Duration: 10/01/2012 to 09/30/2017

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

In the United States, cash receipts from marketing of milk during 2010 totaled $31.4 billion (NASS, 2011) and it is estimated that U.S. consumers spend approximately 11% of their food dollars on dairy products (IDFA 2009). However, the dairy industry continues to experience significant monetary drain through the losses associated with common diseases. Bovine mastitis is the most costly infectious disease currently affecting dairy cattle. Recent estimates suggest that economic losses due to clinical and subclinical mastitis are in the range of $200 per cow per year (Hogeveen et al., 2011). These losses are primarily due to lost milk production, increased veterinary costs, increased cow mortality, and discarded milk. While significant advances have been made in controlling some types of mastitis, the complex etiology of the disease and ongoing changes in dairy practices dictate that new and more effective methods for control and treatment be developed over time. Single site studies are often limited in terms of expertise and cattle numbers. A multi-state project provides advantages in terms of increased numbers of herds and cattle as well as multiple levels of expertise. The purpose of NE-1028 is to coordinate multidisciplinary research efforts on mastitis that are being conducted at various laboratories throughout the United States. The magnitude and scope of attempting to solve these problems extend far beyond the ability of any one institution. The ability to cooperate on a regional and national basis allows the integration of resources and knowledge to address this problem. Recognition of the need for a coordinated effort to study resistance of the dairy cow to mastitis resulted in the design and initiation of multi-State Project NE-1028. The NE-1028 project has provided a forum for new and established researchers to develop collaborative relationships, and to share resources and expertise. The NE-1028 project is comprised of three objectives 1) characterization of host mechanisms associated with mastitis susceptibility and resistance, 2) characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses, and 3) assessment and application of new technologies that advance mastitis control, milk quality, and dairy food safety. Accomplishments in the last 5 years are listed below by objective. Objective 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance. Achievements include determination of antibody response following vaccination against Staphylococcus aureus, as well as Escherichia coli; understanding the risk for E. coli mastitis during a S. aureus infection; evaluation of neutrophil function in the peripartum period and during vitamin E supplementation; determination of the relationship between feeding behavior, animal activity and intramammary infection; understanding the host response during challenge with Enterococcus faecium; determination of the role of lactoferrin in Streptococcus uberis internalization by epithelial cells and the role of dietary phosphorus in immune function; evaluation of the role of superantigens in the differentiation of monocytes into dendritic cells; determination of the effect of lipoxygenase metabolites on platelet activating factor in E. coli mastitis; evaluation of the role of CXCR1 in mastitis susceptibility and host binding in S. uberis infections; discovery of associations between genetic susceptibility to mastitis and altered actin expression in neutrophils; an evaluation of the factors associated with cytokine levels and acute phase proteins; evaluation of the relationships between the metabolic dynamics of fat mobilization (especially during the periparturient period) and immune responses focusing on the impact of fat mobilization on oxidative stress and the ensuing impact on mononuclear cell membrane lipids/inflammatory responses; and determination of association between hepatic retinol binding protein and key pro-inflammatory markers in dairy cows. Objective 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses. A major achievement for this objective is the continued examination of the role of coagulase-negative staphylococci on mastitis. More detailed examination of this diverse group of bacteria, including genotypic identification, has revealed that different species have surprisingly different effects on pathogenesis. Some species cause little or no decline in milk production. Additional progress is being made on using PCR-based techniques to identify pathogens in high SCC milk that had previously been declared as no growth using traditional culture techniques. This will be of great, widespread importance in mastitis treatment and research. Continued work exploring the development of bacterial resistance to commonly used antimicrobials is providing needed information on this critical treatment of mastitis. Antimicrobial resistance patterns of bovine and human isolates appear similar, and recent work suggests that mastitis therapeutics do not seem to induce or select for resistant bacteria (Barlow, 2011). Additional achievements include further identification of pathogen virulence factors and their association with mastitis pathogenesis, and detailed in vitro studies on bacterial adherence to, and penetration into mammary epithelial cells. Objective 3: Assessment and application of new technologies that advance mastitis control, milk quality, and dairy food safety. Achievements include evaluation of S. aureus and E. coli mastitis vaccines; development and application of milk residue testing; development of control programs for S. aureus and Mycoplasma spp.; evaluation of extended therapy for S. aureus and S. uberis mastitis and assessment of methods for on-farm and laboratory diagnosis of mastitis and food-borne pathogens. Improvements in diagnostic technologies include development and application of PCR to detect Mycoplasma mastitis; to identify and speciate pathogenic Prototheca (algae) as a screening technique for bulk tank filters; and to validate 16S gene sequence analysis for mastitis pathogen detection. These molecular techniques are critically important tools in mastitis treatment. Other achievements include developing and assessing techniques for evaluating milk quality in sheep and goats; and evaluation of an in-line milk analysis system for somatic cell count and other quality assessment components; and studies of colostral quality assessment methods. Finally, several workers have reported on alternative methods of preventing (probiotic) and treating (botanical/novel intramammary infusions) mastitis. In addition, the group has completed portions and has work in progress on several other collaborative studies which include determination of antibiotic residues in heifers treated with intramammary antibiotics before parturition (coordinated by CT), understanding virulence factors and host adaptation to Klebsiella spp. (NY, VA, Moredun), evaluation of environmental streptococcal mastitis (IA, IL, TN, WA, VA), evaluation of the role of vaccination in mastitis prevention (GA, MI, MO), evaluation of genetic selection for milk production on innate immune response (VT and MN), and the standardization of definitions for intramammary infection (PEI, ONT). Through the concerted and collaborative efforts of its member stations, the NE- 1028 has met the milestones detailed in the project description and will continue to work on similar collaborative projects in the next 5 years. Data generated under the auspices of the NE-1028 project have been used by member stations to gain intramural and extramural funding for mastitis research. In addition, a subcommittee of NE-1028 submitted a proposal for the USDA Coordinated Agricultural Project (CAP) program in July 2010. Although the proposal was not funded, the group members are eager to re-submit for related requests for proposals. As a multi-State, multi-institution project, the NE- 1028 is uniquely situated to apply for CAP funding should mastitis and milk quality become a targeted area of research. The mastitis research workers group has met in conjunction with the NE-1028 annual meeting for many years, and in recent years, the mastitis research workers topics have been included in NE-1028 minutes, showing current active areas of research by NE-1028 members. International visitors and collaborators are often included in these presentations. In addition to the mastitis research workers conference, the NE-1028 members provide technology transfer to the scientific community and industry stakeholders. In the last 4 years, members of the project have collectively published multiple book chapters, in excess of 192 peer-reviewed journal articles, over 300 abstracts and proceedings, and presented numerous oral and poster presentations related to mastitis, milk quality, and food safety. Venues for oral and poster presentations have included the National Mastitis Council regional and annual meetings (attendees include researchers, veterinarians, dairy producers, and representatives from industry), Conference for Research Workers in Animal Diseases, American Association of Bovine Practitioners annual meetings, International Dairy Federation meetings, American Dairy Science Association meetings, World Buiatrics Congress meetings, American Society of Microbiology meetings, Conference on Production Diseases in Farm Animals, Plant and Animal Genome Conference, Agriculture and Agri-Food Canada - Food Safety meetings, American College of Veterinary Internal Medicine annual forum meetings, and several regional extension and veterinary continuing education meetings. The continuation of the NE-1028 multistate project is of utmost importance to foster research in mastitis leading to the provision of science based information to dairy producers and the dairy industry. The impact of the European Union's strict enforcement of import regulations on milk quality highlights the need to continue efforts to reduce the incidence of mastitis. These new regulations require milk export companies to certify that any farm contributing milk must show a bulk milk cell count below 400,000 cells/mL. This regulation has recently been supported by the National Mastitis Council as a goal for all US dairies. Mechanisms leading to improvement in milk quality, dairy animal welfare, and appropriate use of antimicrobial therapeutics form the basis of research conducted in the NE-1028 multistate project. Given that approximately 25% of US bulk milk shipments exceeded 400,000 cells/mL in 2010, it is clear that continued mastitis research and education are required to maintain the global competitiveness of the US dairy industry (USDA APHIS, 2011). Furthermore, the animal agriculture industry in general is under closer scrutiny than ever before by various interest groups. The work of the NE-1028 is clearly focused on reducing mastitis, the most significant animal health issue in the dairy industry. In summary, the NE-1028 project is a productive group of collaborators that has provided new and meaningful information to all levels of the dairy industry from the bench scientist to the dairy producer with regard to bovine mastitis control, treatment and prevention. In the next 5 years we will continue to pursue collaborative projects under our 3 stated objectives which will lead to new information of value to the management of dairy cattle mastitis. Mastitis is an evolving disease syndrome, as is the science that studies mastitis; therefore, continued research efforts are needed.

Related, Current and Previous Work

The Multi-State Mastitis Research Project (MMRP) has a strong history of productivity in applied mastitis research. The project was begun in 1977 as NE-112, then renewed in 1982, 1987, 1992, 1997, in 2002 as NE-1009, and in 2007 as NE-1028. A substantial percentage of international mastitis research is conducted by MMRP members and affiliates. Members of MMRP collaborate extensively within the project and with other national and international research groups that have interests in bovine mastitis. The 2002-2007 iteration of the MMRP had 3 main objectives pertaining to the host, the pathogen, and the environment. In the current proposal we intend to continue work and begin new studies using these 3 objectives. The following are brief reviews, listed by objective, of current and previous work conducted during the last 5 years by the MMRP. In this summary, we focus on the most recent accomplishments. Multiple stations have contributed to the various objectives and are listed following each sub-objective. Objective 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance. (i) Environment, Nutrition, and Management Related Host Factors Associated with intramammary infections (IMI) (Alberta, CT, ID, IL, MI, OH, Ontario, PEI, VA, WA) The risk of mastitis increases during the transition period from late-pregnancy to early lactation. During this period, cows are under the hormonal influence of pregnancy, and are most likely in negative energy balance during the early part of lactation. Research has, therefore, focused on developing dietary strategies aimed at improving the immune response during this time, as well as better understanding the relationship between negative energy balance, other nutritional factors, or pregnancy related hormones versus immunity (Rezamand et al., 2007; Weiss et al., 2009; Piantoni et al., 2010). More recent work is also examining these factors at a more global level by evaluating gene transcription profiles of the liver and its function versus varying levels of nutrition (Moyes et al., 2010) as well as associations among behavioral and acute physiological response to lipoprotein lipase (LPL)-induced mastitis (Zimov et al., 2011). Members of MMRP have conducted experiments to determine the effects of antimicrobial treatments on heifer mastitis (Andrew et al., 2009); the effects of fatty acids on gene expression of metabolic factors and immune response during pro-inflammatory responses (Bionaz et al., 2011); adherence and efficacy of external teat sealants (Lim et al., 2007a and 2007b); effects of elevated concentrations of non-esterified fatty acids (NEFA) on immune/inflammatory functions in periparturient dairy cows (Contreras et al., 2010; Contreras & Sordillo, 2011); understanding standing/lying behavior patterns versus incidence of IMI in dairy cows, as well as the effect of udder health management practices on herd somatic cell count (Devries et al., 2011; Devries et al., 2010: Dufour et al., 2011; Olde Riekerink et al., 2011). Research efforts have focused on understanding the survival ability of common mastitis-causing pathogens during the ensiling process (Petersson-Wolfe et al., 2011) as well as determination of factors associated with cytokine and acute phase proteins in dairy cows with naturally occurring clinical mastitis (Wenz et al., 2010). (ii) Host-Pathogen Interactions at the Cellular Level. (CA, TN, IL, NY, VT) Members of the MMRP have investigated the molecular epidemiology of, and interactions between, mastitis-causing organisms and host response at gene and protein levels (Kerro Dego et al., 2011; Almeida et al., 2011; Loor et al., 2011; Zadoks et al., 2011; Richards et al., 2011; Fisher et al., 2011; Schukken et al., 2011; Bruno et al, 2010). Research efforts have included understanding the association between the single nucleotide polymorphism at in the interleukin-8 receptor (CXCR1) gene versus disease incidence, milk production, reproductive performance, and survival in Holstein cows (Pighetti and Elliott, 2011; Pighetti et al., 2012). Production of enterotoxin, its gene distribution, and genetic diversity of Staphylococcus aureus in milk obtained from cows with subclinical mastitis has also been studied (Oliveira et al., 2011). Vermont has studied the innate immune response, as measured by the activity of isolated dermal fibroblasts, as well as mammary epithelial cell cultures, after in vitro challenge with mastitis-causing pathogens (E. coli or S. aureus), their toxins, or inflammatory cytokines (Green et al., 2011; Kandasamy et al. 2011). California studied host/pathogen interactions during Streptococcus spp. mastitis, including transcription of immune mediators (Bruno et al, 2010). (iii) Candidate Genes of Mastitis Susceptibility (TN, VT) A number of projects within the MMRP have focused on studying genes related to mastitis susceptibility (Green et al., 2011; Kandasamy et al., 2011; Pighetti et al., 2012). Their work may allow consideration of selective breeding for mastitis resistance, which may prove valuable to the dairy industry as a whole. Objective 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses. (i) Characterization of Pathogen Virulence Factors (TN, WA, WI) Characterization of virulence factors is an essential step to developing strategies to prevent or treat mastitis. Considerable progress has been made regarding understanding virulence factors of S. uberis (Almeida et al., 2011). For example, deletion of the sua gene that encodes the S. uberis adhesion molecule (SUAM) markedly reduced the ability of the bacteria to adhere to and internalize into bovine mammary epithelial cell (Chen et al., 2011). The development of a vaccine based on the SUAM molecule is now in progress (Prado et al., 2011). Members of the MMRP are further characterizing virulence factors of other pathogens including coagulase negative staphylococci (Park et al. 2010), S. epidermidis (Madhusoodanan et al., 2011), and S. aureus (Oliveria at el., 2010). (ii) Antimicrobial Resistance (LA, TN, VT) The impact of antibiotic use for mastitis control on the development of antimicrobial resistance has recently been reviewed (Oliver et al. 2011). These authors concluded that although some degree of resistance is present in mastitis-causing pathogens, this has been observed for decades, and no evidence has been published to indicate that this is an emerging or progressing phenomenon. Another recent review evaluated whether testing mastitis pathogens for antimicrobial susceptibility was of use in predicting outcomes of antibiotic treatment (Barlow, 2011). The authors conclusion, based on data from 17 peer-reviewed publications, was that no clear evidence exists to indicate that such testing had predictive value for cure or non-cure outcomes in the treatment of mastitis. However, the importance of antibiotic treatment in dairy animal welfare and production, and its potential relation to the development of antibiotic resistance in bovine and human strains, indicates that further monitoring and research are warranted. (iii) Use of Molecular Epidemiology & Diagnostic Tools (NY, TN, ME, MI, UT) Members of the MMRP have had a great impact on advancing the field of DNA-based characterization of mastitis pathogens, also known as molecular epidemiology. A recent review by Zadoks et al. (2011) highlights a number of key advances that have been made through the use of molecular techniques in understanding of sources, transmission routes, and prognosis for many bovine mastitis pathogens. For example, it is now quite clear that a number of recurrent cases of E. coli mastitis are caused by the same bacterial strain rather than infection by two different environmental strains. This suggests that recurrent infection may actually be a case of a persistent or chronic infection more typical of Gram-positive organisms such as S. aureus. This may lead to a change in management practices toward culling persistently infected cows. Substantiating the concept of a chronic E. coli infection is the finding that strains of E. coli recovered from chronic cases of mastitis were better able to survive within mammary epithelial cells than were strains recovered from acute cases of mastitis (Almeida et al., 2011). Molecular techniques have also been used to further understand S. uberis-induced mastitis. The entire genome sequence of this organism was recently published (Ward at al., 2009), which has enabled members of the MMRP to conduct genome-wide, micro-array based comparison of an additional 21 isolates (Lang et al., 2009). This generated data to indicate that approximately 82.5% of the genome can be considered the core genome of this species, and that substantial strain variability exists outside of this core region. This pathogen appears to be primarily an opportunistic environmental pathogen, whereas the major contagious mastitis-causing pathogen is S. aureus. Molecular strategies have increased understanding of this pathogen including its mode of transmission, source, and adaptations to prevent host detection. For example, molecular typing techniques have revealed that in most herds with S. aureus mastitis many cows are infected by a single strain (Middleton, 2002). The ability to accurately identify the causative strain has led to studies showing the existence of relatively distinct bovine-associated and non-bovine-associated strain types (van den Borne et al., 2010). A new PCR-based technique to detect Prototheca strains isolated from milk will greatly speed research into the epidemiology of this pathogen (Ricchi et al., 2010). Likewise, the epidemiology of Mycoplasma mastitis is now being examined with a PCR-based detection strategy that has been validated by comparison with much more time-consuming conventional culture techniques (Justice-Allen et al., 2011). Objective 3. Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. Technology applications within the last 5 years have included extensive evaluation of relatively simple tools, such as teat treatments (dips, sealants, dry cow treatments); application of strategies such as on-farm culture systems; evaluation of bedding systems, technologies for training producers and professionals, and diagnostic/milk quality test development and implementation strategies. As well, as animal welfare becomes more important, behavioral monitors have been developed that can have diagnostic utility as well as enhancing marketability of dairy products due to welfare certification needs for selected markets. Research in this objective has proven useful in evaluation of multiple teat treatments, including innovative ones (Baskaran et al, 2012). Teat sealant use has been evaluated by several centers, including Missouri, where intramammary location of the material was identified using imaging. Misapplication of teat sealants may be an important limitation to their effectiveness. The application of established antibiotic residue tests for detection of alternative antimicrobial compounds, including some marketed products, is an important tool for organic producers; work conducted by the Connecticut station focused on this objective. Use of technology either developed during this project, or applied to mastitis research, has contributed to our understanding of the epidemiology of major mastitis pathogens. For instance, Vermont used pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) to identify strains of Staphylococcus aureus and to follow the transmission dynamics of this pathogen in 2 commercial dairy herds. Improved detection and speciation of mycoplasma, an important mastitis pathogen for which detection and culling may be a control strategy, was accomplished during this 5-year period at several stations (Justice-Allen et al: 2011; Wilson et al, 2011; Boonyayatra et al, 2012; Punyaportwithaya et al, 2010 and 2011). Using real-time PCR for detection of mastitis-causing organisms for which few treatments exist (mycoplasma, prototheca; Barker 2010; Ricchi et al, 2010) may allow more efficient control of these pathogen species. Culture-based diagnosis of these relatively slow-growing pathogens requires special media and longer times (up to 14 days), whereas real-time PCR techniques may require as little as four hours. Application of these techniques to either bulk tank filters or to milk samples directly may be highly sensitive and specific methods for detecting mastitis-causing pathogens. California worked with Sandia National Laboratory on a novel diagnostic platform to detect chemical, nuclear and biological agents of interest in raw milk (Derzon et al, 2008; Moeller et al, 2009). This station also investigated testing waste milk fed to calves to detect mastitis pathogens on farms, uses a risk assessment model to emphasize detecting S. aureus in herds, and works with the LA station on mycoplasma diagnostics (Heidinger et al, 2009; Ruzante et al, 2008). In summary, the current and past five years work conducted within the framework of this MMRP has resulted in over 192 refereed publications and over 300 presentations at various scientific and stake-holder forums. We are continuing to build on our past findings to reduce the incidence of mastitis through additional research and extension activities. Mastitis is clearly a multi-faceted disease that will require continued efforts to not only ensure the production of safe, high quality food, but to do so in a sustainable fashion and with continued improvements in dairy animal welfare and reductions the use of antimicrobial drugs.

Objectives

1. Characterization of host mechanisms associated with mastitis susceptibility and resistance
   
2. Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses
   
3. Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety
   

Methods

Objective 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance. One major goal is to develop nutritional strategies to enhance innate resistance to mastitis. This involves evaluating the effect of potential nutraceutical compounds on the functional capacity of white blood cells in-vitro as well as evaluating the in-vivo immune response of cattle fed diets or supplements designed to enhance disease resistance. The ultimate goal is to develop nutritional and therapeutic strategies to reduce systemic inflammatory responses and increase mastitis resistance (DE). Research on nutritional factors affecting immune function such as bovine retinoid metabolites and retinol binding protein (RBP) are limited for dairy cows during the periparturient period and in response to intra-mammary infection. There is a need to better understand how alterations in retinoid metabolism and retinol transport (RBP) affect immune function relevant to incidence and severity of new intramammary infections (ID). An evaluation of the role of oral immunostimulants on the bovine immune system will be completed. A yeast extract containing B vitamins and proprietary ingredients is being tested in heifer calves, and continuing through their first lactation. The goal is to demonstrate that daily feeding of this supplement will boost the animal's immune system and prevent the occurrence of diseases such as mastitis in developing heifers (GA). One group will study the relationships between the metabolic dynamics of fat mobilization in dairy cattle, especially during the periparturient period, and immune responses. In particular, the impact of fat mobilization on oxidative stress and the ensuing impact on mononuclear cell membrane lipids and inflammatory responses will be studied (MI). Ohio will characterize the effects of metabolic diseases on host defenses against bovine intramammary infections. In particular, they will determine effects of subacute acidosis on endotoxin tolerance in cattle, effects of acute acidosis on fecal shedding of potential mastitis causing bacteria in the cow's environment. They will also test products and procedures to mitigate the negative effects of metabolic disease on host defenses and to reduce pathogen exposure in bedding, will characterize pathogen populations in the cow's environment, and will determine the ecological impact of recycling manure and sand on pathogen exposure and distribution of antimicrobial resistance genes (OH). Vermont will use in vitro fibroblast and mammary epithelial cell cultures to study host immune responses to Gram-positive as compared to Gram-negative mastitis pathogens and will conduct in vivo intramammary challenge experiments to assess association of fibroblast and whole animal responses to infection (VT). Virginia will identify mechanisms by which a bovine feed additive enhances immune cell function using a mouse model, and will study proteomic analysis of commensal and pathogenic bovine S. aureus strains to identify immune suppressing/enhancing antigens. Further, they will identify early indicators of mastitis and metabolic diseases using novel activity monitors and daily milk component monitoring (VA). Other studies include determination of the effect of stress on the innate immune response to experimental challenge by M. bovis. As well, this group will study how this affects critical immune response factors. This group will test the differential effects of various disinfectants, used as a pre- and/or post-dips, have on colonization of the teal canal, teat skin, and on intramammary infections by coagulase negative staphylococci (WA). Objective 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses. (i) Characterization of Pathogen Virulence Factors Continued studies on pathogen virulence factors will be carried out at a number of stations (NY, TN, VA, MN, UT). The TN station will undertake a promising approach to determine virulence factors associated with S. uberis. In this approach, a mutant library will be screened against bovine mammary epithelial cells to locate mutants with altered adhesion. The library will also be screened for ability to evade killing by neutrophils. Such an approach has already uncovered the importance of the SUA gene that encodes for S. uberis adhesion molecule (SUAM). Future studies will evaluate the efficacy of a SUAM-based vaccine in preventing or ameliorating severity of S. uberis infection. A joint project between NY and VA will evaluate the contribution of capsule expression by host-adapted Klebsiella strains to its ability to evade neutrophil killing. Mastitis due to coagulase negative staphylococcal species cause variable levels of elevation in milk SCC, and the WA station will investigate if strain differences in virulence factors such as fibronectin binding protein and enterotoxins are associated with the differential SCC response. A new proteomic technique, stable isotope labeling with amino acids in cell culture (SILAC), will be employed (VA) to compare the expression profiles of commensal and pathogenic strains of S. aureus with the goal of identifying differentially expressed virulence factors. Once identified, selected virulence factors will be knocked-out through genetic modification to evaluate their role in pathogenesis. The iron acquisition system is critical to S. aureus pathogenesis, and detailed studies of this system will be carried out at NJ. In particular, this station will examine a novel mechanism by which S. aureus builds, protects, and maintains iron-sulfur clusters, as well as how it senses and responds to changes in intracellular iron pools. (ii) Antimicrobial Resistance Continued monitoring of antimicrobial resistance of mastitis-causing pathogens is of utmost importance to determine if such resistance is emerging or progressing. Work in this area will be carried out at several stations (KY, LA, VT). LA and VT will continue to evaluate the impact of agricultural use of antibiotics on bacterial resistance to antibiotics. Comparison of resistance patterns between bovine and human associated isolates will be performed (LA). Additionally, VT will examine potential associations between carriage of antimicrobial resistance genes and response to antimicrobial therapy among different strain types of S. aureus and S. uberis. The effect of ensiling on antibiotic resistance of enterococcal isolates will be examined by VA. The minimum inhibitory concentrations of various cephalosporin compounds for several mastitis pathogens will be evaluated (WI) with the goal of improving treatment options. In addition, WI will characterize genotypic and phenotypic patterns of resistance for a large number of mastitis pathogens collected from a cross section of large Wisconsin dairy farms. (iii) Use of Molecular Epidemiology & Diagnostic Tools Considerable progress has been made on the development of molecular tools to investigate Klebsiella species. Genomic sequencing of Klebsiella species will be undertaken by NY with subsequent comparison of genomic data and pathogenicity determined. Genomic sequencing of selected strains of coagulase negative staphylococci will be undertaken at MO to evaluate potential between-strain differences in virulence. The use of molecular tools to identify pathogens in mastitic milk samples that are culture negative will continue to be carried out by IA. This new metagenomic approach could reveal a range of pathogens that are difficult to culture using standard microbiological techniques. Efforts will be made by GA to develop enhanced diagnostic tools to differentiate non-hemolytic S. aureus isolates from coagulase-negative staphylococci. Monitoring of mycoplasma mastitis will be carried out at several stations (LA, MI, UT) with emphasis on development and validation of new PCR-based diagnostic tools. Objective 3: Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. Twenty-two stations have stated that they have been, or will be working on Objective 3-related projects, although many projects are relevant to more than one objective. Antimicrobials, including injectable and extra-label use antibiotics, will be evaluated at the Georgia station, along with a method to evaluate goat milk SCC. ID will evaluate teat dips, continue the collaboration with MN and CA studying dry cow treatments, and begin a mastitis vaccine evaluation trial. LA will assess the impact of dairy practices on antibiotic resistance in the human population. CT will collaborate with Yale University to study antibiotic resistance of bacteria in dairy farm manure, will identify farm-specific critical control points associated with risk of mastitis, and will study the use of alternative therapies, such as essential oils, against mastitis pathogens. KY will study new technologies for mastitis detection, including Precision Dairy Farming Technologies methods (MilkLine P4C individual quarter conductivity, IceRobotics IceQube lying behavior monitors, DVM Systems temperature boluses and SCR Rumination Tags). Use of multiple methods will help characterize physiological and behavioral changes associated with health events in dairy cattle. As well, KY is developing a simulation model to evaluate the economic impact of mastitis on the farmer. ME will validate and expand the use of it's prototheca PCR detection method, and work on development of on-farm prototheca detection methods. MI will evaluate a mycoplasma PCR detection method for herd surveillance, integrating evaluation of both PCR technology and sampling methods. MN will continue to prepare on-line teaching materials, and will continue a collaboration with ID and CA on dry cow treatments. MO will evaluate a novel treatment for chronic S. aureus mastitis, and will investigate dry period mastitis prevention methods, novel approaches to intramammary treatment, and using advanced imaging techniques in studying mastitis prevention and treatment. NY will continue a focus on reducing pathogens of public health significance in milk. OH will test products and procedures that will improve host immunity, and reduce pathogens in dairy bedding materials. TN will design inline milk sensors for addition to existing milking systems to allow improved quality monitoring in an affordable manner, as well as a lab-on-a-chip approach for on-farm pathogen detection. They will also work with producers to help characterize factors associated with adoption of successful mastitis-control strategies. UT will provide surveillance for dairy cow infectious disease, using bulk tank milk sampling, and will continue to study mycoplasma species in cows with mastitis. As well, UT will evaluate bulk milk testing for Bovine Viral Diarrhea (BVD), and will assess the Johne's Disease (JD) status of dairy herds to determine if these impact mastitis incidence. Vermont will study transmission dynamics of contagious mastitis, and methods for control in automated milking systems. Virginia will study the use of novel activity monitors and daily milk component monitoring for mastitis indicators. WA will evaluate disinfectant pre- and post-dip effects on teat colonization and CNS infection. WA also will implement an innovative outreach program to reduce mastitis in the western United States.

Measurement of Progress and Results

Outputs

• Peer reviewed publications
• Presentations at national and local meetings
• Non-peer reviewed publications
• Extension publications and meetings
• Bacterial isolate collections with epidemiological data
• Joint projects/collaborations

Outcomes or Projected Impacts

• Models developed for mastitis transmission on dairies.
• Risks and benefits of antibiotic use in first lactation heifers evaluated.
• Experimental intramammary treatments, both conventional and organic, studied.
• On-farm culture and other cow-side methods of reducing antibiotic use on dairies evaluated.
• Effectiveness of teat dips, teat sealants, bedding types and treatments and dry cow treatments in deterring mastitis evaluated.
• New diagnostic tests for select organisms, including high-risk human pathogens and non-bacterial causes of mastitis, developed.
• Nutritional effects, including silage inoculants, on mastitis evaluated.
• Strategies developed to improve immune responses during the dry and transition period, using molecular analyses of host responses.
• Use positional/behavioral patterns to predict IMI in dairy cattle.
• Describe the molecular epidemiology of mastitis pathogens.
• Describe host cytokine and other genetic predictors for mastitis susceptibility, milk production, reproductive performance, and survival.
• Describe selected mastitis pathogens toxin production, gene distribution, and genetic diversity in milk.
• Develop potential mastitis vaccine candidates.

Milestones

(2012): Submission for publication of findings in studies of metabolic dynamics and their relationship to dairy cattle immune responses.

(2013): Submission for publication of findings in studies of retinoid metabolism effects on mammary gland health during the periparturient period.

(2014): Submission for publication of findings in studies of vaccine candidates, such as the Strep. uberis sua gene. Submission for publication of findings in regards to the impact of nutritional factors, such as oral immunostimulants, on prevention of mastitis.

(2015): Submission for publication of findings in studies of metabolic disease, such as acidosis, impacts on mastitis.

(2016): Completion and submission for publication of the remaining studies on focused research objectives, as well as summaries of surveillance studies.

Projected Participation

View Appendix E: Participation

Outreach Plan

Multiple centers have described outreach projects in their plans for the upcoming period. As well, collaborative proposals are in preparation for current USDA-AFRI requests for proposals to address changes in the SCC requirement for the dairy industry. As an example, Tennessee will develop more effective extension-based strategies to aid producers in generating high quality milk. A number of both East and West coast stations are collaborating on Extension-based proposals to improve milk quality. For instance, the Experiment Stations of ID, WA, CA, and UT are working on the formation of a collaborative project that will assess and then address the weaknesses in mastitis control on western dairy farms considering communications, labor relations and process management. These projects, and thus the MMRP, will be characterized by their emphasis on producer communications, and on including experts in communication, sociology, economics, dairy management, mastitis, and milk quality programs. The MMRP has, and continues to, involve numerous Cooperative Extension members. This factor, and the willingness of the dairy industry to seek new tools for improvement, enhance the current and future effectiveness of the MMRP. In the words of William Owens of Louisiana, "A major impact of the multistate projects is the credence or impact that a prestigious organization gives to data it generates. The reputation and long history of this project and the many years of scientific expertise that it represents greatly increased the weight of its recommendations. Many of the scientists participating in this project have been continuously involved with this project since the 1980s. This long history allows a continuity of purpose that provides valuable leadership and helps maintain the focus of the group. This in turn makes the outputs of the projects more focused and more valuable."

Organization/Governance

Technical Committee: *Official representative coordinating the research: California: J.C. Cullor*; Connecticut: S. Andrew*; Delaware: T.F. Gressley*; Georgia: S. Nickerson*; Idaho: P. Rezamand*; Illinois: J.J. Loor*; Iowa: L. L. Timms*; Kansas: B. Schultz*, R. Gehring; Kentucky Cooperative Extension: J. Bewley*; Louisiana: W. E. Owens*; Maine: A.B. Lichtenwalner*; Michigan R. S. Erskine*, L. Sordillo; Minnesota: S. Godden*; Missouri: J.R. Middleton*; New Jersey: J.M. Boyd; New York: Y.H. Schukken*, P. Moroni, A.A. Gurjar; Ohio: J.S. Hogan*; Utah: D.J. Wilson*; Pennsylvania: B. Jayaroa*; South Dakota: D. Scholl*; Tennessee G. Pighetti*; S. Oliver, R.A. Almeida; Vermont: D.E. Kerr*; J. Barlow; Virginia: C.S. Petersson-Wolfe*; I.K. Mullarky; Washington: L. K. Fox*; Wisconsin: P. Ruegg*; International members (Canada): Ontario: K. E. Leslie*, D.F. Kelton, R. Dingwell; Alberta: H.W. Barkema; PEI: G Keefe; Moredun Research Institute: R.N. Zadoks. Mastitis is a problem throughout the United States, and indeed the World. Therefore, participants were selected from outside the region on the basis of common interest and special competence in the various research areas applicable to the project. The organization of this project will be in accordance with that set forth in the Manual for Cooperative Multistate Research. A technical committee that includes the project participants from each of the participating stations will administer the project. An executive committee will consist of the Chairman, Vice-Chairman, Secretary, and the Administrative Advisor. The officers will serve one year after which the Vice-Chairman automatically becomes Chairman and the Secretary becomes Vice-Chairman. This executive committee will conduct business between meetings. An annual meeting will be called by the Administrative Advisor and will be held in conjunction with the Mastitis Research Worker's Conference. At these meetings, research accomplishments will be reviewed, updates and summaries of joint projects will be presented, and new projects will be planned and a project coordinator selected. The Secretary will call for annual reports of research data from each station. These reports will be compiled and sent to each participant prior to the annual meeting. The responsibility for multi-State summaries and publications will be assigned at the meetings. If no annual report is submitted for two consecutive years from a participating station, or if no one from a station is present at the MMRP meeting for two years out of five, then this station will be eliminated from the project. The Administrative Advisor will contact the member station before the member is formally eliminated from the roster to ensure that extenuating circumstances do not exist. International members will be expected to submit an annual report and to have representation at annual meetings, as per stations in the United States. However, these participants will not be required to submit Appendix E forms detailing formal FTE commitments.

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Attachments

Land Grant Participating States/Institutions

CA, CT, DE, FL, GA, IA, ID, KS, KY, LA, MD, ME, MI, MN, MO, MS, NJ, NY, OH, OR, PA, TN, UT, VA, VT, WA, WI

Non Land Grant Participating States/Institutions

University of Calgary, University of Montreal, University of Prince Edward Island, University of Saskatchewan