Brief Summary of Minutes of Annual Meeting

NE-1009 ANNUAL REPORT - 2006 COMPLETED RESEARCH OBJECTIVE 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance. CONNECTICUT In a retrospective study, associations were determined among indices of negative energy balance, plasma lipid and lipid-soluble vitamin concentrations, plasma acute phase protein status, and occurrence of new intramammary infection (IMI) during the periparturient period for 15 Holstein and 15 Jersey dairy cows. Jersey cows without a new IMI had greater plasma ²-carotene at wk 9 compared to Jersey cows with a new IMI and Holstein cows regardless of IMI status. Holstein and Jersey cows with greater tissue energy stores prepartum and reduced plasma proteins and ±-tocopherol had a greater risk for developing a new IMI during the periparturient period. ILLINOIS 12 Holstein cows in mid-lactation were fed an ad libitum or restricted diet to determine the effect of negative energy balance on gene expression in mammary tissue during intramammary infection. Cows were inoculated with Streptococcus uberis 0140J in one rear quarter. A variety of immune function and energy parameters were measured in milk and blood. Eighteen hours after inoculation, the inoculated mammary quarter and the non-inoculated rear (control) gland were biopsied. Gene expression events in mammary tissue and in neutrophils will be characterized using a bovine-specific high-density microarray platform. IOWA 1) The uterocalin (Lcn2) protein was expressed in bacteria and purified. This protein when injected into rabbits yielded excellent antibodies that detected bovine Lcn2. 2) Lcn2-knockout animals were bred against a Balb/c background, and results showed higher cytokine response to LPS in lung and liver of the KO compared with the wild-type mice. 3) The Lcn2 promoter was cloned and a truncation series prepared. The series was inserted upstream from the Luciferase gene and tested in HC11 and Eph4 mammary epithelial cells to identify the transcription elements that are important for basal expression in these cells. Two transcription elements were found to be important for expression: NFºB amd C/EBP. 4) 45% of 120 Ghanaian women had subclinical mastitis as defined by a sodium:potassium ratio > 1. MINNESOTA The level of genetic diversity of K. pneumoniae isolated among dairy herds from clinical mastitis cases was described and the genotypes most commonly associated with disease were identified. NEW YORK We studied the characteristics of chronic coliform mastitis isolates in comparison to isolates from transient infections. We evaluated the use of SCC diagnostic assays for on-farm use. Klebsiella infections were studied to further understand the within host and within farm dynamics of this infection. TENNESSEE Clearance of bacterial infections such as mastitis often depends upon the effectiveness of neutrophils.This white blood cell is activated and recruited to the infection site by interleukin-8. This small peptide binds two receptors in most species: CXCR1 and CXCR2. Our research has identified the correct annotation of the bovine CXCR1 gene and determined the CXCR2 coding sequence which was previously unavailable. Recent research also has determined that prior association of polymorphisms in the CXCR1 sequence with impaired neutrophil function are partly tied to defects in receptor binding of interleukin-8, as well as altered signaling pathways downstream of the receptor. VERMONT The genomic response of the bovine mammary gland to intramammary LPS challenge was characterized in vivo. Many genes associated with innate immunity were markedly induced within four hours. Genomic response of mammary epithelial cells in culture to LPS was subsequently characterized. Differences between these model systems are now being evaluated. WASHINGTON The Staphylococcal toxin C1 (SEC1) was modified by deleting amino acid sequence 94 to 106 and combining an amino acid sequence at the deleted portion to produce modified toxin acting as a vaccine, which forms antibodies for humoral immune responses as well as elicits non-specific cellular immune responses to protect against superantigenic properties of SEC1. Cows (n=40) vaccinated against this modified toxin had different T cell (CD4/CD8) responses when compared to controls, and responses appear to be dose and time dependent. OBJECTIVE 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defense. MISSOURI A study was completed evaluating the in vivo susceptibility of different bovine mammary Staphylococcus aureus strain-types to two treatment protocols. One strain of S. aureus predominated and that strain had an overall lower cure rate than the next most prevalent strain. A study is currently underway to evaluate the influence of coagulase negative staphylococcal species and genotype on elevated milk somatic cell count. NEW YORK Worldwide S.uberis isolates were studied, comparative genomics were used to identify host adapted and non host adapted isolates. Within host dynamics of E.coli infections were studied using mathematical modeling. ONTARIO Work was performed to characterize SCV (small colony variant) Staphylococcus aureus. A SCV phenotype was isolated and demonstrated to survive longer without experiencing cell damage in bovine aortic endothelial cells than Newbould 305. These results suggest that SCV may be an important contributor to better understand the pathogenesis of S. aureus mastitis and report for the first time the isolation of S. aureus SCV from persistent bovine mastitis. PENNSYLVANIA Isolation and Characterization of Bovine class II MHC-Bound Microbial Peptides from Cows with High Somatic Cell Counts: A Proteomics-based Approach to Identifying Naturally Processed Microbial Peptides. It is our hypothesis that screening quarter milk samples from cows with mastitis could reveal a vast array of uncharacterized bacterial antigens of both contagious and environmental mastitis pathogens. This investigation will result in better understanding of the control and expression of diagnostically relevant antigens of bovine mastitis pathogens. TENNESSEE Streptococcus uberis is an important cause of mastitis in dairy cattle throughout the world. Research has demonstrated that lactoferrin, a component of milk, serves as a bridging molecule between S. uberis adhesion molecule (SUAM) and mammary epithelial cells. A S. uberis mutant library also was generated to serve as a tool to identify other pathogenic factors. A retrospective comparison of 96 S. uberis isolates collected over the past 16 years determined that antimicrobial susceptibility patterns were similar over time. Studies also were initiated to determine the prevalence and source of coagulase-negative Staphylococcus species isolated from the dairy environment. A separate study also indicated the presence of at least one enterotoxin or toxic shock syndrome gene in most (88.5%) Staphylococcus aureus isolates from milk of cows with mastitis. WASHIINGTON 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 mycoplamsa mastitis in a dairy herd, colonization of other body sites with the mastitis strain was quite common immediately after the outbreak, with nearly half of all livestock (cows and replacements) were shedding the pathogen. But, within 3 months after the outbreak less than 3% of livestock were shedding the clone of mycoplasma that caused the initial outbreak. WISCONSIN We completed a study that characterized antimicrobial resistance of Gram-positive mastitis pathogens recovered from 20 Wisconsin dairy farms. We developed a method to quantify antimicrobial exposure and compared the relationship between antimicrobial exposures and antimicrobial resistance. For several antimicrobials used on dairy farms we identified a dose-response relationship between antimicrobial exposures and resistance but for other mastitis pathogens no relationship was found. In a separate study, we evaluated antimicrobial resistance before and after exposure to a selected intramammary antimicrobial. OBJECTIVE 3: Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. CONNECTICUT The effect of an acidic clay-based bedding additive for sawdust on teat end environmental pathogen load was evaluated using sixteen mid-lactation Holstein dairy cows in a 21-day cross-over two-period design. The bedding conditioner reduced environmental bacteria counts in bedding and on teat ends during period 2 when teat and bedding cleanliness was reduced and environmental humidity was increased. ILLINOIS Six Holstein cows were used in a 3-way cross-over study to determine the influence of milking frequency and timing of intramammary antibiotic administration on milk antibiotic concentrations. Cows were milked twice (2X) or 3 times (3X) daily and cephapirin administered into one rear mammary gland after the first and second or first and third milking of the day. Milk yield from the treated gland was measured and samples submitted for quantification of cephapirin and desacetylcephapirin concentrations by HPLC-MS-MS. Milk antibiotic concentrations will be compared with published MIC50 and MIC90 values for common mastitis pathogens. Time to achieve the regulatory tolerance concentration for cephapirin in milk will also be determined. IOWA 1) An on-farm culture system (and CMT at calving for initial screening) was used to delay treatment decisions for 24 hrs until culture results were obtained for clinical and fresh cow subclinical mastitis, respectively. Using these systems (delay treatment 24 hrs.) resulted in a 50 and 67% reduction in treatments compared to controls treated initially (clinical or CMT + at calving), respectively, and 50% of controls showed no growth on culture. Cure rates within treatments were similar. Partial budgets showed a 67% saving with clinical mastitis while fresh cow subclinical groups were economically equivocal due to the added costs of discarded milk by waiting 24 hrs to treat. MINNESOTA In a study to evaluate the test characteristics of two assays (Kirby-Bauer and Mastik test) to determine antimicrobial susceptibility of mastitis pathogens, as compared to the Sensititre MIC test method (Trek Diagnostics), preliminary results suggest that the Kirby-Bauer test has poor test performance, but the Mastik test has reasonably good test performance, as compared to the Sensititre test. Final analysis is being completed. Cow enrollment continued in an ongoing 3-year multistate multiform study to describe the efficacy and costbenefit of implementing on-farm culture tools for diagnosis and strategic treatment of clinical mastitis and subclinical mastitis fresh cows (MN, WI, Ontario). Study showed that the ability of various bedding materials to support environmental bacteria growth, ranked in order from least (or no growth) to most growth supported, included i) new sand, ii) shavings, iii) recycled sand, and iv) digested manure solids. Laboratory analysis of bedding materials for pH and organic material content is ongoing. MISSOURI A study was performed using a modified commercial enzyme-linked immunosorbent assay (ELISA) to detect IgGA, IgG1, IgG2, and IgM against Staphylococcus aureus to characterize the milk antibody response to vaccination with a commercial S. aureus mastitis bacterin. Results showed that the modified ELISA was capable of detecting different antibody isotypes in milk of an infected cow, but no differences in milk antibody content were found between vaccinates and controls. NEW YORK Molecular epidemiology is being developed. Applications include new diagnostic tools, better understanding of pathogen epidemiology and treatment evaluation. Intramammary Coxiella infections were studied in great detail. ONTARIO Work was done to characterize the difference in infections between mastitis diagnosis made by single versus duplicate milk samples. There was large agreement between the two sampling strategies. Duplicate samples were more sensitive to identify species specific infections. Single quarter milk samples were comparable in sensitivity and specificity to identify truly negative quarters. The error associated with a single sample to correctly call a quarter uninfected was 7%. An inline SCC measurement device from New Zealand was assessed. The CellSense ® unit takes a spot sample of milk and mixes it with a reagent in line to measure the viscosity associated with an increased SCC. The CellSense® test characteristics indicate that it would be useful to identify cows for further action, such as laboratory milk culture. We also evaluated the method of infusion of Orbeseal ® to enhance recovery of the product at calving. There was no difference in recovery when either a rapid versus a slow and occluded method of infusion was used. PENNSYLVANIA Role of select viral etiological agents in lactating cows with high somatic cell counts The inability to identify the etiological agent(s) in milk samples with >200,000 somatic cells/ml is a dilemma, as this makes it difficult to treat infected cows and implement mastitis prevention and control practices in dairy herds. Historically, mastitis research has focused on bacterial pathogens, while viruses have received little attention. We have recently standardized and validated real time PCR assays for BHV-1, BHV-2, BHV-4, and BVDV viruses directly from raw milk. We are now studying several herds in Pennsylvania to determine the incidence of viral infection as a direct cause or predisposing factor for mastitis in dairy cows. Web-based software tool using expert systems for determining bulk tank milk quality The existing web-based software tool (http://www.vetsci.psu.edu/jayarao/) is being upgraded. The new version will permit the user to not only interpret bulk tank milk quality but also track and monitor milk quality over time. TENNESSEE Certain types of mastitis-causing bacteria can escape the immune system by surviving inside mammary epithelial cells. A new model system to evaluate the effectiveness of antibiotics in killing intracellular bacteria was developed and demonstrated the ability of penethamate hydriodide to kill intracellular mastitis pathogens without affecting mammary cell viability. A separate study evaluated the ability a synthetic protein, BTEA1, derived from a bactericidal protein, b-defensin, which is produced by mammary epithelial cells to kill veterinary and foodborne pathogens. This synthetic peptide was capable of killing several Streptocococcus species, Escherichia coli, Listeria monocytogenes, and certain strains of Salmonella when used at relatively low concentrations (20-40 mg/ml). VERMONT The direct and indirect effects of lactation therapy were evaluated using a mathematical modeling approach. Predictions from the model suggest there is an additional indirect benefit of lactation therapy for control of contagious pathogens that should be considered in assessment of the potential benefits of antimicrobial therapy of chronic subclinical infections during lactation. WASHINGTON Research studying the reduction of the length of the dry period has largely ignored the effects on udder health. Four herds have been enrolled where cows are allocated to either 30 day, or greater than 45 day, dry periods. Preliminary data suggests no untoward effects of a reduction in the dry period length on prevalence or rate of new intramammary infections. WISCONSIN We initiated a study that is evaluating the use of SPR technology to detect S. aureus and other mastitis pathogens. Field testing of a candidate test to rapidly differentiate Gram-positive and Gram-negative clinical mastitis pathogens was performed. WORK IN PROGRESS OBJECTIVE 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance. CONNECTICUT Two manuscripts are submitted to the Journal of Dairy Science. Submit a grant to the USDA-NRI to fund a study to evaluate nutritional protein status on immune function in periparturient dairy cows. ILLINOIS Conduct microarray analysis on mammary tissue from S. uberis-inoculated cows in positive and negative energy balance. Dr. Loor has received a USDA NRI grant to continue and expand upon this work. The new study will focus on the impact of dry period feeding strategies on early postpartum mastitis. Drs. Morin and Hurley are collaborators. IOWA 1) Determine levels of Lcn2 in the milk as a function of mastitis, identify the cell target of Lcn2 for cytokine production, and identify the promoter elements that are utilized for regulating Lcn2 expression during involution and in mastitis. (VT) 2) Analyze prevalence and longitudinal and cross sectional studies evaluating effect of subclinical mastitis on child growth and performance in Ghanaian women.(USDA) MINNESOTA Determine to what extent biological factors such as cows milk, (possibly mammary gland cells), fecal and bedding material affect the expression of fimbriae 1 gene (fimH). Complete work describing the level of genetic diversity of K. pneumoniae isolated from clinical mastitis cases and among dairy herds. MISSOURI A study is underway to investigate the relationship between bovine leukosis virus infection, persistent lymphocytosis, and chronic subclinical mastitis in dairy cattle. NEW YORK Further studies on chronic E.coli infections and the associated immune response will be conducted. OHIO An in vitro study was conducted to examine the growth of enterococcal isolates in mammary secretion from various stages of lactation. These results suggest the source of enterococcal isolates and the bacterial species significantly contribute to the ability of that organism to adapt to mammary secretion from various stages of lactation. TENNESSEE Identify the basis (causative mutation) for impaired intracellular signaling in cows with greater genetic susceptibility to mastitis and impaired neutrophil functions. Identify mammary epithelial and neutrophil based responses that contribute to pathogenesis of Streptococcus uberis and Escherichia coli. VERMONT Evaluate the genomic response of bovine milk cells to LPS challenge. Describe the direct and indirect effects of lactation therapy of subclinical mastitis caused by major gram-positive pathogens, comparison of a theoretical model to results of a prospective field trial. (Cornell). Describe impact of antimicrobial use on population transmission dynamics of major mastitis pathogens and antimicrobial resistance in dairy herds. (Cornell) WASHINGTON Determine if a vaccine using modified Staphylococcal Enterotoxin C can be used to protect against S. aureus mastitis. Sixty cows will be allocated to various vaccination groups and then challenged with intramammary instillations of S. aureus. OBJECTIVE 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defense. MISSOURI In the coming year we will continue to work on characterizing the relationship between coagulase negative staphylococcal species and genotype and milk somatic cell count. NEW YORK Klebsiella infections will be studied in more detail, particularly molecular genetics and antimicrobial resistance. OHIO An in vivo study was conducted to examine the physiologic effects of 4 E. faecium isolates in 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 secretion.. ONTARIO Work on Small colony variant S. aureus will be continuing PENNSYLVANIA Isolation and Characterization of Bovine class II MHC-Bound Microbial Peptides from Cows with High Somatic Cell Counts: A Proteomics-based Approach to Identifying Naturally Processed Microbial Peptides. TENNESSEE Identify virulence factors of Streptococcus uberis and Escherichia coli activated when in the presence of mammary epithelial cells and neutrophils. Phenotypic screening of Streptococcus uberis mutants for growth and virulence factors. Prevalence and sources of coagulase-negative staphylococci that cause mastitis. Evaluate the efficacy of a vaccine against the Streptococcus uberis adhesion molecule (SUAM). 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. WISCONSIN Further studies on the relationship between antimicrobial exposure and the development of resistance are planned with emphasis on identifying temporal relationship between exposure and changes in minimum inhibitory concentrations of antimicrobials. OBJECTIVE 3: Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. CONNECTICUT One manuscript is submitted to the Journal of Dairy Science. During a sabbatical leave, a mastitis-control program focused on control of Mycoplasma bovis will be developed. The major objectives of this project will be to identify farms with Mycoplasma bovis pathogens in milk and develop intervention strategies to eliminate/control the pathogen. Also, to incorporate components of mastitis control programs that have been successful in controlling the Mycoplasma bovis pathogen and finally, to conduct a milk quality workshop with presentations and demonstrations by nationally-recognized experts to train dairy farmers, veterinarians, and support personnel in the implementation of effective mastitis control program on farms. ILLINOIS Perform HPLC-MS-MS analysis on frozen milk samples and pharmacokinetic analysis on the data to determine the effect of milking frequency (2X vs 3X) and timing of antibiotic administration on the disposition of cephapirin and desacetylcephapirin in milk. Pursue additional studies using different antibiotics, a longer duration of antibiotic treatment, or cows with mastitis if results of this pilot study are significant. IOWA 1) Complete and analyze 2 trials evaluating the effects of 2 new teat dips on teat end and skin health and mastitis. 2) Initiate new trials to develop and/ or evaluate novel internal and external sealants for prevention of mastitis during the dry period and precalving. (U of Guelph) 3) Complete analysis of studies evaluating dairy tools for detection of subclinical mastitis in lactating women. MINNESOTA Conclude cow enrollment and begin data analysis/reporting of results for an ongoing 3-year multistate multiform study to describe the efficacy and costbenefit of implementing on-farm culture tools for diagnosis and strategic treatment of clinical mastitis and subclinical mastitis fresh cows. 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) (Kirby-Bauer) the Sensititre MIC test method (Trek Diagnostics). NEW YORK Development of bulk milk monitoring as it relates to animal health, milk quality and food safety will be developed and implemented. OHIO Genotypic evaluation was characterized using pulsed-field gel electrophoresis analysis of SmaI restriction patterns. These data suggest enterococci of bovine origin are genetically diverse while isolates of various sources appear to cluster together. ONTARIO New technologies and different applications of new technologies will continue in the coming year, and as the opportunities arise. PENNSYLVANIA Epidemiology of select viral agents in quarter milk samples with high somatic cell counts. Web-based software tool using expert systems for determining bulk tank milk quality WASHINGTON Continue study on the effect of shortened dry period on mastitis at parturition. WISCONSIN We will be field testing an entirely new methodology for detection of selected mastitis pathogens.