Duration: 10/01/2002 to 09/30/2007

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Bovine mastitis is the most costly 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. Single site studies are often limited in terms of expertise and cattle numbers. A multistate project provides these advantages. In the United States, the dairy industry contributes in excess of 65 billion dollars per year to the national economy, and provides jobs for 1.1 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 dairy cows on every dairy farm and approximately 38% of the 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 morbidity and mortality, and discarded milk. The purpose of NE-112 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 mastitis related 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 on solutions for mastitis. Recognition of the need for a coordinated effort to study resistance of the dairy cow to mastitis resulted in the design and initiation of Multistate Project NE-112. The NE-112 project 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 through the auspices of the NE-112 project. These have included a study of the epidemiology of Streptococcal bacteria, an investigation of associations between cow factors at dry-off and during the dry period and intramammary infections (IMI) in the subsequent lactation, and a collaborative heifer mastitis project. In the current project, we will continue to develop and execute joint studies on current topics through this group. The mastitis research workers group has met in conjunction with the NE-112 annual meeting for many years, and in recent years, the mastitis research workers topics have been included in NE-112 minutes, showing current active areas of research by NE-112 members. International visitors and collaborators are often included in these presentations. In the present proposal, we have included the mastitis group from the Ontario Veterinary College, Canada, as a member.

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 NE-112 Multistate Project. The NE-112 Regional Project was originated in 1977 and was revised in 1982, 1987, 1992 and again in 1997. Research conducted under the project has contributed substantially to enhance 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. The current NE-112 project continues to contribute research advances in all areas covered by its objectives. Currently, the NE-112 project has three collaborative research programs in varying stages of completion. We will refer to these throughout this document and briefly introduce them here. 1. Dry cow study. A project studying the importance of dry cow procedures on teat closing and the subsequent risk of new infections. 2. Environmental streptococci study. A project in six herds where the dynamics of intramammary infections of environmental streptococci are studied. This project also results in a shared epidemiologic data base and a shared strain collection. 3. Heifer mastitis study. A project currently in the design phase where mastitis in heifers will be studied. This project also results in a shared epidemiologic data base and a shared strain collection. The tremendous number of publications cited in this review demonstrates the unique productivity of this group. The members of NE-112 collaborate extensively within the project and with other national and international research groups that have interests in bovine mastitis. The previous project had 4 objectives. In this proposal, these will be consolidated into 3 objectives, pertaining to the host, the pathogen, and the environment. The following are brief reviews by objective of previous and related research taken from MEDLINE and other sources. Objective 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance. During this 5-year project, we will emphasize three areas of host mechanisms influencing mastitis susceptibility and resistance: (i) host factors associated with IMI during the dry and transition periods; (ii) identification of candidate genes for resistance to mastitis; and (iii) host-pathogen interactions at the cellular level. i) Host factors associated with IMI during the dry and transition periods. The dry period offers a chance for control of intramammary infection with contagious pathogens, but is a risk period for new infections, primarily with environmental pathogens. Intramammary infection during the late dry period may result in reduced colostral volume and total IgG1, but does not appear to effect the concentration of IgG1, fat, or protein (Maunsell et al., 1998). NE-112 researchers are collaborating on a study to determine the associations between new IMI during the dry period and udder characteristics such as teat end integrity, rate of involution, existing IMI, and level of milk production at the time of dry-off. A total of 290 cows enrolled by 4 stations had data collected weekly throughout the early dry period and following calving. Preliminary results show that a high percentage of cows have at least one teat remaining open six weeks into the dry period and higher milk production at the time of dry off was a risk factor for new IMI at calving. Members of NE-112 have undertaken further studies on host factors and their associations with IMI. The initial barrier to IMI is the teat duct, acting as a mechanical barrier to infection. Teat end biopsies have shown that tissue changes are associated with cold stress on the tissue (Timms et al., 1998). Teat end changes, such as cracking can occur or change rapidly (1-2 days) when exposed to temperature changes. Salves and glycol teat dips do not appear to influence degree of teat chapping when cows are exposed to cold weather environments (Timms, 2001). NE-112 members have been involved in the development and testing of both conventional and barrier-type teat dip products as aids to mastitis control (Boddie and Nickerson, 1997a, 1997; Boddie et al., 1998; Oliver et al., 1999, Boddie et al., 2000; Oliver et al., 2001). ii) Candidate genes of mastitis susceptibility. Selection of dairy cows for enhanced disease resistance without compromising production traits is a very appealing concept that until the last decade was primarily a theoretical fantasy. However, excellent molecular techniques have been developed, resulting in the identification of new genetic markers for characterization of genes responsible for production traits and host immunity. Studies comparing in vivo and in vitro functional capacities of leukocytes from non-parturient and periparturient dairy cows have provided substantial evidence that systemic and local mammary immune defenses are deficient around parturition (Kehrli and Harp, 2001). This evidence has lead to the hypothesis that immune deficiency underlies the heightened mastitis susceptibility of periparturient cows. A thorough understanding of leukocyte biology during periparturition would seem a critical goal for development of effective mastitis prevention strategies. Combinations of DDRT-PCR and cDNA microarray technologies can be used to interrogate bovine leukocyte RNA for global gene expression changes occurring at and around parturition (Burton et al., 2001; Madsen et al., 2002; Yao et al., 2001; Weber et al., 2002). Over 25 novel candidate genes of periparturient immunosuppression have already been discovered, most of which are involved in the normal growth, metabolism, and immune surveillance function of blood leukocytes. Major histocompatibility complex (MHC) genes also have been investigated to determine their involvement in host immunity (Dietz et al., 1997; Gillespie et al., 1999; Sharif et al., 2000). iii) Host-pathogen interactions at the cellular level. While the teat surface is commonly contaminated with pathogenic bacteria, IMI is relatively infrequent. This is a consequence of various defense systems collectively referred to as "resistance of the mammary gland to mastitis". These defense systems include the teat duct, immune cells such as neutrophils, macrophages, and lymphocytes, immunoglobulins, and nonspecific defense mechanisms. The effectiveness of these host defense systems may be influenced by genetic, nutritional, physiological, and environmental factors. While these host defense systems may offer unique approaches to mastitis control, our knowledge of the systems and their interactions is incomplete. Active areas of research in this area include, but are not limited to; active phase proteins, such as uterocalin, that are induced during the inflammatory response (Liu et al., 1997); non-specific host defenses such as selenium, alpha-tocopherol and lactoferrin (Barrett et al., 1997; Pighetti et al, 1998; Fang and Oliver, 1999); and the potential immunogentic effects of ferric citrate and ferric enterobactin receptors (Lin et al., 1998a; Lin et al., 1998b; Lin et al., 1999a; Lin et al., 1999b). Objective 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing the host defenses. 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 immune functions, production of capsule and slime layers that protect bacteria from host defenses, and antibiotic resistance (Ebling et al. 2001, Calvinho et al. 2001, Fitzgerald et al. 2001, Yazdankhah et al. 2000, De Oliviera et al. 2000, Lin et al, 1999, Ferens et al. 1998). Mammary epithelial cell culture techniques have been used to show that in the early stages of infection, Streptococcus uberis, S. dysgalactiae, E. coli and S. aureus invade mammary epithelial cells and adhere to and enter bovine epithelial and myoepithelial cells in culture (Almeida and Oliver 2001, Dopfer et al. 2001, Dopfer et al. 2000, Cifrian et al., 1994). Streptococcus uberis and recently Streptococcus dysgalactiae were shown to elaborate unique proteins in culture and to possess a capsule that contributes to its virulence (Almeida and Oliver 2001, Song et al. 2001). Staphylococcal alpha and beta toxins and especially Staphylococci expressing superantigens (i.e. entorotoxin c1) were shown to be associated with pathogenicity of intramammary infections (Ferens et al. 2000, Ebling et al. 2001, Ferens et al. 1998, Fitzgerald et al. 2001). Rapid genotyping techniques have been developed for streptococci, staphylococci (Raimundo et al. 1999), Escherichia coli and also mycoplasma (Yazdankhah et al. 2000, Pinnow et al. 2001). A PCR assay based on the nuc gene of Staphylococcus aureus was developed to detect Staphylococcus aureus in milk (Kim et al., 2001). The long term goal is to rapidly identify pathogens from milk samples without the need for time consuming and labor intensive culture techniques. In some situations this has become a reality (Riffon et al. 2001). Gene polymorphism in S. aureus and other mammary pathogens is also being utilized to subtype and identify S. aureus isolates for epidemiologic studies (Raimundo et al. 1999, Lee et al. 1998). Recent results from Washington State University indicated that the magnitude of mammary secretory epithelial cell damage caused by an IMI with a certain strain of S.aureus was sufficient to cause lost milk production, unlike other strains of S. aureus isolated in the same herd. Hence, it appears that there may be differences in pathogenicity between strains of S. aureus causing IMI in the bovine (Smith et al. 1998). 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 in intramammary pathogens in US dairy herds (De Oliviera et al. 2000). 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). Population dynamics of intramammary infections in dairy populations has become an important area of research. Application of modern diagnostic tools allows a much better understanding of infection dynamics (Zadoks et al. 2002). Mastitis in heifers continues to be an important area of research. Studies have shown that up to 90% of heifers are infected in some herds, (Nickerson, 1994). Dry cow therapy prepartum has been shown to be highly effective in eliminating these infections, with cure rates greater than 90% observed (Oliver et al. 2002). The etiology of these infections is unknown, and it was initially suspected that infected cows could be the primary source of S. aureus infections in heifers (Zadoks et al. 2001). However, preliminary evidence from coagulase gene typing, RNA ribotyping, and biochemical finger printing indicate that the organisms infecting heifers probably arise from the environment (Zadoks et al. 2002). Objective 3. Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. This proposed objective will replace objectives 3 and 4 from the previous project and will be addressed as a combination of evaluation of techniques for modulation of the host defense system and adaptation of new technologies to control mastitis and to promote dairy food safety. Application of emerging technologies for mastitis control provides the dairy industry with tools for effective mastitis control while maintaining or enhancing human food safety (Mckewan et al., 1991).The areas of emphasis for this new combined objective will be to evaluate the application of new technologies that have been developed to reduce the pathogenicity of organisms, to promote host defense systems, and to enhance milk quality and human food safety of dairy foods. A major emphasis of this objective is to disseminate the information derived from this regional project to appropriate stakeholders; including milk quality specialists and dairy farm personnel. NE-112 members have addressed these objectives during the last five years as described below. i) Reducing pathogenicity of mastitis organisms. Environmental streptococci are commonly reported causes of clinical and subclinical IMI in dairy herds (Watts, 1988; Todhunter et al, 1995). 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 Streptococcus uberis (eg, Hoblet et al, 1991; VanEenennaam et al, 1995; Cattell, 1996; Hoeben et al, 1999; Zadoks et al, 2001). A multi-site NE-112 research project investigated the impact of naturally occurring environmental Streptococcus IMI on cow health, milk production, and milk quality. Preliminary findings indicate that environmental Streptococcus IMI at freshening or during lactation resulted in increased somatic cell counts (SCC) at the next DHIA test (Morin, et al., 2001a). Approximately 14% of glands with environmental Streptococcus IMI at freshening had clinical mastitis, and 12 to 25% of clinical mastitis samples contained environmental Streptococcus spp. Preliminary analyses did not identify adverse short or long-term impacts of environmental Streptococcus IMI on milk production, milk fat or protein production, or survival (Morin et al., 2001b). At this time, little is known about differences in pathogenicity and epidemiology among different environmental Streptococcus spp. in naturally occurring bovine mastitis. Mastitis associated with Mycoplasma sp. is a growing concern in the diary industry. However, risk factors associated with the prevalence of Mycoplasma sp. are largely unknown. The effect of the presence of a contagious pathogenic organism in bulk milk on the risk of identifying a different contagious organism was evaluated on 463 dairies in the Pacific Northwest (Fox et al., 2001). Results indicated that a bulk tank culture that was positive for S. aureus was not a risk factor for the presence of Mycoplasma sp. in the same sample. The risk factors identified for mycoplasma in bulk milk were herd size and the presence of environmental mastitis pathogens. Seventy percent of herds first positive for Mycoplasma sp. were negative within 1 month, and all herds were negative within one year. ii) Therapies to promote host defense systems. Optimizing the effectiveness of antimicrobial therapies has the potential for improving milk quality, reducing milk losses and ultimately reducing the risk of antibiotic residue contamination of commingled milk (McKewen et al., 1991). In cows with naturally occurring clinical mastitis, an aggressive antibiotic treatment regimen, combined with supportive treatment, resulted in higher clinical and bacteriologic cure rates, less severe clinical disease, fewer recurrences of clinical mastitis, reduced lactational milk yield loss, and reduced overall cost, compared with supportive treatment alone (Shim et al., 2000). Extended intramammary antibiotic therapy was no more effective than label therapy for clinical cases of environmental streptococci (Virginia). Hypertonic saline infusion was of no clinical benefit in cows with experimentally induced coliform mastitis (Haddad et al., 2001). Several non-antibiotic therapies have shown potential for enhancing the host defense system. Transgenic mice expressing lysostaphin in their milk have been developed and shown to be resistant to staphylococcal mastitis (Kerr et al., 2001). Efficacy of Escherichia coli J5 vaccines containing novel adjuvants were tested in an intramammary challenge trial. Addition of cholesterol to bacterins did not alter the immune response to antigen or clinical severity of experimentally induced coliform mastitis (Hogan et al., 2001). A Staphylococcus aureus bacterin that comprised both an autogenous and an experimental encapsulated strain have been developed to determine the potential synergism between immunization and antimicrobial therapy in eliminating intramammary infections caused by this pathogen (Michigan). Infusion of quarters with bispecific antibodies at dry-off reduced the number of bacteria present following experimental challenge with S. aureus when compared to phosphate buffered saline (PBS) injected quarters (Tomita et al., 2000). Nutrition plays a role in the immune system of the cow. Several studies have shown a direct relationship between particular nutrients and susceptibility to mastitis (Hogan et al., 1992; Torre et al., 1995). The effect of energy status on immune system function is currently under investigation. Preliminary results indicate that elevated plasma and milk ketone concentrations are associated with suppression of the immune system (Suriyasathaporn et al., 2000). iii) Enhancing milk quality and human food safety of dairy foods. There is a potential for pathogenic bacteria present in infected mammary glands to contaminate milk and meat, resulting in human illness. The heightened public awareness concerning human health issues gives added emphasis to these potential areas of concern. Surveillance methods to detect antibiotic residues in milk and meat are currently being evaluated to improve their specificity and enhance the ease of their use on the farm. Improved accuracy is needed in this area to both prevent unnecessary waste of saleable milk and protect the public from exposure to antibiotic residues. Very little information is available concerning the relationship between bovine mastitis and these human and food safety issues; therefore, a need exists to answer these questions and determine what steps, if any, need to be taken to insure that mastitis control and treatment procedures do not adversely impact public safety. Effective mastitis control programs that promote dairy food safety are based on identifying the pathogens present, developing effective tools to control mastitis pathogens, and utilizing practices that reduce the risk of antibiotic contamination of commingled milk. Pathogens that have a significant human health hazard, such as S. aureus, E. coli O157:H7, and Salmonella spp., may be present in milk and must therefore be controlled. Antibiotic residue screening tests can be used to screen milk from individual cows that were treated with an antibiotic. However, the specificity of the tests can vary depending on test format and milk composition (Cullor, 1994). High concentrations of milk protein, fat, and components in colostrum can adversely affect antibiotic residue test performance (Andrew, 2000, 2001). Further evaluation of the effect of screening tests and milk composition on antibiotic residue screening test sensitivity and specificity will continue. A comparative study on the phylo-genetic tree of Streptococcus agalactiae isolates from bovine and human origin is underway in New York. The results indicate a strong species separation (Sukhnanand et al. 2001). Very few human strains fall into a category where most dairy cattle strains are located. Additional work in this area, including sequencing of genes important in pathogenesis, is currently being performed.

Objectives

1. Characterization of host mechanisms associated with mastitis susceptibility and resistance.
   
2. Characterization and manipulation of virulence factors of mastitis pathogens for enhancing the host defenses.
   
3. Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety.
   
4. 
   

Methods

Joint research trials have been, and will continue to be, conducted by NE-112 researchers. The NE-112 forum allows groups of researchers to formalize their collaboration, maximize cow numbers to increase statistical power, and provides diverse expertise to address important issues related to mastitis prevention and control. We are completing data analysis on joint NE-112 projects related to Streptococcal mastitis and to dry cow management. We have planned, and are currently initiating, a study on heifer mastitis. During this project (2002-2007), we intend to include a discussion of emerging and important mastitis issues in our annual meeting. From these discussions, we will identify a topic of importance and interest to the group and design a study to address the selected issue. A project leader will be identified to oversee the final study design, trial implementation, and data collection and analysis. Results of the research will be presented at the next annual meeting. This procedure will be followed at each annual meeting. Prominent examples of joint planning and coordination by the cooperating stations are as follows: 1) development and implementation of intramammary experimental challenge protocols for both contagious and non-contagious pathogens, and models such as toxin infusion; 2) methods of identification of mastitis pathogens, most notably coagulase-negative staphylococci and coliform bacteria; 3) the exchange of reagents (i.e., common monoclonal and polyclonal antibodies as well as various bovine immunoglobulin isotypes, cell surface proteins, and bacterial toxins); and 4) biotechnology reagents that include various cytokines and genetic probes. SPECIFIC METHODS BY OBJECTIVE Objective 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance. i) Host factors associated with IMI during the dry and transition periods. A major collaborative project recently undertaken by NE-112 members under Objective 1 was a study of host factors during the dry period and their effect on mastitis in the subsequent lactation (Guelph, Cornell, Iowa). In this project, each station began collecting daily milk production information at the time of dry off. Quarter milk samples were taken at the time of dry off, at calving, and for any case of clinical mastitis. Beginning at the time of dry off, and extending 6 weeks into the dry period, weekly evaluations of mammary involution, teat closure, and teat end condition were made. Milk production and episodes of clinical mastitis were recorded for the first 30 days of the subsequent lactation. A total of 290 cows were enrolled. Preliminary data results were summarized in the previous and current work section of this proposal. During the next year, data analysis and publication will be completed. Clinical trials will be conducted in Iowa, Louisiana, and Washington to evaluate therapeutic approaches to improve teat end condition and prevent the negative teat end changes associated with cold weather. Several stations (Minnesota, Illinois, Wisconsin, Indiana & Pennsylvania) will collaborate on studies to investigate novel controls for dry cow mastitis centering on preventing bacterial entry to the teat canal. This work will include adherence and efficacy studies on teat sealants and persistent barrier dips. ii) Candidate genes of mastitis susceptibility. Periparturient cows are at risk for new IMI and for clinical episodes of mastitis. This is in part due to the immunosuppression that occurs during this period. When investigating potential marker genes, it is hypothesized that the physiology of parturition negatively affects gene expressions in blood leukocytes. Candidate genes identified to date are involved in the normal growth, metabolism, and immune surveillance functions of blood leukocytes. Identification of differentially expressed genes will allow greater insight into potential therapeutic or preventive strategies for the control of mastitis. The identification of candidate genes in isolated populations of blood leukocytes and bone marrow cells will continue. The candidacy of these genes as markers of mastitis susceptibility / resistance will be tested using several strategies. The expression of genetic haplotypes will be compared between cows with and without IMI. The University of Tennessee has identified, and will continue to identify, mastitis resistant and susceptible dairy cows and cow families. In conjunction with Michigan State University, differential gene expression by mononuclear cells will be compared between resistant and susceptible cows following stimulation with major mastitis pathogens. Finally, genetically identical clones with and without mastitis resistance will be exposed to various environmental conditions to evaluate gene expression with the cow effect nullified. This work will complement and extend efforts to investigate the immune state of cows during the periparturient period and the association with clinical or subclinical mastitis. iii) Host-pathogen interactions at the cellular level. NE-112 researchers will work both collaboratively and independently to increase our understanding of the role of cellular and non-specific host defense systems in mastitis prevention. Specific plans include further characterization of the role of the mammary gland humoral response to ferric-regulated protein of gram-negative bacteria (Ohio), defining the influence of milking frequency on inflammatory responses of the mammary gland following bacterial challenge (Ohio), identifying the role of uterocalin, an acute phase protein, in neutrophil behavior (Iowa), and investigating the cellular immune response to J5 coliform vaccination focussing on the importance of gamma-delta T cell populations during intramammary infection (New York). Objective 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defense. i) Characterizing genomic markers of virulence factors of mastitis pathogens. At Tennessee and New York, factors affecting bacterial adherence to mammary epithelial cells, including bacterial cell surface components, epithelial cell surface components, and toxins, will be studied using the mammary epithelial cell model recently developed through NE-112 collaborative studies. Bacterial proteins with affinity for host cell receptors are also expressed and probably function to fine-tune the process and achieve more efficient adherence and entry into host cells. These results explain important aspects of the disease, such as persistent and recurrent infections. Persistent and recurrent infections were detected in natural cases of mastitis in the NE-112 collaborative streptococci study (Morin et al . 2001). Measures directed towards the control of mastitis should continue to focus on pathogenic mechanisms of mastitis pathogens. At Vermont, the lysostaphin gene has been cloned and modified for epithelial cells. The expression of the gene and secretion of gene products will be studied. Also at Vermont, adenovirus and retrovirus vectors are being studied and developed as a means for gene introduction into bovine mammary epithelial cells. ii) New epidemiological techniques to evaluate population dynamics of intra-mammary infections. Studies will be conducted at Tennessee and Vermont on rapid non-culture methods of identifying mastitis pathogens. PCR fingerprinting for staphylococci and streptococci will be refined, as will ribosomal RNA ribotyping and phage typing for identification of staphylococci (Vermont and Louisiana). In all such studies, bacterial strains and isolates will continue to be shared freely among NE-112 members. The etiology of heifer mastitis will continue to be investigated at Washington, Louisiana, Vermont, Tennessee, Virginia, Guelph and New York. Fomites and reservoirs of S. aureus mastitis in heifers will be characterized and management strategies developed to block transmission. Strains of S. aureus from heifers will be identified using recently developed techniques in PCR fingerprinting and ribotyping. * Environmental Streptococci. In the NE-112 collaborative environmental streptococci project, 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 would result in the greatest adverse effects on the cow and economic loss for the producer. Most environmental Streptococcus 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 environmetal streptococci isolates. Studies in the next five years in Pennsylvania, Virginia, Iowa, Washington, Wisconsin, Tennesee 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. * Mycoplasma mastitis. Further collaborative studies on the epidemiology of mycoplasma mastitis will be conducted in Washington, Iowa and New York. Mycoplasma infected herds will be identified and intensively studied to identify sources of mycoplasma. Mycoplasma isolated 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. iii) Environmental regulation and expression of virulence factors (i.e. antibiotic resistance in intramammary pathogens). Further studies on the development and mechanisms of antibiotic resistance will be performed. At Michigan, an effort to develop a protocol for evaluating antibiotic resistance development in intramammary pathogens will be spearheaded in collaboration with the National Mastitis Council. Further longitudinal analysis of data collected in New York, as part of their milk quality programs, will be used to exemplify the protocol proposed by the consortium. iv) Integration of basic knowledge into applied technology. Louisiana, Minnesota and Ohio will continue to evaluate new vaccine products and regimens for efficacy against S. aureus mastitis in heifers and cows. Determination of when to initiate vaccination in heifers will be a focal point. Development of new vaccines for the different types of bovine mastitis is a major area of work. Vaccines for both S. aureus and streptococcal mastitis would provide a major breakthrough for control of these difficult infections. Similar vaccine success with the J5 coliform vaccine has already had a major impact on coliform mastitis, with a return of $57/cow/lactation. It is currently estimated that 25% of the US dairy herd is now being vaccinated with gram-negative core antigen vaccines with an expected efficacy of 70%. In Ohio expression of virulence factors by E. coli in mammary secretions obtained during the dry period and lactation will be compared. Vaccines containing purified outer membrane proteins of E. coli will be evaluated for efficacy in preventing clinical mastitis in challenge and natural exposure trials. In Louisiana, researchers will evaluate the efficacy of various adjuvants used with a commercial S. aureus bacterin. Other studies on cytokines and immune modulators could result in major changes in both therapy and vaccine protocols, and in vaccine and treatment delivery systems. 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 - NE-112 joint project. A major focus of this objective will be to conduct a multi-site study to determine the effect of intramammary antibiotic therapy on incidence of mastitis, milk production and composition, and reproductive status of primipartum heifers. A collaborative approach will provide the necessary sample size for meaningful statistical comparisons, and university herds (Michigan, Minnesota, Ohio, Wisconsin, Kansas, Indiana, Pennsylvania & Iowa) will allow close monitoring to prevent antibiotic residues. Heifers from NE-112 station research herds will be enrolled 14 days prior to expected calving over a one year period. Each heifer will be randomly assigned to a treatment (intramammary infusion of an antibiotic in all quarters) or no-treatment control group. Quarter milk samples will be collected at calving and 1,2, and 3 weeks post-calving and cultured to determine the presence of IMI. Ancillary data on milk production, SCC, and reproductive performance will be collected throughout the first lactation of each heifer. A composite milk sample will be collected at the seventh milking postpartum to check for antibiotic residues. Milk from study animals will not be shipped for human consumption until antibiotic residue tests are negative. Composite milk samples collected at weeks 1 and 2 of lactation will be tested for ketones, and this data correlated with reproductive performance. Data will be summarized across study sites. Environmental Streptococcus - NE-112 joint project. The data analyses for the collaborative environmental Streptococcus project conducted by members of the NE112 regional project 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. 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 (Washington), the effect of management practices on clinical mastitis incidence (multiple sites), hornfly control to reduce mastitis (Louisiana), the efficacy of milking frequency as a treatment for mastitis (Virginia), and the effects of a mastitis control program on the incidence of environmental organisms (Connecticut). ii) Technologies to promote host defense systems. Environmental and nutritional factors affecting the immune system of the bovine during the periparurient period will be investigated at Illinois, Guelph, New York and Connecticut. Research at Illinois will determine the influence of photoperiod length during the dry period on immune responses and susceptibility to Strep. uberis mastitis during the periparturient period. The effect of the energy status of cows and ketosis on immune function will be investigated at Guelph, New York and Connecticut. Several stations will investigate methods that will potentiate the efficacy of antimicrobial therapies. Antimicrobial gene expression in transgenic animals will continue at Vermont. Illinois will identify cow and milk factors that act as covariates for antibiotic elimination from milk in cows with naturally occurring clinical mastitis. 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. Studies to determine antimicrobial resistance patterns in organic and conventional dairy herds will be conducted in New York. Tennessee and New York researchers will investigate the dynamics and risk factors of Listeria monocytogenes infections and 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.

Measurement of Progress and Results

Outputs

• The results of NE-112 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 NE-112 have a long-standing record of productivity and dissemination of data through a variety of avenues (See Appendix 1: Publications - 1997 - 2001).
• 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 will be amassed and made available to NE-112 members for research projects. This represents a resource that could not be compiled by one researcher or in one study.
• NE-112 members will continue to share expertise, experimental protocols, reagents, and mastitis pathogen isolates.

Outcomes or Projected Impacts

• A better understanding of dry cow factors associated with IMI will allow targeted 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.
• Outcome/Impact 6. 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. Outcome/Impact 7. A better understanding of the immunity involved in J-5 coliform mastitis vaccination. Dairy cows in the US are often vaccinated using a J-5 coliform mastitis vaccine (approximately 25% of cows are vaccinated at least twice per year). A better understanding of the mechanisms behind this vaccine enhances our abilities to optimize the vaccine and the vaccination strategies. Outcome/Impact 8. Determination of the etiology of heifer mastitis will provide important information for control of new infections in younger animals. This collaborative NE-112 project will also result in a strain collection of pathogens involved in intramammary infections in heifers. Outcome/Impact 9. A better understanding of the epidemiology of Streptococcal mastitis will allow targeted control strategies to be designed and validated. Outcome/Impact 10. New technologies to control mastitis will be devised and field-tested. Outcome/Impact 11. 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

(2002): Environmental Streptococci, joint project data analysis. Dry cow factors associated with IMI, joint project data analysis. Endemic Mycoplasma herds will be identified to allow studies of this pathogen to be initiated.

(2003): Environmental Streptococci, joint project presentation and publication submission. Dry cow factors associated with IMI, joint project presentation and publication submission. Heifer mastitis project, joint project trial implementation and data collection. An antimicrobial resistance evaluation protocol will be completed.

(2004): Heifer mastitis project, joint project pathogen and data analysis. Identification and evaluation of both host resistance and pathogen virulence genes will continue. Analysis of existing antimicrobial resistance data bases will be completed. New group project (based on results of projects in 2002-2004) designed and initiated.

(2005): Heifer mastitis project, joint project presentations and publication submission. Completion of data collection for 2004 joint research project.

(2006): 2004 joint project data analysis, presentation and publication submission.

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

All of the members of NE-112 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 NE-112 members in recent years. Future plans include similar publications. In addition, the NE-112 and Mastitis Research Workers have established a home page on the internet (http://w3.aces.uiuc.edu/AnSci/USDA/NE-112) to increase our availability to the public. 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 of the nation.

Organization/Governance

Administrative Advisor: K. M. Kerr USDA/CSREES Representative: D.L. Morris Technical Committee: * Official representative coordinating the research, 1 New stations Connecticut S. Andrew*, L. Hinckley Illinois D. E. Morin*, W. L. Hurley Indiana M. Schutz* Iowa L. L. Timms*, M. Nilsen-Hamilton Kansas J. Sargeant*, J. Roberson Louisiana W. E. Owens* Michigan R. S. Erskine*, P. Sears, J. Burton Minnesota1 S. Godden* New York R. N. Gonzalez*, D. Wilson, Y.H. Schukken Ohio K. L. Smith*, J. Hogan Pennsylvania L. M. Sordillo*, R. L. Vallejo Tennessee S. Oliver*, G. Pighetti Vermont A. J. Bramley*, D. Kerr, J. Barlow Virginia E. Hovingh* Washington L. K. Fox*, W.C. Davis Wisconsin1 P. Ruegg* International members: Guelph1 K. E. Leslie*, D.F. Kelton 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 chairman. These reports will be compiled and sent to each participant prior to the annual meeting. The responsibility for multistate 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 NE-112 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

Objective 1 Barrett, J.J., J.S. Hogan, W.P. Weiss, K.L. Smith, and L.S. Sordillo. 1997. Alpha-tocopherol concentrations after intramammary infusion of Escherichia coli or lipopolysaccaride. J. Dairy Sci. 80:2826-2832. Boddie, R.L., and S.C. Nickerson. 1997a. Evaluation of two iodophor teat germicides: Activity against Staphylococcus aureus and Streptococcus agalactiae. J. Dairy Sci. 80:1846-1850. Boddie, R.L., and S.C. Nickerson. 1997b. Efficacies of teat germicides containing 0.5% chlorhexidine and 1% iodine during experimental challenge with Staphylococcus aureus and Streptococcus agalactiae. J. Dairy Sci. 80:2809-2814. Boddie, R.L., S.C. Nickerson, R.W. Adkinson. 1998. Germicidal activity of a chlorous acid-chlorine dioxide teat dip and a sodium chlorite teat dip during experimental challenge with Staphylococcus aureus and Streptococcus agalactiae. J. Dairy Sci. 81: 2293-2298. Boddie, R.L., S.C. Nickerson, R.W. Adkinson. 2000. Efficacies of chlorine dioxide and iodophor teat dips during experimental challenge with Staphylococcus aureus and Streptococcus agalactiae. J. Dairy Sci. 83:2975-2979. Burton, J.L., P.S. D. Weber, J.B. Wells, S.A. Madsen, J. Yao, P.M. Coussens. 2001. Immunogenomics approaches to understanding periparturient mastitis susceptibility in dairy cows. Acta Vet. Scand. 42 (in press). Dietz, A.B., N.D. Cohen, L.L. Timms, M.E. Kehrli. 1997. Bovine lymphocyte antigen class II allelles as risk factors for high somatic cell counts in milk of lactating dairy cows. J. Dairy Sci. 80:406. Fang, W. and S. P. Oliver. 1999. Identification of lactoferrin-binding proteins in bovine mastitis-causing Streptococcus uberis. FEMS Microbiol. Lett. 176:91-96. Gillespie, B.E., B.M. Jayarao, H.H. Dowlen, S.P. Oliver. 1999. Analysis and frequency of bovine lymphocyte antigen DRB3.2 alleles in Jersey cows. J. Dairy Sci. 82:2049-2053. Kehrli, M.E., Jr, and J.A. Harp. 2001. Immunity in the mammary gland. Vet. Clin. North. Am. Food Anim. Pract. 17:495-516. Liu Q, J. Ryon, M. Nilsen-Hamilton. 1997. Uterocalin, a mouse acute phase protein expressed in the uterus around birth. Molecular Reproduction and Development 46, 507-514. Madsen, S.A., P.S.D. Weber, and J.L. Burton. 2001. Altered blood neutrophil gene expression in periparturient dairy cows. Vet. Immunol. Immunopathol. (accepted). Maunsell, F.P., D.E. Morin, P.D. Constable, W.L. Hurley, G.C. McCoy, I. Kakoma, R.E. Isaacson. 1998. Effects of mastitis on volume and composition of colostrum produced by Holstein cows. J. Dairy Sci. 81:1291-1299. Oliver, S.P., M.J. Lewis, B.E. Gillespie, S. Ivey, L. Coleman, R.A. Almeida, W. Fang, K. Lamar. 1999. Evaluation of a postmilking teat disinfectant containing phenol for the prevention of bovine mastitis during lactation. J. Food Prot. 62:1354-1357. Oliver, S.P., B.E. Gillespie, M.J. Lewis, S.J. Ivey, R.A. Almeida, W. Fang, D.A. Luther, D.L. Johnson, K.C. Lamar, H. Moorehead, H.H. Dowlen. 2001. Efficacy of a premilking teat disinfectant containing a phenolic combination for the prevention of mastitis in lactating dairy cows. J. Dairy Sci. 84:1407-1412. Lin, J., J.S. Hogan, K.L. Smith. 1998a. Inhibition of in vitro growth of coliform bacteria by a monoclonal antibody directed against ferric enterobactin receptor FepA. J. Dairy Sci. 81:1267-1274. Lin, J., J.S. Hogan, M. Aslam, K.L. Smith. 1998b. Immunization of cows with ferric enterobactin receptor from coliform bacteria. J. Dairy Sci. 81:2151-2158. Lin, J., J.S. Hogan, K.L. Smith. 1999a. Growth responses of coliform bacteria to purified immunoglobulin G from cows immunized with ferric enterobactin receptor FepA. J. Dairy Sci. 82:86-89. Lin, J., J.S. Hogan, K.L. Smith. 1999b. Antigenic homology of the inducible ferric citrate receptor (FecA) of coliform bacteria isolated from herds with naturally occurring bovine intramammary infections. J. Clinic. Diagnost. Lab. Immunol. 6:966-972. Pighetti, G.M., M.L. Eskew, C.C. Reddy, L.M. Sordillo. 1998. Selenium and vitamin E deficiency impairs transferrin receptor internalization but not interleukin 2, interleukin 2 receptor, or transferrin receptor expression. J Leuk Biol. 63: 131. Sharif, S., B.A. Mallard, J.M. Sargeant. 2000. Presence of glutamine at position 74 of pocket 4 in the BoLA-DR antigen binding groove is associated with occurrence of clinical mastitis caused by Staphylococcus species. Vet. Immunology and Immunopathology. 76: 231-238. Timms, L.L., 2001. Field trial evaluation of a persistent barrier teat dip for preventing dry period mastitis and as a potential alternative / adjunct to dry cow antibiotic therapy. J. Dairy Sci.84 (Suppl. 1 MW-ADSA):67. Timms, L.L., M.A. Faust, M.E. Kehrli. 1998. Characterizing of winter teat end lesions for lactating dairy cows. J. Dairy Sci. 81(4):1196. Yao, J., J.L. Burton, P. Saama, S. Sipkovsky, P.M. Coussens. 2001. Generation of EST and cDNA Microarray Resources for the Study of Bovine Immunobiology. 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Oliver. 1998. Factors influencing adherence of Streptococcus dysgalactiae to bovine mammary epithelial cell monolayers. Zentralbl Veterinarmed [B]. 45:161-70. Cifrian, E., A.J. Guidry, C.N. Hambleton, S.C. Nickerson, W.M. Marquardt. 1994. Staphylococcus aureus adherence to cultured bovine mammary epithelial cells. J. Dairy Sci. 77:970. De Oliveira A.P., J.L. Watts, S.A. Salmon, F.M. Aarestrup. 2001. Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Europe and the United States. J Dairy Sci. 83:855-62. Dopfer D, R.A. Almeida, T.J. Lam, H. Nederbragt, S.P. Oliver, W. Gaastra. 2000. Adhesion and invasion of Escherichia coli from single and recurrent clinical cases of bovine mastitis in vitro. Vet Microbiol. 74:331-43. Dopfer D, H. Nederbragt, R.A. Almeida, W. Gaastra. 2001. Studies about the mechanism of internalization by mammary epithelial cells of Escherichia coli isolated from persistent bovine mastitis. Vet Microbiol. 80:285-96. Ebling T.L., L.K. Fox, K.W. Bayles, G.A. Bohach, K.M. Byrne, W.C. Davis, W.A. Ferens, J.K. Hillers. 2001. Bovine mammary immune response to an experimental intramammary infection with a Staphylococcus aureus strain containing a gene for staphylococcal enterotoxin C1. J Dairy Sci. 84:2044-50. Ferens W.A., W.L. Goff , W.C. Davis, L.K. Fox, C. Deobald, M.J. Hamilton, G.A. Bohach. 1998. Induction of type 2 cytokines by a staphylococcal enterotoxin superantigen. J Nat Toxins. 3:193-213. Ferens WA, and G.A. Bohach. 2000. Persistence of Staphylococcus aureus on mucosal membranes: superantigens and internalization by host cells. J Lab Clin Med. 135:225-30. Review. Fitzgerald J.R., S.R. Monday, T.J. Foster, G.A. Bohach, P.J. Hartigan, W.J. Meaney, C.J. Smyth. 2001. Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens. J Bacteriol. 183:63-70. Garrison, L.L., Y.H. Schukken, B. Hilton. 2000. Antibiotic Susceptibility Patterns for Various Bacterial Intramammary Pathogens Over 15 Years: Results and Analysis of a Database., Proc 39th annual meeting of Nat Mastitis Council, Atlanta, GA pages 215-216. Gilbert F.B., D.A. Luther, S.P. Oliver. 1997. Induction of surface-associated proteins of Streptococcus uberis by cultivation with extracellular matrix components and bovine mammary epithelial cells. FEMS Microbiol Lett. 156:161-4. Guidry A, A. Fattom, A. Patel, C. O'Brien, S. Shepherd, J. Lohuis. 1998. Serotyping scheme for Staphylococcus aureus isolated from cows with mastitis. Am. J. Vet. Res. 59:1537-9. Kim CH, Khan M, Morin DE, Hurley WL, Tripathy DN, Kehrli M, Oluoch AO, Kakoma I. Optimization of the PCR for detection of Staphylococcus aureus nuc gene in bovine milk. J Dairy Sci 2001 Jan 84:1 74-83. Lee S.U., M. Quesnell, L.K. Fox, J.W. Yoon, Y.H. Park, W.C. Davis, D. Falk, C.F. Deobald, G.A. Bohach. 1998. 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Application of pulsed-field gel electrophoresis and binary typing as tools in veterinary clinical microbiology and molecular epidemiologic analysis of bovine and human Staphylococcus aureus isolates. J Clin Microbiol 38:1931-9 Zadoks, R.N. 2002. Dynamics of intramammary infections of S.aureus and S.uberis in dairy herds. PhD. Thesis, University of Utrecht. Objective 3: Andrew, S. M. 2000. Effect of fat and protein content of milk from individual cows on the specificity rates of antibiotic residues screening tests . J. Dairy Sci. 83:2992-2997. Andrew, S.M. 2001 . Effect of composition of colostrum and transition milk from Holstein heifers on the specificity rates of antibiotic residue tests. J Dairy Sci. 2001 84:100-6. Cattell, M.B. 1996. An outbreak of Streptococcus uberis as a consequence of adopting a protocol of no antibiotic therapy for clinical mastitis. Proc. 35th Annual Meeting of the National Mastitis Council, pp. 123-127. Cullor, J.S., A. van Eenennaam, I. Gardner, L. 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Attachments

Land Grant Participating States/Institutions

CT, IA, IL, KS, LA, MI, MN, MO, NY, OH, PA, TN, UT, VA, VT, WA

Non Land Grant Participating States/Institutions

University of Glasgow