Duration: 10/01/2007 to 09/30/2012

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Bovine mastitis is the most costly infectious disease currently affecting dairy cattle. 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.

In the United States, the dairy industry contributes in excess of 65 billion dollars per year to the national economy, and provides jobs for over 1 million Americans. The single most costly disease of dairy cattle and a major monetary drain on the dairy industry is bovine mastitis. Mastitis is defined as an inflammation of the mammary gland that is almost always associated with bacterial infection. Mastitis affects every dairy farm and approximately 38% of dairy cows in the United States. The National Mastitis Council estimates that this devastating disease complex costs the dairy industry more than 2 billion dollars per year or approximately $180.00 per cow. These losses are primarily due to lost milk production, increased veterinary costs, increased cow mortality, and discarded milk.

The purpose of the Multi-State Mastitis Research Project (MMRP; formerly designated NE-112 and most recently designated NE-1009) 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 the MMRP. The MMRP has provided a forum for new and established researchers to develop collaborative relationships, and to share resources and expertise.

In the past several years, we have initiated joint projects, which were conceived, developed, and run under the auspices of the MMRP. Examples of these projects and linkages between stations include completion of 2 multi-State projects evaluating prepartum intramammary antibiotic treatment of heifers on the prevalence of early lactation mastitis and first lactation performance parameters (Summarized below). In addition, the group has completed portions and has work in progress on several other collaborative studies which include evaluation of duplicate versus single samples for the diagnosis of intramammary infection (coordinated by ONT), determination of antibiotic residues in heifers treated with intramammary antibiotics before parturition (coordinated by CT), evaluation of cow and mammary quarter level risk factors at dry-off for the development of mastitis during the dry period (IA, NY, ONT, PEI), evaluation of external teat sealants during the dry period (IA, ONT, PEI), evaluation of environmental streptococcal mastitis (IA, IL, WA, VA), and evaluation of on-farm culture technologies and their use in making treatment decisions for clinical and subclinical mastitis (IA, MN, ONT, PEI, WI). Through the concerted and collaborative efforts of its members, the MMRP has been highly productive and will continue to work on similar collaborative projects in the next 5-years.

Examples of Completed Multi-State Projects: Heifer Prepartum Treatment Trials

Research data accumulated over the past 20 years suggest that a high proportion of dairy heifers develop intramammary infections (IMIs) prior to calving. Because of the high prevalence of prepartum IMIs in heifers, several single institution studies have evaluated prepartum treatment of heifers to decrease the prevalence of IMI and one such study has evaluated the economic viability of such treatment by measuring first lactation performance parameters. However, large multi-site studies were lacking. Hence, during the last 5 years members of the MMRP have undertaken two collaborative multi-State studies to evaluate the effect of prepartum treatment of dairy heifers on first lactation udder health and performance. Study 1 (Middleton et al., 2005) involved two centers in the MMRP (IA & MO) and Study 2 (Borm AA et al., 2006) involved 7 centers (WA, ONT, OH, CT, TN, NY, & LA). Study 1 included 183 dairy heifers on 2 farms and Study 2 included 561 heifers on 9 farms. Heifers were divided into treated and untreated control groups with treated heifers receiving a single intramammary dose of either pirlimycin (Study 1) or cephapirin (Study 2) in each mammary quarter approximately 10-21 days prior to parturition. Samples were collected before treatment and after calving for microbiologic assessment and post-calving production parameters were recorded and evaluated. Both studies found that the majority of prepartum IMIs were caused by coagulase negative staphylococci (CNS) and treated quarters had a significantly higher cure rate than untreated quarters. Treatment did not significantly affect milk production in either study; however, there was a significant treatment by herd interaction for milk production in Study 2. In Study 1, one herd realized a significant reduction in milk somatic cell count in treated quarters while no difference was detected between groups in the other herd. In Study 2, quarters cured of either a CNS or major mastitis pathogen IMI had a lower milk somatic cell count in the first 200 days of lactation. In addition, Study 2 found no significant effect of treatment on services per conception or days open. Given these results, use of prepartum intramammary antibiotic therapy in heifers as a universal strategy to increase first lactation performance may not be warranted. Results from these studies have led to new collaborative studies evaluating milk residue detection and test kit validation in treated heifers and studies to examine methods of speciating CNS and characterizing differences in pathogenicity between CNS species and genotypes.

Data generated under the auspices of the MMRP project has been used by member stations to gain intramural and extramural funding for mastitis research. While the majority of research conducted by the group is funded by intramural and extramural grants and corporate contracts awarded to individual participating stations, our Canadian colleagues and collaborators/participants in the MMRP were recently awarded a multi-year, multi-million dollar grant to fund the Canadian Bovine Mastitis Research Network. In addition, a subcommittee of MMRP members met in December 2003 and February 2004 to outline and discuss a proposal for the USDA Coordinated Agricultural Project (CAP) program. However, the 2004 and 2005 requests for proposals were in areas other than milk quality and food safety. As a multi-State, multi-institution project the MMRP is uniquely situated to apply for CAP funding should mastitis and milk quality become a targeted area of research. Examples of current jointly funded research are listed below.

1) Pathogenesis of chronic E. coli mastitis. Y.H. Schukken (NY) & S.P. Oliver (TN). New York Vitamin Settlement Grant

2) Diagnostics for coagulase negative staphylococci. P. Ruegg (WI) & R.N. Zadoks (NY). Formula Funds

3) On-farm culture and treatment protocols. R. Erskine (MI) & S. Wagner (ND). Mastitis Research Foundation

4) Mathematical and molecular epidemiology of mastitis. J. Bramley, J. Barlow (VT) & Y.H. Schukken, R.N. Zadoks (NY). USDA-NRI

5) Presence of mastitis pathogens in culture-negative milk samples from cows with clinical mastitis. H.W. Barkema, R. Olde Riekerink (CAN) & R. N. Zadoks (NY). Mastitis Research Foundation

6) On-farm mastitis diagnostics. S. Godden (MN) & K.E. Leslie (CAN) & P. Ruegg (WI). MN Ag Experiment Station

The mastitis research workers group has met in conjunction with the MMRP annual meeting for many years, and in recent years, the mastitis research workers topics have been included in MMRP minutes, showing current active areas of research by MMRP members. International visitors and collaborators are often included in these presentations. In addition to the mastitis research workers conference, the MMRP members provide technology transfer to the scientific and lay communities. In the last 5 years, members of the project have collectively published several book chapters, approximately 290 peer-reviewed journal articles, approximately 270 abstracts and proceedings, and presented numerous oral and poster presentations related to mastitis, milk quality, and food safety (Appendix 1: Publications 2002-2007). 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.

In summary, the MMRP 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 on 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 which studies mastitis and therefore continued research efforts are needed.

Related, Current and Previous Work

A MEDLINE search with the keywords mastitis and bovine revealed that the majority of the research being conducted in the United States on bovine mastitis continues to be done by members of the MMRP. The Multi-State Mastitis Research Project (MMRP) originated as the NE-112 Project in 1977 and was revised in 1982, 1987, 1992, and 1997. In 2002, the project was designated the NE-1009 Project. Research conducted under the project has contributed substantially to enhancing our understanding of bovine mastitis and has significantly impacted development of control and therapy measures for bovine mastitis that are now in practice throughout the United States and the World.

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 under these 3 objectives. The following are brief reviews by objective of current and previous work conducted within the MMRP.

Objective 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance.

(i) Environment, Nutrition, and Management Related Host Factors Associated with IMI

The ability of a cow to defend against bacterial invasion of the mammary gland is affected by a multitude of factors such as the environment, nutrition, and management decisions. Research by members of the MMRP has contributed significantly to our knowledge regarding these factors. For example, the ability of bacteria to gain entry into the mammary gland requires the organism to penetrate the teat end. Recent studies have taken a more in depth look at cow-related factors that contribute to either a weakened teat end through chapping and cracking such as occurs with extreme temperature variation or an open teat end that does not close efficiently upon cessation of milking prior to the dry period (Dingwell et al., 2004; Rajala-Schultz et al., 2005). A 5 kg increase in milk yield at dry off above 12.5 kg, increased the odds of a cow having an environmental IMI at calving by 77% (Rajala-Schultz et al., 2005). The risk of mastitis also increases during the transition period near calving when cows are under the hormonal influence of pregnancy and 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 and pregnancy related hormones with immunity. Dietary supplementation with various antioxidant supplements (vitamin E, selenium, vitamin C) during the transition period have been examined for their ability to improve neutrophil functional activity during this transition period (Weiss and Hogan, 2007, Weiss and Hogan 2005). Following calving, cows often enter into negative energy balance which can lead to ketosis. Cows that develop ketosis are 4.6 times more likely to develop a new IMI than cows that did not develop ketosis (CT). More recent work is also examining these factors at a more global level by evaluating transcription profiles of the liver and its function with varying levels of nutrition prepartum (Loor et al., 2006). This research will form the basis of future studies which seek to address the role of these changes in relation to disease resistance.

(ii) Host-Pathogen Interactions at the Cellular Level

The ability of organisms to cause infection does not occur in a vacuum, but is a complex interplay of the organism with host immune cells. Whether or not an infection occurs depends upon many factors. Thus greater understanding of how the organism and the host interact: how genes and proteins are turned off and on in response to cues from the other is critical to developing new strategies aimed at reducing the incidence and severity of mastitis. Research conducted by members of the MMRP has involved both in vitro and in vivo experiments to elucidate these interactions. This research includes the role of mammary epithelial cells which are capable of responding directly to invading pathogens or toxins to elicit an inflammatory response (Pareek et al., 2005, Wellnitz and Kerr, 2004; Zheng et al., 2006), pathogens such as S. uberis and E. coli which can invade and survive within mammary epithelial cells (Almeida and Oliver, 2006; Dogan et al, 2006), and the interaction of specific antibodies against the fecA (ferric citrate receptor) of E. coli with bacterial growth, neutrophil function, and pathogenesis (Takemura et al., 2002; Takemura et al., 2004; Wise et al., 2003). Other work has shown that experimental infection with S. aureus leads to a reduction in minerals zinc, copper, and iron (Middleton, et al., 2004). Previous studies also indicate that S. aureus can induce the production of host coagulation proteins (Zavizion, White et al. 1997; Veltrop, Beekhuizen et al. 1999) by the production of coagulase, fibrinogen-binding protein, and clumping factors. However, there is still controversy as to the role of fibrin, be it protective or detrimental, during this infection. Utilizing fibrin(ogen)-deficient mice, we have shown a protective role for fibrin formation during both toxoplasmosis (Johnson, Berggren et al. 2003) and listeriosis (Mullarky, Szaba et al. 2005).

(iii) Candidate Genes of Mastitis Susceptibility

The search for genes and gene markers associated with mastitis susceptibility or resistance has been a long standing goal in the dairy industry. By understanding the interaction of the immune response with invading pathogens, as well as the influence of environmental, nutrition, and management factors, the identification of candidate genes associated with mastitis susceptibility has been possible. One potential candidate gene is the uterocalin (Lcn2) gene, a potential protective factor against inflammation (IA; Nilsen-Hamilton, 2003). Using the mRNA sequence from a cDNA library (MI), the protein has been expressed using a bacterial expression vector and was observed to exist as both a monomer and dimer. This protein has been used to develop bovine specific antibodies which will be used in future studies. In addition, studies in knockout mice indicated a greater cytokine response to LPS in the lung and liver when compared to wild type mice - supporting an anti-inflammatory role for this protein.

Separate studies have indicated the existence of single nucleotide polymorphisms in the bovine CXCR1 gene associated with mastitis susceptibility and altered neutrophil function in dairy cattle (Youngerman, 2004; Rambeaud 2005, Rambeaud 2006, Rambeaud 2007). This polymorphism is currently being evaluated for its potential as a genetic marker allowing for the selection of sires and cows more resistant to mastitis. In addition, this marker is serving as a marker for in vitro and in vivo studies aimed at identifying the cause for greater mastitis susceptibility. This may be, in part, due to impaired affinity of the CXCR1 receptor for interleukin-8, a prominent cytokine produced during mastitis that induces neutrophil migration and activation (Rambeaud 2007). Additional studies have indicated that other mechanisms independent of the CXCR1 receptor are related to impaired neutrophil function and most likely tied to an intracellular signaling event.

Objective 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses.

(i) Characterization of Pathogen Virulence Factors

A variety of virulence mechanisms have been identified in recent years that allow bacteria to enter and persist in the mammary gland and cause mastitis. These include adherence to bovine skin and mammary epithelial cells, elaboration of various toxins that destroy host tissues and alter immune function, production of capsule and slime layers that protect bacteria from host defenses, and antibiotic resistance (Erskine et al. 2002; Almeida et al. 2003; Fox et al. 2005; Zadoks et al. 2005a; Almeida et al. 2006; Dogan et al. 2006; Srinivasan et al. 2006; Pol & Ruegg 2007). In vitro models demonstrate that mastitis pathogens such as Streptococcus uberis and Staphylococcus aureus can penetrate and persist within host cells (Tamilselvam et al. 2006), in part, explaining their persistence in the mammary gland. Hence, certain unique pathogen virulence factors such as S. aureus enterotoxin C (WA) and the Strep. uberis adhesion molecule (TN) are being investigated as potential pluripotent vaccine antigens to protect cattle against mastitis with these pathogens. Based on preliminary work at Ohio State on the epidemiology of enterococcal mastitis, the group at Virginia Tech will be evaluating differences in pathogenicity of enterococci that cause bovine mastitis in the next 5 years. In addition, research collaborations between New York and Tennessee have led to identification of virulence factors associated with E. coli (Dogan et al. 2006) and this work will be continued over the next 5 years.

(ii) Antimicrobial Resistance

Antibiotic resistance in mammary pathogens is becoming an important topic for the dairy industry. Recent studies indicate that antibiotic resistance is an important risk factor for a failure to cure after therapy (Osteras et al. 1999). Antimicrobial resistance is certainly present among intramammary pathogens in US dairy herds. Initial historical analysis of antibiotic resistance data showed an increase in antibiotic resistance in mastitis pathogens in the last 15 years (Garrison et al. 2000). In contrast, more recent work by the group at Michigan (Erskine et al. 2002) showed that among 2778 bacterial isolates collected from cases of mastitis between 1995 and 2000 there was not an overall trend towards resistance against antibiotics commonly used to treat dairy cattle. Similarly, Pol & Ruegg (2007) reported a dose-response effect between antimicrobial exposure and in vitro susceptibility for some pathogen-antimicrobial combinations, but exposure to antimicrobial drugs commonly used for prevention and treatment of mastitis was not associated with antimicrobial resistance. More studies evaluating the relationship between drug exposure and antimicrobial resistance are planned for the coming years.

(iii) Use of Molecular Epidemiology & Diagnostic Tools

Over the last 5 years members of the MMRP have used modern molecular and epidemiologic tools to improve our understanding of several important mastitis pathogens including Strep. uberis (Zadoks et al. 2003; Zadoks et al. 2005 a, b), Klebsiella pneumoniae (Munoz et al. 2006), S. aureus (Middleton et al. 2002 a, b, c, 2005, 2007), Mycoplasma spp. (Fox et al. 2003; Biddle et al. 2005), and Enterococcus spp. These studies have led to a better understanding of the ecology of mastitis pathogens on dairy farms and ultimately have led or will lead to improved mastitis control procedures.

Staphylococcus aureus has been a prevalent cause of contagious mastitis in dairy cattle for many years. However, recent research using molecular fingerprinting techniques has demonstrated that most herds have a predominant and sometimes highly contagious strain of S. aureus which cause mastitis (Middleton et al. 2002 a, b, c). In addition, studies have demonstrated that herds which purchase replacement heifers are more likely to introduce new strains of S. aureus and have more strains of S. aureus isolated from milk than closed herds which rear their own replacement heifers (Middleton et al. 2002c). Furthermore, some strains of S. aureus appear to be more pathogenic than others. In one study, a predominant, highly contagious, strain of S. aureus caused a significant reduction in milk production compared with other strains of S. aureus isolated from milk of cattle in the same herd. More recent studies have shown that some strains of S. aureus appear to respond better to intramammary antibiotic therapy than others (Luby & Middleton 2005; Middleton et al. 2007), and in some herds intramammary antibiotic therapy is not very useful in reducing the prevalence of S. aureus mastitis.

Historically, mastitis pathogens have been grouped based on their reservoir of infection as contagious or environmental. However, recent data using molecular fingerprinting techniques suggest that some environmental pathogens, particularly Strep. uberis (Zadoks et al. 2003) and Klebsiella pneumoniae (Munoz et al. 2007), may also be harbored in the mammary gland and transmitted from cow-to-cow during milking. Additional work using molecular fingerprinting has demonstrated that fecal shedding of both Strep. uberis and Klebsiella pneumoniae likely contribute to cases of mastitis, particularly in herds where bedding materials are culture negative for these organisms (Zadoks et al. 2005b; Munoz et al. 2006).

Mycoplasmas are emerging as a major cause of mastitis in some herds. A recent study showed that herds which ship more milk, i.e., larger herds are more likely to have Mycoplasma in bulk tank milk samples, and 60% herds which detect Mycoplasma in bulk tank milk will be Mycoplasma negative on a subsequent sample (Fox et al. 2003). Similarly, all of the bulk tank positive herds in the study were Mycoplasma bulk milk culture negative within one year of the initial positive culture. The presence of other contagious mastitis pathogens in bulk milk was not associated with the presence of Mycoplasma suggesting that Mycoplasma transmission may differ from other contagious mastitis pathogens. Using molecular fingerprinting techniques it has been shown that cows colonized with Mycoplasma in the mammary gland tend to have a body site colonized with the same strain of Mycoplasma, and over 60% of Mycoplasmas isolated from body sites have the same strain-type as those found in the mammary gland suggesting that the mammary gland may become infected from an internal site (Biddle et al. 2005). Further work in a herd with a Mycocplasma IMI prevalence of less than 1% has shown that the same strain of Mycoplasma which causes a single IMI can be frequently isolated from body sites on animals in the same herd at the time Mycoplasma is isolated from the mastitis case. However, frequency of isolation from body sites of herd members is low when no cases of Mycoplasma IMI are detected (Punyapornwithaya & Fox 2006).

Enterococcus spp. are another emergent mastitis pathogen. Research at Ohio State University has shown that the source and species of enterococcal isolates can make a significant contribution to how the organism adapts to mammary secretions harvested from various stages of lactation. Further research at Ohio State evaluated the physiologic effects of 4 Enterococcus faecium isolates on the bovine mammary gland in early and late lactation. All 4 tested E. faecium isolates were able to establish intramammary infection. However, poorly adaptive isolates were more likely to cause infection and cause clinical signs in vivo than the isolates that were highly adaptive to in vitro growth in mammary secretions

While members of the MMRP will continue to characterize the pathogens listed above, our collaborative studies on prepartum treatment of heifer mastitis led to the need for a better understanding of coagulase negative staphylococcal mastitis. Historically, CNS have been grouped together as minor mastitis pathogens. However, they are the most prevalent organism isolated from dairy cattle milk and in some herds appear to be the only cause of high SCC milk. Using some of the molecular tools used to characterize the aforementioned pathogens, members of the MMRP are currently establishing techniques for accurate speciation of CNS, evaluating virulence factors associated with each species of CNS, evaluating longevity of infection, and using udder health parameters to evaluate the pathogenicity of different species and strains within species of CNS. These studies will be a focus area for the next 5 years of the MMRP.

Molecular tools have not only been useful in better describing mastitis pathogen epidemiology, but are being used to more efficiently diagnose mastitis. Recently Gillespie and Oliver (2005) designed a multiplex polymerase chain reaction (PCR) test that can simultaneously detect the presence of S. aureus, Strep. uberis and Strep. agalactiae DNA in milk samples with the intent of more rapidly diagnosing IMIs caused by these pathogens. Research at Washington State University is exploring a multiplex PCR for the rapid diagnosis of Mycoplasma mastitis. Current Mycoplasma culture techniques can take 7-10 days before a diagnosis can be made.

Objective 3. Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety.

(i) Reduction of Pathogenicity of Mastitis Organisms

Heifer mastitis - MMRP joint project. We conducted two multi-site studies to determine the effect of intramammary antibiotic therapy on incidence of mastitis, milk production and composition, and reproductive status of primiparous heifers. This collaborative approach provided the necessary sample size for meaningful statistical comparisons. Participants in the first study included Michigan, Minnesota, Ohio, Wisconsin, Kansas, Indiana, Pennsylvania, New York and Iowa. Heifers were enrolled 14-21 days prior to expected calving over a 1-year period. Each heifer was randomly assigned to a treatment (intramammary infusion of an antibiotic in all quarters) or no-treatment control group. Quarter milk samples were collected at calving and at 1, 2, and 3 weeks post-calving and cultured to determine the presence of IMI. Data on milk production, SCC, and reproductive performance were collected throughout the first lactation of each heifer. The results of the trial were published in both peer-reviewed journals and in scientific meeting proceedings and popular press (Borm et al. 2006, Fox et al. 2005 a, b). The results indicate that blanket pre-partum treatment of heifer is not warranted, however in herds with a high prevalence of major mastitis pathogens, pre-partum treatment of heifers was efficacious. A second study with partners from Missouri and Iowa came to a very similar conclusion (Middleton et al. 2005).

Environmental streptococci (Hillerton and Berry 2003) were studied in another collaborative multi-State project. Project participants included Illinois, Iowa and Washington. Many of our assumptions about the impact of environmental Streptococcus IMI have been derived from results of clinical mastitis surveys, experimental inoculation trials, reports of investigations in problem herds, or studies focusing specifically on Strep. uberis (Zadoks et al. 2003, Dingwell et al. 2004). This multi-site MMRP research project investigated the impact of naturally occurring environmental streptococcal IMI on cow health, milk production, and milk quality. Approximately 14% of glands with environmental streptococcal IMIs at freshening had clinical mastitis, and 12 to 25% of clinical mastitis samples contained environmental Streptococcus spp. This project is currently being completed with the support from other MMRP stations (NY, WA).

(ii) Technologies to Promote Host Defense Systems

Under this objective, multiple stations performed research into the natural host defense of the cow against mastitis and on the biology and efficacy of vaccines against mastitis. Michigan continued to investigate the impact of immunization of lactating dairy cattle with J5 E. coli bacterins (Burton and Erskine, 2003, Chaiyotwittayakun et al. 2004). In a recently completed project involving over one-thousand lactations, it was determined that cows immunized with 6 doses of a J5 core-antigen bacterin had higher serum immunoglobulin G2 responses than did cows receiving only 3 doses. Additionally, hyperimmunized (6-dose) cows had a reduced incidence of severe mastitis from 40 to 120 days in milk. This field study was a continuation of preliminary studies in Holstein steers that determined 1) that hyperimmunization with J5 bacterin increased serum responses of immunoglobulin G1 and G2, 2) that the antibodies were cross-reactive to a wide variety of gram-negative pathogens, 3) that serum from hyperimmunized animals enhanced in vitro neutrophil phagocytosis of E. coli, and 4) immunoglobulin G2 antibodies from hyperimmunized steers recognized different antigens of protein lysates of J5 E. coli, as determined from Western blot gel analysis (Chaiyotwittayakun et al. 2004).

Missouri studied the efficacy of two experimental S. aureus mastitis bacterins and a currently marketed five-isolate-based S. aureus bacterin (Lysigin, Boehringer Ingelheim Vetmedica, Inc.) with unvaccinated controls. All groups were challenged with a heterologous strain of S. aureus. There was no evidence of a difference between vaccinates and control with regard to S. aureus clearance rates post-challenge. Cattle vaccinated with Lysigin had a lower mean duration of clinical mastitis and lower total mastitis score post-challenge than controls. Hence, there was no evidence that the vaccines reliably prevented S. aureus IMI, but Lysigin showed benefit in reducing the clinical severity and duration of clinical disease post-challenge. Neither of the experimental bacterins appeared to perform better than Lysigin (Middleton et al. 2006; Luby et al. In Press). Mammary humoral immunity to this vaccine is currently being studied.

(iii) Mastitis Control and Dairy Food Safety.

Several projects were completed within this objective. Studies to determine antimicrobial resistance patterns in organic and conventional dairy herds were conducted in New York (Tikofsky et al. 2003). Tennessee and New York investigated the dynamics and risk factors of Listeria monocytogenes infections (Nightingale et al. 2004, 2005; Nguyen et al. 2004, Oliver et al. 2005, Srinivasan et al. 2005). Pennsylvania and New York investigators developed bulk milk surveillance systems (Jayarao et al. 2004, 2006; Straley et al. 2006). Minnesota, Wisconsin and Guelph (CAN) started a 3-year multi-State study to describe the efficacy and cost-benefit of implementing on-farm culture tools for diagnosis and strategic treatment of clinical mastitis and subclinical mastitis fresh cows. A valuable development was the introduction of a new journal on "Foodborne Pathogens and Disease" where the editor-in-chief is Dr. S.P. Oliver from the Tennessee station. This journal particularly highlights the importance of pathogen control on the dairy and thus dairy food safety.

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. (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). (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. Through a series of collaborative efforts (MI, MN, NY, TN, VA, VT), researchers from this project will identify factors that allow mammary-adapted strains of E. coli, K. pneumoniae, and Strep. uberis to evade host defenses - specifically bovine mammary epithelial cells and neutrophils. To accomplish this, the ability of transient and chronic strains of these bacteria will be assessed for their ability to adhere, invade, and survive in mammary epithelial cells. Additionally, these strains also will be evaluated to determine if mammary adapted strains have an increased ability to evade milk and blood neutrophil phagocytosis and killing. Identifying the potential mechanisms by which these better adapted bacterial strains evade host defenses will aid in identifying potential targets for preventive and therapeutic strategies. Other studies will screen a Strep. uberis mutation library for specific mutations which modify the ability of milk and/or blood neutrophils to phagocytose or kill the mutant Strep. uberis strain relative to the wild type strain. The mutated clones that exhibit altered functional activity then will be sequenced to determine the Strep. uberis gene that was altered. Once identified additional studies will be conducted to determine the role of the identified gene in bacterial pathogenesis using both in vitro and in vivo challenge models. A separate series of studies will use a combination of in vivo and in vitro challenge models to assess the genes which are activated in bovine mammary cells and neutrophils in response to bacteria such as Strep. uberis and toxins such as lipopolysachharide. In order to maximize our understanding of the responses activated, gene expression will be examined using bovine specific microarrays developed by University of Illinois, Michigan State University, or Affymetrix. Genes observed to change significantly with infection will be confirmed by real time RT-PCR. In addition, the expression of critical acute phase and infection related cytokines will be evaluated and include tumor necrosis factor and interleukin-1, 6, 8, 10, and 12. Several studies also will evaluate neutrophil functional activity prior to and following in vivo challenge with Strep. uberis in relation to nutritional status of the animal. Subsequent changes in mammary and neutrophil gene expression will be related to observed alterations in neutrophil function and disease severity. The prevention of hemorrhage and containment of the infection occurs through the deposition of fibrin. However, this process can also lead to abscess development. A series of experiments will test the hypothesis that identification of the mechanisms regulating fibrin deposition during infection will provide new targets to prevent dissemination and abscess formation during staphylococcal infections. Two primary questions will be addressed: does coagulation function protectively or pathologically during S. aureus infection and how does S. aureus alter coagulation to promote bacterial dissemination and growth during mastitis. To accomplish this, levels of mRNA and activity of fibrin-forming and -degrading proteins during the course of S. aureus infection will be evaluated. The relationship of these levels to acute phase cytokines also will be assessed. The mechanisms by which S. aureus induces production of coagulatory factors by mammary endothelial and myoepithelial cell lines also will be evaluated. Furthermore, the isolation of bovine neutrophils from both milk and blood will allow for the ex-vivo study of pathogen-immune cell interactions and the resulting activation of the coagulation cascade. Future studies will focus on utilizing the bovine mastitis challenge model to evaluate the role of both the pathogen and the host in regulating fibrin deposition during infection. These studies will provide insight into the role of coagulation during S. aureus mastitis and provide new targets for the prevention of bacterial dissemination and abscess formation. (iii) Identification of Candidate Genes for Resistance to Mastitis Our search for beneficial genes for use in standard breeding schemes is currently focused on the dairy cows' innate immune system. This work involves both in vivo experiments, in which cows are challenged with mastitis causing pathogens, and in vitro experiments, in which isolated bovine mammary epithelial cells and neutrophils are similarly challenged. In both cases, the response to the infection is closely monitored through measurement of proteins, or their genes, that are associated with the inflammatory response. The goal is to more fully understand the inflammatory response and thus be able to identify critical control points that may be targets for genetic selection. The availability of cattle genetic information is at an unprecedented level. Completion of the bovine genome, identification of quantitative trait loci associated with various health traits, generation of high definition comparative maps, and bovine specific microarrays that can assess thousands of genes simultaneously has opened many doors to enable identification of candidate genes and markers associated with mastitis susceptibility. Each of the studies mentioned in the above section will provide candidate genes that can be assessed for their ability to serve as biomarkers or therapeutic targets to minimize the incidence and/or severity of disease. In addition, these genes provide candidates that can be evaluated for the expression of single nucleotide polymorphisms (SNP) that are associated with disease incidence and can be used as genetic markers for selecting cattle more resistant to disease. The overall objective of these experiments will be to identify a series of single nucleotide polymorphisms indicative of mastitis susceptibility that can be used as markers to select a more mastitis-resistant cattle population. To accomplish this, DNA from a panel of 24 grandsires will be used to identify SNP in candidate genes and loci previously shown to contain genomic regions associated with mastitis, somatic cell counts, or other diseases. All identified SNPs will be evaluated to determine which combination of SNPs best represents the genetic variation observed in that gene region. This select group of SNPs will be examined for their potential association with mastitis and related traits using one of the following designs: granddaughter, daughter, and/or phenotypic tail. The expected outcome of this research would be identification of a composite genetic marker for mastitis susceptibility that could be utilized to improve mastitis resistance in the dairy population by more accurately selecting sires or cows. Furthermore, identification of specific markers will provide basic model systems to better understand host mechanisms that impact immune function and subsequent disease resistance. Prior research has identified a polymorphism in the CXCR1 gene which is associated with a 15% difference in mastitis incidence and altered neutrophil activity. Future studies will seek to identify the mechanisms that cause altered neutrophil migration, reactive oxygen species generation, and apoptosis between cows with different genetic backgrounds relative to the polymorphism. Objectives include identification of the genetic structure, expression, and functionality of the CXCR1 and its companion receptor CXCR2 under normal and infection conditions in dairy cows. This information is critical as very little is known regarding these receptors in cows despite their key role in neutrophil function and disease resistance. Currently, full length cDNAs for CXCR1 and its companion receptor CXCR2 are being prepared for in vitro experiments, as well as specific antibodies for in vivo assessment of function during infection. Additionally, current research also has indicated the polymorphism is a marker for a defect in downstream signaling events such as phosphorylation. The goal for this research is to identify genetic-based differences in intracellular signaling that impact neutrophil functionality. This will be accomplished using a quantitative proteomics based approach using MS-MS technology. A separate study will evaluate the role of a separate candidate gene - uterocalin (bLcn2). Future experiments will test the hypothesis that uterocalin is protective against mastitis and its negative consequences (IA, MI). Studies will include: characterizing antibacterial/anti-inflammatory properties of uterocalin in vitro, identifying the target cell to which uterocalin binds and influences cytokine production, identifying the promoter elements used to regulate uterocalin expression during mammary gland involution and mastitis, determine uterocalin expression in milk during mastitis and correlate with SCC, and directly test hypotheses using a mouse model with strains that differ in uterocalin expression. To aid in the identification of viable genetic markers, the possible creation of a National Mastitis Database using information from this regional project will be assessed. The overall goal is to develop a National mastitis database that includes a combination of mastitis field and experimental challenge data with an associated DNA bank inclusive of sires and cows. The proposed site for the database and DNA repository would be the University of Tennessee. Objective 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses. (i) Characterization of Pathogen Virulence Factors Studies within the MMRP to date have led to a large bank of mastitis pathogen isolates. This Nationwide collection of isolates continues to be used for studies on mastitis pathogen virulence. In the next 5 years members of the MMRP will continue studies evaluating virulence factor genes and their phenotypic expression by E. coli, Streptococcus uberis, Staphylococcus aureus, Mycoplasma and an emerging area of interest the coagulase negative staphylococci. Tennessee will continue to characterize the role of the Strep. uberis Adhesion Molecule (SUAM), which they discovered, on adherence an internalization of Strep. uberis into bovine mammary epithelial cells using a SUAM knock-out mutant. Studies will be conducted to see if SUAM and the SUAM gene (sua) can be detected in multiple strains of Strep. uberis and other species of streptococci. The SUAM molecule will be further characterized to locate biologically active domains and additional epitopes such that antibodies may be developed to block Strep. uberis adhesion thus blocking infection. A chromosomal library representing interrupted genes will be developed to allow studies on the molecular mechanisms associated with Strep. uberis infection of the bovine mammary gland. Furthermore, studies will be conducted to identify other virulence factors of Streptococcus uberis and E. coli which are activated in the presence of mammary epithelial cells and neutrophils. Virginia will initiate studies evaluating the bacterial traits of enterococci which contribute to their pathogenicity. Isolates from various sources including the mammary gland and the cow's environment will be examined for differences in the presence of certain virulence factor genes coding for molecules associated with bacterial adhesion and evasion of host defense mechanisms. (ii) Antimicrobial Resistance Studies evaluating the relationship between antimicrobial usage and changes in susceptibility of mastitis pathogens will be continued at Wisconsin. Specific aims of this study include 1) assessment of temporal changes in the proportion of Gram-positive mastitis pathogens that are resistant to selected antimicrobials in cohorts of cows defined by exposures to antimicrobials; 2) determine if exposure to intramammary antibiotics routinely administered at the end of lactation (dry cow therapy) is associated with minimum inhibitory concentrations of selected antibiotics for Gram-positive mastitis pathogens isolated in subsequent lactations; and 3) determine if changing the class of antibiotic used for dry cow therapy from the first to the second dry period is associated with the MIC of the antibiotics for Gram positive mastitis pathogens isolated in subsequent lactations. Studies on coagulase negative staphylococci will include an evaluation of antimicrobial susceptibilities of various species and genotypes of CNS (LA, NY, WI, MO). In addition, a study conducted in Missouri will evaluate differences in antimicrobial susceptibility between different strains of S. aureus and evaluate differences between in vitro susceptibility patterns and actual in vivo cure rates. (iii) Use of Molecular Epidemiology & Diagnostic Tools The epidemiology of CNS mastitis will be studied at various centers (MO, TN, WA, NY, WI, LA) with the goal of determining if there are differences in pathogenicity between different CNS species and genotypes and whether these differences are related to certain virulence factors. The first step in this work will be to define methods for accurately differentiating CNS isolates into species. Work is underway using PCR techniques to define species and these methods are being compared to more traditional biochemical typing techniques. Molecular epidemiologic tools will be used to different strains (genotypes) within a species of CNS. Staphylococcus aureus mastitis remains a problem on many small farms. Work in Missouri and at other member stations will continue to investigate the relationships between S. aureus strain-type and pathogenicity in the udder. These studies will include characterization of differences in responsiveness of certain strains to antimicrobial therapy. MMRP collaborators at Guelph, Ontario have discovered a small colony variant of S. aureus and they will study this strain in the context of udder health. In the last 5 year period members of the MMRP initiated a collaborative study on environmental streptococcal mastitis. A variety of environmental Streptococcus spp. were isolated from mammary secretions of lactating and dry cows. The dry period was an important time for acquisition of new environmental Streptococcus IMI, with approximately 12% of glands infected at freshening. Although most environmental Streptococcus IMI were short-lived, one third of cows had IMI lasting over 30 days. These persistent IMI resulted in the greatest adverse effects on the cow and economic loss for the producer. Most environmental streptococcal IMI episodes were subclinical. While a smaller proportion of infections resulted in clinical mastitis, these were often preceded by subclinical IMI. This collaborative project has resulted in a large well documented set of environmental streptococci isolates. Studies in the next five years in Pennsylvania, Virginia, Iowa, Washington, Wisconsin, Tennessee and New York will focus on the genetic differences between streptococcal isolates that result in subclinical versus those that result in clinical mastitis cases. Both genotyping and closely followed experimental infections will allow a better understanding in the differences between these two types of environmental streptococci. Studies spearheaded by Washington have led to new knowledge on Mycoplasma mastitis. Further collaborative studies on the epidemiology of mycoplasma mastitis will be conducted in the coming years. Mycoplasma infected herds will be identified and intensively studied to identify sources of mycoplasma. Mycoplasma isolates will be fingerprinted to determine strains most apt to result in intramammary infections. Methods of controlling and/or preventing mycoplasma mastitis can then be established and later tested. Objective 3: Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. (i) Reduce Pathogenicity of Mastitis Organisms. Heifer mastitis: MMRP joint project. This project has been a great collaborative effort of MMRP members. A common protocol was developed and executed on several dairy farms around the country. The results were analyzed and published. Currently the isolates of all confirmed cases are banked for further study. In the next 5 years a number of the stations will use this biobank and perform further studies on the bacterial organism. Results of residue testing will be published in the next study period Environmental streptococcal mastitis - MMRP joint project. The data analyses for the collaborative environmental streptococcal mastitis project conducted by members of the MMRP will be completed and the results will be published and disseminated to appropriate target groups. Results from this study will be used to direct further research in this area. Several stations have a strong interest in streptococcal mastitis and will continue research in this area (NY, TN, VT). Additional studies in this area will include an investigation of the effect of Mycoplasma sp. on herd milk quality as measured by protein, fat, and bacterial counts in commingled milk (WA), and the effects of a mastitis control program on the incidence of environmental organisms (CT). (ii) Technologies to Promote Host Defense Systems Environmental and nutritional factors affecting the immune system of the bovine during the periparturient period will be investigated at Illinois, Guelph, New York and Connecticut. The effect of the energy status of cows and ketosis on immune function will be investigated at Guelph, New York and Connecticut. Teat disinfectant studies will be conducted in IA and LA. Iowa will complete and analyze 2 trials evaluating the effects of 2 new teat dips on teat end and skin health and mastitis, and initiate new trials to develop and/ or evaluate novel internal and external sealants for prevention of mastitis during the dry period and pre-calving. Louisiana will perform in vitro evaluations of teat dips using the excised teat model and A.O.A.C. procedures will continue. Several stations will be working on the development, optimization or evaluation of mastitis vaccines. Missouri will complete work characterizing the mammary humoral immune response to a commercial S. aureus bacterin. Washington is evaluating Staphylococcal enterotoxin C vaccine for S. aureus mastitis. Michigan will continue investigations to determine the effect of injection site and number of J5 bacterin doses on serum immunoglobulin G1 and G2 responses and on lymphocyte cytokine responses when stimulated with J5 E. coli antigen. (iii) Mastitis Control and Dairy Food Safety Organisms of human health concern will be the focus of several studies. Louisiana will determine the presence of E. coli 0157:H7 in dairy lagoon effluent. Tennessee and New York researchers will develop a risk-based system to gather relevant data on the epidemiology of food-borne pathogens for the identification of critical control points during harvesting and storing raw milk. Factors affecting antibiotic screening test performance will be determined at Connecticut. Tennessee is developing the Tennessee Milk Quality Initiative (2007-2012). Similarly, Connecticut is developing a mastitis-control program focused on control of Mycoplasma bovis. The major objectives of these projects will be to identify farms with pathogens in milk and develop intervention strategies to eliminate and / or control the pathogens. Minnesota, Wisonsin and Guelph (CAN) will conclude cow enrollment and begin data analysis/reporting of results for an ongoing 3-year multi-State multiform study to describe the efficacy and cost-benefit of implementing on-farm culture tools for diagnosis and strategic treatment of clinical mastitis and subclinical mastitis in fresh cows. Minnesota will use this field trial to further evaluate the test characteristics of cephapirin discs using the Kirby-Bauer test to determine antimicrobial susceptibility of mastitis pathogens, as compared to cephalothin (model drug) using the Sensititre MIC test method (Trek Diagnostics). Illinois will Perform HPLC-MS-MS analysis on frozen milk samples and pharmacokinetic analysis on the data to determine the effect of milking frequency (2X versus 3X) and timing of antibiotic administration on the disposition of cephapirin and desacetylcephapirin in milk. This may lead to additional studies using different antibiotics or a longer duration of antibiotic treatment on cows with mastitis if results of this pilot study are significant. New York and Pennsylvania will be developing and implementing bulk milk monitoring protocols (NY) and web-based software tools using expert systems (PA) as it relates to animal health, milk quality and food safety.

Measurement of Progress and Results

Outputs

• The results of MMRP initiated studies will be disseminated through presentations at National meetings and through peer-reviewed journals, proceedings, abstracts, book chapters, theses, and extension publications. The results will also be used to develop short-courses for presentation at National meetings, such as the American Association of Bovine Practitioners and National Mastitis Council. The members of MMRP have a long-standing record of productivity and dissemination of data through a variety of avenues (See Appendix 1: Publications 2002-2007).
• Joint projects and collaborative initiatives have resulted in a large collection of isolates of bacterial pathogens that cause mastitis and a large amount of epidemiological data. From these projects, an isolate bank and an epidemiological data set is being amassed and made available to MMRP members for research projects. This represents a resource that could not be compiled by one researcher or in one study.
• MMRP members will continue to share expertise, experimental protocols, reagents, and mastitis pathogen isolates.

Outcomes or Projected Impacts

• A better understanding of dry and transition cow, particularly nutritional factors, associated with IMI will allow targeted mastitis intervention strategies to be designed and validated. Evaluation of new teat dip products will provide new and innovative means of mastitis control and teat chap prevention.
• Identification of candidate genes will continue throughout this project. The marker genes will be evaluated to determine their association with mastitis resistance / susceptibility. Continued identification of mastitis susceptible cows and cow families will provide a resource for evaluating gene function.
• A better understanding of cellular and nonspecific host mechanisms will provide insight into potential technologies to control mastitis. This may eventually provide effective non-antibiotic approaches to the treatment and control of mastitis.
• A better understanding of the genetic determinants of adhesion and invasion in environmental streptococci and E. coli may eventually allow these genetic factors to be targeted for vaccination.
• Currently, mycoplasma mastitis is not well understood and there is no effective therapy for this type of mastitis. A better understanding of the factors involved in the population dynamics may elucidate potential preventive practices that can be used on farms to inhibit transmission of this pathogen.
• A generic protocol for the evaluation of antimicrobial resistance in intramammary pathogens will be developed. Both nationally and internationally, antimicrobial resistance in farm animals is an important concern. In dairy cows, the majority of antibiotics are used to treat or prevent intramammary infections. Hence the ability to study the presence and the development of antimicrobial resistance is a key component of our understanding of this phenomenon.
• A better understanding of the epidemiology of streptococcal, staphylococcal, enterococcal, Klebsiella and E. coli mastitis will allow targeted control strategies to be designed and validated.
• New technologies such as vaccines and targeted treatment protocols will be devised and field-tested.
• Recommendations to optimize the use of antibiotic residue screening tests will be developed. A risk-based quality control program will be developed to reduce the risk of food-borne pathogens in milk and dairy beef. This will positively impact on human health.

Milestones

(2007): Completion and submission for publication of the environmental streptococcal mastitis study. Completion and submission for publication of the duplicate versus single sample techniques for detection of IMI.

(2008): Completion and submission for publication coagulase negative staphylococcal speciation techniques.

(2009): Completion and submission for publication of 3-year study to evaluate cost-benefit of on-farm culture and strategic treatment of clinical and subclinical mastitis in fresh cows.

(2010): Completion and submission for publication of some of the studies on epidemiology, and pathogenesis, and intracellular survival of major mastitis pathogens.

(2011): Completion and submission for publication of the remaining studies on epidemiology, and pathogenesis, and intracellular survival of major mastitis pathogens.

Projected Participation

View Appendix E: Participation

Outreach Plan

All of the members of the MMRP are closely associated with cooperative extension programs at their respective Universities. Many have joint extension appointments and all participate with their local extension personnel in various informational programs designed to disseminate information directly to producers. Previous examples of such efforts are illustrated by the large number of popular press articles and audio visual tapes produced by MMRP members in recent years. Future plans include similar publications. Also, information about the project is available on the internet: http://www.nimss.umd.edu/homepage/home.cfm?trackID=1294. This document will be presented at this web site along with timely information and new developments in mastitis control and prevention. The unique blend of basic and applied research that make up this project ensure that major advancements will be made in this important area, and that this information will be quickly made available to the dairy producers, dairy scientists, milk processors, regulatory personnel, and veterinarians of the Nation and World.

Organization/Governance

Administrative Advisor: Cameron Faustman

USDA/CSREES Representative: Gary Sherman

Technical Committee: *Official representative coordinating the research
Connecticut: S. Andrew*; Illinois: D. E. Morin*, W. L. Hurley; Iowa: L. L. Timms*, M. Nilsen-Hamilton; Kansas: Bruce Schultz*; Louisiana W. E. Owens*; Michigan R. S. Erskine*; Minnesota: S. Godden*; Missouri: J.R. Middleton*; New York: R. N. Gonzalez*, Y.H. Schukken, F.L. Welcome, R.N. Zadoks, H. Schulte; Ohio: J.S. Hogan*; Pennsylvania: B. Jayaroa*; Tennessee S.P. Oliver*, G. Pighetti; Vermont: A. J. Bramley*, D.E. Kerr, J.W. Barlow; Virginia: Christina Petersson-Wolfe*, Isis Mullarky; Washington: L. K. Fox*, W.C. Davis; Wisconsin: P. Ruegg*

International members (Canada): Guelph: K. E. Leslie*, D.F. Kelton, R. Dingwell; Montreal: D. Scholl, Calgary: H.W. Barkema; PEI: G Keefe.

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. The project will be administered by a technical committee which includes the project participants from each of the participating stations. 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. Annual reports of research data from each station will be called for by the Secretary. 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.

Literature Cited

Statement of Issues and Justification:

Middleton JR, Timms LL, Bader GR, Lakritz J, Luby CD, Steevens BJ. 2005. Effect of prepartum intramammary treatment with pirlimycin hydrochloride on prevalence of early first-lactation mastitis in diary heifers. J Am Vet Med Assoc. 227:1969-1974.

Borm AA, Fox LK, Leslie KE, Hogan JS, Andrew SM, Moyes KM, Oliver SP, Schukken YH, Hancock DD, Gaskins CT, Owens WE, Norman C. 2006. Effects of prepartum intramammary antibiotic therapy on udder health, milk production, and reproductive performance in dairy heifers. J Dairy Sci. 89:2090-2098.

Objective 1:

Almeida RA, Oliver SP. 2006. Trafficking of Streptococcus uberis in bovine mammary epithelial cells. Microb Pathog. 41(2-3):80-9.

Dingwell RT, Leslie KE, Schukken YH, Sargeant JM, Timms LL, Duffield TF, Keefe GP, Kelton DF, Lissemore KD, Conklin J. 2004. Association of cow and quarter-level factors at drying-off with new intramammary infections during the dry period. Prev Vet Med. 30;63(1-2):75-89.

Dogan B, Klaessig S, Rishniw M, Almeida RA, Oliver SP, Simpson K, Schukken YH. 2006. Adherent and invasive Escherichia coli are associated with persistent bovine mastitis. Vet Microbiol. 116(4):270-82.

Johnson, L. L., K. N. Berggren, et al. (2003). "Fibrin-mediated protection against infection-stimulated immunopathology." J Exp Med 197(6): 801-6.

Loor JJ, HM Dann, NA Janovick Guretzky, RE Everts, R Oliveira, CA Green, NB Litherland, SL Rodriguez-Zas, HA Lewin and JK Drackley. 2006. Plane of nutrition prepartum alters hepatic gene expression and function in dairy cows as assessed by longitudinal transcript and metabolic profiling. Physiol Genomics 27:29-41, 2006.

Middleton JR, Luby CD, Viera L, Tyler JW, Casteel S. 2004. Influence of Staphylococcus aureus intramammary infection on serum copper, zinc, and iron concentrations. J Dairy Sci. 87(4):976-9.

Mullarky, I. K., F. M. Szaba, et al. (2005). "Infection-stimulated fibrin deposition controls hemorrhage and limits hepatic bacterial growth during listeriosis." Infect Immun In Press.

Nilsen-Hamilton M, Liu Q, Ryon J, Bendickson L, Lepont P, Chang Q. 2003. Tissue involution and the acute phase response. Ann N Y Acad Sci. 2003 May;995:94-108.

Pareek, R., O. Wellnitz, R. Dorp, V, J. Burton, and D. Kerr. 2005. Immunorelevant gene expression in LPS-challenged bovine mammary epithelial cells. J. Appl. Genet. 46:171-177.
Rajala-Schultz PJ, Hogan JS, Smith KL. 2005. Association between milk yield at dry-off and probability of intramammary infections at calving. J Dairy Sci. 88(2):577-9.

Rambeaud M, Clift R, Pighetti GM. 2006. Association of a bovine CXCR2 gene polymorphism with neutrophil survival and killing ability. Vet Immunol Immunopathol. 15;111(3-4):231-8.

Rambeaud M, Pighetti GM. 2005. Impaired neutrophil migration associated with specific bovine CXCR2 genotypes. Infect Immun. 73(8):4955-9.

Rambeaud M, Pighetti GM. 2007. Differential calcium signaling in dairy cows with specific CXCR1 genotypes potentially related to interleukin-8 receptor functionality. Immunogenetics. 59(1):53-8.

Takemura K, Hogan JS, Lin J, Smith KL. 2002. Efficacy of immunization with ferric citrate receptor FecA from Escherichia coli on induced coliform mastitis. J Dairy Sci. 85(4):774-81.

Takemura K, Hogan JS, Smith KL. 2004. Growth responses of Escherichia coli to immunoglobulin G from cows immunized with ferric citrate receptor, FecA. J Dairy Sci. 87(2):316-20.

Veltrop, M. H., H. Beekhuizen, et al. (1999). "Bacterial species- and strain-dependent induction of tissue factor in human vascular endothelial cells." Infect Immun 67(11): 6130-8.

Weiss WP, Hogan JS, Smith KL. 2004. Changes in vitamin C concentrations in plasma and milk from dairy cows after an intramammary infusion of Escherichia coli. J Dairy Sci. 87(1):32-7.

Weiss WP, Hogan JS. 2005. Effect of selenium source on selenium status, neutrophil function, and response to intramammary endotoxin challenge of dairy cows. J Dairy Sci. 88(12):4366-74.

Weiss WP, Hogan JS. 2007. Effects of dietary vitamin C on neutrophil function and responses to intramammary infusion of lipopolysaccharide in periparturient dairy cows. J Dairy Sci. 90(2):731-9.

Wellnitz O, Kerr DE. 2004. Cryopreserved bovine mammary cells to model epithelial response to infection. Vet Immunol Immunopathol. 101(3-4):191-202.

Wise AJ, Hogan JS, Takemura K, Smith KL. 2003. Opsonic activity of serum and whey from cows immunized with the ferric citrate receptor. J Dairy Sci. 86(1):146-51.

Youngerman, S. M., A. M. Saxton, S. P. Oliver, and G. M. Pighetti. 2004. Association of CXCR2 polymorphisms with subclinical and clinical mastitis in dairy cattle. J. Dairy Sci. 87:2442-2448.
Zavizion, B., J. H. White, et al. (1997). "Staphylococcus aureus stimulates urokinase-type plasminogen activator expression by bovine mammary cells." J Infect Dis 176(6): 1637-40.

Zheng, J., A. D. Watson, and D. E. Kerr. 2006. Genome-wide expression analysis of lipopolysaccharide-induced mastitis in a mouse model. Infect Immun 74:1907-1915.

Objective 2:

Almeida RA, Luther DA, Nair R, Oliver SP. 2003. Binding of host glycosaminoglycans and milk proteins: possible role in the pathogenesis of Streptococcus uberis mastitis. Vet Micorbiol. 94:131-141.

Almeida RA, Luther DA, Park HM, Oliver SP. 2006. Identification, isolation, and partial characterization of a novel Streptococcus uberis adhesion molecule (SUAM). Vet Microbiol. 115:183-191.

Biddle MK, Fox LK, Evans MA, Gay CC. 2005. Pulsed-field gel electrophoresis patterns of Mycoplasma isolates from various body sites in dairy cattle with Mycoplasma mastitis. J AM Vet Med Assoc. 227:445-459.

Dogan B, Klaessig S, Rishniw M, Oliver SP, Simpson K, Schukken YH. 2006. Adherent and invasive Escherichia coli are associated with persistent bovine mastitis. Vet Microbiol. 116:270-282.

Erskine RJ, Walker RD, Bolin CA, Bartlett PC, White DG. 2002. Trends in antibacterial susceptibility of mastitis pathogens during a seven-year period. J Dairy Sci. 85:1111-1118.

Fox LK, Hancock DD, Mickelson A, Britten A. 2003. Bulk tank milk analysis: factors associated with appearance of Mycoplasma sp. In milk. J Vet Med B. 50:235-240.

Fox LK, Zadoks RN, Gaskins CT. 2005. Biofilm production by Staphylococcus aureus associated with intramammary infection. Vet Microbiol. 107:295-299.

Garrison LL, Schukken YH, Hilton B. 2000. Antibiotic Susceptibility Patterns for Various Bacterial Intramammary Pathogens Over 15 Years: Results and Analysis of a Database. Proc 39th Annual Meeting Natl Mast Counc, Atlanta, GA. Pages 215-216.

Gillespie BE, Oliver SP. 2005. Simultaneous detection of mastitis pathogens, Staphylococcus aureus, Streptococcus uberis, and Streptococcus agalactiae by multiplex real-time polymerase chain reaction. J Dairy Sci. 88:3510-3518.

Luby CD, Middleton JR. 2005. Efficacy of vaccination and antibiotic therapy for Staphylococcus aureus mastitis. Vet Rec. 157:89-90.

Middleton JR, Fox LK. 2002a. Influence of Staphylococcus aureus strain on mammary quarter milk production. Vet Rec. 150:411-413.

Middleton JR, Fox LK, Gay JM, Tyler JW, Besser TE. 2002b. Influence of Staphylococcus aureus strain-type on mammary quarter milk somatic cell count and N-acetyl-B-D-glucosaminidase activity in cattle from eight dairies. J Dairy Sci. 85:1133-1140.

Middleton JR, Fox LK, Gay JM, Tyler JW, Besser TE. 2002c. Use of pulsed-field gel electrophoresis for detecting differences in Staphylococcus aureus strain populations between dairy herds with different cattle importation practices. Epidemiol Infect. 129:387-395.

Middleton JR, Bader GR, Corbett RB. 2007. Use of pulsed-field gel electrophoresis to evaluate the success of treatment of Staphylococcus aureus mastitis. Proc 46th Annual Meeting Natl Mast Counc. San Antonio, TX. January 23rd. Pages 202-203.

Munoz MA, Ahlstrom C, Rauch BJ, Zadoks RN. 2006. Fecal shedding of Klebsiella pneumoniae by dairy cows. J Dairy Sci. 89:3425-3430

Munoz MA, Zadoks RN, Welcome F. 2007. Klebsiella pneumoniae clinical mastitis on a dairy farm in New York State  A case report. Proc 46th Annual Meeting Natl Mast Counc. San Antonio, TX. January 23rd. Pages 206-207.

Murinda SE, Ebner PD, Nguyen LT, Mathew AG, Oliver SP. 2005. Antimicrobial resistance and class 1 integrons in pathogenic Escherichia coli. Foodborne Pathogens & Disease. 2:348-352.

Osteras O, Martin SW, Edge VL. 1999. Possible risk factors associated with penicillin-resistant strains of Staphylococcus aureus from bovine subclinical mastitis in early lactation. J Dairy Sci. 82:927-938.

Pol M, Ruegg PL. 2007. Relationship between antimicrobial usage and antimicrobial susceptibility of Gram-positive mastitis pathogens. J Dairy Sci. 90:262-273.

Punyapornwithaya V, Fox LK. Colonization by Mycoplasma spp. on mucosal body sites in cows and replacements: The relationship to mastitis, a preliminary report. Proc 45th Annual Meeting Natl Mast Counc. Tampa, FL. January 24th. Pages 236-237.

Srinivasan V, Sawant AA, Gillespie BE, Headrick SJ, Ceasaris L, Oliver SP. 2006. Prevalence of enterotoxin and toxic shock syndrome toxin genes in Staphylococcus aureus isolated from milk of cows with mastitis. Foodborne Pathogens & Disease. 3:274-283.

Tamilselvam B, Almeida RA, Dunlop JR, Oliver SP. 2006. Streptococcus uberis internalizes and persists in bovine mammary epithelial cells. Microbial Pathogenesis. 40:279-285.

Zadoks RN, Gillespie BE, Barkema HW, Sampimon OC, Oliver SP, Schukken YH. 2003. Clinical, epidemiological and molecular characteristics of Streptococcus uberis infections in dairy herds. Epidemiol Infect. 130:335-349.

Zadoks RN, Schukken YH, Wiedmann M. 2005a. Multilocus sequence typing of Streptococcus uberis provides sensitive and epidemiologically relevant subtype information and reveals positive selection in the virulence gene pauA. J Clin Microbiol. 43:2407-2417.

Zadoks RN, Tikofsky LL, Boor KJ. 2005b. Ribotyping of Streptococcus uberis from a dairys environment, bovine feces and milk. Vet Microbiol. 109:257-265.

Objective 3

Borm AA, Fox LK, Leslie KE, Hogan JS, Andrew SM, Moyes KM, Oliver SP, Schukken YH, Hancock DD, Gaskins CT, Owens WE, Norman C. Effects of prepartum intramammary antibiotic therapy on udder health, milk production, and reproductive performance in dairy heifers. J Dairy Sci. 2006 Jun;89(6):2090-8.

Burton JL, Erskine RJ. Immunity and mastitis. Some new ideas for an old disease. Vet Clin North Am Food Anim Pract. 2003 Mar;19(1):1-45, v. Review

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University of Calgary, University of Glasgow, University of Guelph, University of Prince Edward Island