram of tissue) will be immediately snap frozen in liquid nitrogen. Mammary tissue will be homogenized, lysed using RIPA buffer, and then centrifuged to remove debris. The total protein concentration of the tissue homogenate samples will be standardized to 1 mg/mL. Mammary cytokines and chemokines will be quantified simultaneously using Luminex® xMAP technology and the Milliplex Bovine Cytokine/Chemokine Magnetic Bead Panel 1 - Immunology Multiplex Assay. This assay is validated for bovine tissue homogenates and plasma and is capable of quantifying 15 commonly measured cytokines and chemokines. The 15 available analytes are IFNγ, IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17A, IL-36RA, IP-10, MCP-1, MIP-1α, MIP-1β, TNFα, and VEGF-A.Blood samples will be collected daily starting on d 1 and ending on d 8. Plasma will be separated and stored at -80°C. As with the mammary tissue, plasma cytokines and chemokines will be quantified simultaneously using Luminex® xMAP technology and the Milliplex Bovine Cytokine/Chemokine Magnetic Bead Panel 1 - Immunology Multiplex Assay. We will quantify plasma IFNγ, IL-1α, IL-1β, IL-4, IL-6, IL-8 (CXCL8), IL-10, IL-17A, IL-36RA (IL-1F5), IP-10 (CXCL10), MCP-1 (CCL2), MIP-1α (CCL3), MIP-1β (CCL4), TNFα, and VEGF-A. In addition to the immunology panel, we will also quantify plasma free fatty acids, beta-hydroxybutyrate, glucose, and haptoglobin.Data will be analyzed using linear mixed models (GLIMMIX, SAS 9.4) with the fixed effect of treatment (CON or TNF for aim 1, CON or MEL for aim 2), time (repeated measure, when applicable), and the interaction of treatment and time, along with the random effect of block and cow. When applicable, covariates recorded prior to treatment application and their interactions with treatment will be tested.Efforts. Scientific findings will be distributed to the stakeholders (dairy farmers, extension agents, nutritionists, and veterinarians) as well as be used to inform course curriculum administered to students. Scientific findings will be published in peer-reviewed scientific journal articles and presented at numerous conferences.Evaluation. The success of this project will be evaluated by the number of publications generated, the number of students trained, and additional grants funded.