Brief Summary of Minutes of Annual Meeting

COMPLETED RESEARCH OBJECTIVE 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance. GUELPH, ONTARIO: A collaborative trial is in the analysis phase to examine the agreement among duplicate samples and the error associated with taking a single sample to diagnosis an intramammary infection. IOWA: 1) The mRNA sequence for bovine Uterocalin (Utc/lcn2) was identified in a cDNA library (Jeanne Burton, Michigan State) and cDNA was subcloned into a bacterial expression vector and the protein expressed. The protein was found to be present in two forms, which appear to be monomer and dimer. 2) Data analysis from a collaborative study (Guelph, PEI, NY, IA) evaluating cow and quarter factors associated with new dry period IMI and the role of external dry cow barrier sealant in teat closure and/ or IMI prevention was completed at PEI. KANSAS: An in vitro bovine mammary epithelial system (BME-UV cells) has been further characterized as an outstanding and cost-effective model to assess modulation of the epithelial barrier by both host and pathogens. There is a rapid redistribution of epithelial cell proteins, especially occludin, that are associated with the epithelial barrier following exposure to elevated luminal (i.e., milk) salt concentrations. Natural or synthetic corticosteroids can regulate the expression of mammary epithelial salt transport mechanisms within less than 4 hours. These mechanisms can be targeted for prophylactic or therapeutic interventions. MICHIGAN: We have cloned and proliferated in culture Green Fluorescent Protein (GFP) Escherichia coli and Klebsiella oxytoca for use with in vitro phagocyte studies. We have determined that both PMN and macrophage phagocytosis was enhanced in the presence of serum collected from steers hyperimmunized with J5 bacterin. NEW YORK: A number of projects are currently being done under this objective. This includes the host response mechanisms to chronic coliform infections and the immune response to acute transient coliform infections. A PhD thesis will be completed on this in late 2005. Several publications are planned out of this thesis. We perform micro array evaluation of chronic and transient E. coli strains. OHIO: Neutrophil function and inflammatory responses did not differ between early lactation cows fed diets with either selenate or selenized yeast. TENNESSEE: During this past year, we enhanced the accuracy of a genetic marker for mastitis by combining three polymorphisms within the CXCR2 gene. Not only will this potentially aid in developing marker-assisted selection strategies, but also provides a model system for understanding why certain cows are genetically more susceptible to mastitis. A possible explanation may be related to the reduction in neutrophil migration and survival which also was observed in cows genetically identified to be more susceptible to mastitis. Subsequent studies have suggested that the causal defect in neutrophil function may be downstream of the CXCR2 receptor. WASHINGTON: Staphylococci that produce enterotoxin C are rare, less than 1 in 300 isolates. However, those that do produce this enterotoxin appear to cause an acute case of mastitis in 8 cows tested. OBJECTIVE 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defense. GUELPH, ONTARIO: Work has been completed on describing the influence of Staphylococcus aureus genotype on dry period cure, and has progressed and continues to look at controlling S. aureus with bacteriophage. NEW YORK: Research in New York is focused around antimicrobial resistance and virulence factors of S. uberis infections. Antimicrobial resistance of S. aureus, S. agalactiae and E. coli is being investigated. Virulence factors of S. uberis that distinguish isolates causing chronic and transient infections are being investigated. TENNESSEE: Research has continued to focus on the interaction a prevalent mastitis pathogen, Streptococcus uberis with host defenses. Recent studies suggest that S. uberis may avoid intracellular bactericidal mechanisms by binding lactoferrin and entering mammary epithelial cells through a caveloae-dependent endocytic pathway. Additional studies have focused on the identification, isolation, characterization, and recombinant expression of a novel S. uberis adhesion molecule (SUAM). Using bioinformatics analysis, this protein is predicted to be a fibrillar, surface-associated protein which should generate an immune response. As this protein also is expressed by geographically diverse strains of S. uberis, it has strong potential as a vaccine candidate to prevent mastitis. WASHINGTON: Mycoplasma strains that cause mastitis also can be found at other body sites as determined by chromosomal digest technology. In a study of an outbreak of mycoplasma mastitis in a dairy herd, colonization of other body sites with the mastitis strain was quite common immediately after the outbreak, but very rare within 6 mos. after the outbreak. OBJECTIVE 3: Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. CONNECTICUT: 1) An 8-University collaborative NE-1009 project was headed by Washington State University to determine the efficacy of prepartum intramammary antibiotic therapy of primiparous heifers on reducing new intramammary infections in early lactation. The results have been summarized by Washington State University. 2) A study was conducted to determine the risk for antibiotic residues occurring in milk resulting from prepartum antibiotic therapy in primiparous heifers. Heifers (n=136) treated with a commercial ²-Lactam intramammary (IM) antibiotic preparation containing cephapirin sodium at 10 to 21 days prior to anticipated parturition resulted in antibiotic residues detected in milk in 28.0, 8.82, and 3.68 percent of heifers in the third, sixth, and tenth milkings following parturition, respectively. An increase in interval between treatment and parturition and milking number postpartum were associated with a decrease in risk of antibiotic residues. Increasing the interval between prepartum IM antibiotic treatment and parturition and testing for antibiotic residues in milk postpartum will reduce the risk of antibiotic contamination resulting from this treatment. 3) A study was performed to determine if plasma concentrations of lipid metabolites, lipid-soluble vitamins, and measures of tissue energy status differed for Holstein and Jersey cows that were selected for new intramammary infection (new IMI) status and to determine factors associated with the risk for developing a new IMI during the transition period. Using a subset of cows from a larger study, 10 Holstein and 10 Jersey multiparous cows, of which 5 Holstein and 4 Jersey cows had developed a new IMI, were monitored from dry-off (wk -9) through 8 wk postpartum. Holstein and Jersey cows differed in plasma concentrations of several lipid metabolites and lipid-soluble vitamins. However, an increase in prepartum body condition score similarly increased the risk for developing a new IMI postpartum for both breeds. Cows that had developed a new IMI had a significant delay in recovery of postpartum plasma ²-carotene to prepartum levels compared to cows which did not develop a new IMI. CALIFORNIA: Progress towards objective 3 has been in the areas of the enhancement of dairy food safety through 1.) characterization of antibiotic resistant bacteria, 2.) new approaches to rapid screening of milk samples, including somatic cell counting, using DNA sensor methodology, and MAP (the Johnes agent) detection using real time PCR and a modified culture method, and 3.) development of new technologies to disinfect milk and dairy wastewater using radio frequency waves. GUELPH, ONTARIO: University of Guelph has evaluated cow-side direct cell counters, and continues to explore possibilities to institute practical on-farm culture into mastitis treatment protocols. Recent teat dip trials have completed as well. IOWA: 1) Prepartum treatment of heifers with pirlimycin (1 tube/ quarter ~ 10 days prepartum) significantly reduced postpartum IMI (particularly CNS), showed a trend for increased milk production (p = .08), and showed no pirlimycin residues (measured by Delvo) at 1-2 days postpartum. 2) Teat dipping in winter with chlorous acid, iodine, and a non iodine barrier teat dips showed no significant differences in teat end health unless milkers failed to fully remove the chlorous acid barrier dip. Teat skin condition was significantly decreased with the non-iodine product. LOUISIANA: Studies on enhancement of recovery of pathogens from milk samples demonstrated that increased volume to 1 ml was most effective. In vitro evaluation of two chlorhexidine teat dips demonstrated >99.9% kill for both products for all organisms tested except for Staph. aureus. MICHIGAN: We will continue a field trial that is to determine the effect of J5 hyperimmunization on the incidence of severe clinical mastitis in lactating cows. Additionally, we are continuing a study of to determine the effect of culture-based therapy on success of antibacterial treatment for clinical mastitis as compared to empirical therapy MINNESOTA: 1) Clinical trial showed that pre-partum intramammary antibiotic treatment of cows and heifers resulted in no improvement in early lactation SCC, DHIA SCC through 15 weeks, or daily milk production through 15 weeks, as compared to controls. Initiated a multistate multifarm study to describe the efficacy and cost benefit of implementing on-farm culture tools for diagnosis and treatment of clinical mastitis and subclinical mastitis in fresh cows (Minnesota, Wisconsin, Iowa, Ontario). MISSOURI: 1) We have developed an antibody capture ELISA for the detection of anti-S. aureus antibody isotypes (total IgG, IgG1, IgG2, and IgM) in which microtiter plates are coated with antigen of S. aureus capsular serotypes 5, 8 and 336 suspended in phosphate buffered saline at pH 7.2. 2) We have used the aforementioned ELISA to evaluate the antibody response in milk and serum of dairy heifers vaccinated with Lysigin (BIVI, St. Joseph, MO) and cattle challenged with S. aureus by intramammary infusion. 3) We have used a similar ELISA to evaluate humoral immunity (IgG1, IgG2, IgA, and IgM) in milk of mature cattle vaccinated with Lysigin. 4) We have evaluated the efficacy of pre-partum intramammary treatment of dairy heifers with pirlimycin hydrochloride in reducing the prevalence of intramammary infection (IMI), lowering the somatic cell count (SCC) during early lactation, and improving 305-day mature equivalent milk production in cooperation with Iowa station (Middleton, Timms et al., 2005). NEW YORK: Food safety research includes work on Coxiella burnetti, Salmonella and Listeria infections in dairy herds. A recent outbreak of Coxiella burnetti is followed and will be published in the next reporting year. OHIO: Bacterial counts did not differ between fresh sand and recycled sand used as lactating cow bedding during winter and summer months. TENNESSEE: A survey of human and animal samples indicated that at least half of the 35 Escherichia coli O26 isolates were resistant phenotypically to ampicillin, streptomycin, tetracycline, and sulfonamides and carried relevant antibiotic resistant genes. Continued surveillance of emerging antimicrobial resistance among zoonotic foodborne pathogens, including E. coli O26, is necessary to ensure public health. A separate study evaluated whether Enterobacteriaceae obtained from the soil of dairy farms could serve as a source of antibiotic resistance genes. Of 81 samples, approximately 59% carried one or more tetracycline resistance genes, while only 15% carried one or more streptomycin resistance genes, thereby indicating these bacteria could serve as a source of antimicrobial resistance. WASHINGTON: Prepartum intramammary treatment of heifers with commercial lactating therapy did reduce intramammary infections at parturition (mostly CNS IMI), but did not lead to improved milk or reproductive performance, nor lowered SCC during the first 200 DIM. However, cures, a combination of treatment and spontaneous, was associated with a significant reduction in heifer SCC postpartum. WORK IN PROGRESS OBJECTIVE 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance. CALIFORNIA: To investigate the cellular immune response in naturally occurring cases of mastitis during the drying-off period via flow cytometry of lymphocyte sub-populations and mRNA cytokine expression. IOWA: 1) Studies are currently in progress to determine the nature of the uterocalin forms and whether they bind bacterial siderophores as do mouse and human proteins. 2) Publish 2nd dry period mastitis prevention / teat closure study (PEI, Guelph, NY, IA). KANSAS: Regulated expression of the epithelial Na+ channel, ENaC, plays a key role in establishing, maintaining, or re-establishing appropriate milk electrolyte concentrations. This channel has three independently-synthesized subunits that are required for full functionality. In the coming year, this station will focus on determining extracellular and intracellular mediators that modulate the expression of each ENaC subunit. MICHIGAN: Preliminary data suggests that six immunizations with J5 bacterin decreases the incidence of severe mastitis in lactating cows from three to five months in lactation, as compared to three immunizations. Cows that were hyperimmunized (6x) had significantly higher levels of anti-J5 E. coli IgG2 antibodies in serum. NEW YORK: Work in New York will continue on the host response to chronic and transient coliform infections. Chronic and transient E. coli strains will be further characterized. The response of the host to these two types of strains will be investigated in vitro and in vivo OHIO: Challenge trials continue to determine the association of stage of lactation with susceptibility of cows to intramammary infections and clinical mastitis caused by Enterococcus spp. TENNESSEE: Validate the 3 polymorphism combination in the CXCR2 gene in a larger population, potentially in a sire based design. Further delineation of the mechanisms associated with impaired neutrophil function and disease susceptibility in cows with specific CXCR2 genotypes by evaluating gene structure, receptor expression, and signal transduction. WASHINGTON: Determine if a vaccine using modified Staphylococcal Enterotoxin C can be used to protect against S. aureus mastitis. OBJECTIVE 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defense. MISSOURI: We are completing a project to determine differences in in vitro and in vivo susceptibility of different bovine mammary Staphylococcus aureus strain-types to pirlimycin HCl. NEW YORK: Work in New York will continue on antimicrobial resistance of the most important mastitis pathogens. We plan to complete a number of publication on antimicrobial resistance. Work on pathogenicity characteristics of S. uberis will continue. Phylogenetic research into S. uberis will be started. TENNESSEE: Identify the mechanisms by which S. uberis subverts the intracellular processing pathway and is able to persist inside the cell. Evaluate the efficacy of a vaccine against the SUAM protein. WASHINGTON: Continue to follow the dynamics of mycoplasma mastitis, and body site colonization in the outbreak herd. Develop technique to speciate mycoplasma isolates from milk using PCR technology. OBJECTIVE 3: Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. CONNECTICUT: 1) Evaluation of antibiotic residue screening tests using colostrum and transition milk from primiparous cows will continue. The sensitivity, specificity and positive predictive value of four commonly used antibiotic residue screening tests will be evaluated. 2) In order to increase the ability to detect a significant difference, an additional 10 cows (5 Holstein and 5 Jersey cows) will be added to the study to determine the if plasma concentrations of lipid metabolites and lipid-soluble vitamins differed for Holstein and Jersey cows that were selected for new intramammary infection status and to determine factors affecting the risk for developing a new IMI during the transition period. 3) The effect of a shortened dry period on the risk for developing a new intramammary infection postpartum and risk for antibiotic residues in milk to occur in the early subsequent lactation will be determined in two large commercial dairy herds. CALIFORNIA: Re-evaluate the efficacy of milk pasteurization against M. paratuberculosis (Johnes agent) by defining d and z values in an industrial pasteurizer holding tube. Develop a rapid milk bot tox test kit in collaboration with national defense laboratories. Screen dairy milk filters for M. parartuberculosis with a rapid real time PCR assay. GUELPH, ONTARIO: In the coming year, control of S. aureus in the dry period will be explored with novel treatments. As well, on-farm evaluations of direct SCC tools will continue and on-farm culture of clinical mastitis (in collaboration with Iowa & Minnesota) will be studied. Finally, the impact of milk temperature and water quality on Ontario bulk milk quality will be studied. IOWA: 1) Complete analyses of winter teat dip projects, including impacts of chlorous acid dips on decreasing hyperkeratosis. 2) Complete small field trial evaluating CMT and /or on farm culture systems for strategic treatment of fresh cow and clinical mastitis. LOUISIANA: Evaluation of conventional teat dips will continue. Determination of efficacy of therapy for mastitis caused by environmental streptococci is in progress. Evaluation of CNS in heifers will continue. The Mycoplasma mastitis control program in Louisiana will continue. An evaluation of a novel non-antibiotic therapeutic is scheduled. MINNESOTA: 1) Continue a multistate multifarm study to describe the efficacy and cost benefit of implementing on-farm culture tools for diagnosis and treatment of clinical mastitis and subclinical mastitis in fresh cows. Additional objectives include validating an on-farm culture system and describing the relationship between antimicrobial susceptibility patterns and response to antibiotic therapy for clinical and subclinical mastitis cases (Minnesota, Wisconsin, Iowa, Ontario). 2) Complete a field study to apply and refine a microbiological monitoring procedure (bulk tank culture) that can be used as an indicator of increased risk for udder infection, and to develop and implement an early warning system (to be used at the dairy plant level) which will monitor current control programs identifying herd risk factors in need of early interventions to prevent mastitis flare ups. 3) Complete work evaluating the genetic relatedness of Klebsiella pneumonia isolated from clinical mastitis, bedding material and bulk tank milk. MISSOURI: We have a project underway to evaluate the influence of species and genotype of coagulase negative staphylococci on mammary gland inflammation. We will be further evaluating mammary humoral immunity to vaccination with Lysigin. NEW YORK: Further quantification of the milk flow curve and the equipment washing procedures will be studied in New York. We will continue our collaborative research on Salmonella and Listeria infections on Dairy farms. A special focus for 2006 will be on Coxiella Burnetti. TENNESSEE: Identify sources of antimicrobial resistance genes on farming operations WASHINGTON: Complete work on prepartum heifer intramammary therapy.