Brief Summary of Minutes of Annual Meeting

COMPLETED RESEARCH Objective 1. Characterization of host mechanisms associated with mastitis susceptibility and resistance. Connecticut: A study was conducted to determine if severe negative energy balance (NEB) increased the risk of mastitis occurring during transition period in high producing dairy cows. Based on the logistic regression, cows that developed ketosis were 4.61 times more likely to develop a new inramammary infection than cows that did not develop ketosis. Decreasing the amount of days a cow was fed the prefresh ration increased the risk of developing a new IMI. These results support the relationship between the severity of NEB and the development of new IMI and that optimizing body condition and providing adequate dietary energy during the dry period may reduce the severity of NEB and the risk of developing a new IMI during the transition period. Guelph, Canada: 1. The first objective of this study was to estimate the heritability of subclinical mastitis (SCM) caused by major environmental pathogens (ENV), major contagious pathogens (CONT), or both (E+C). The second objective was to estimate the genetic and phenotypic correlations of SCM caused by these major pathogens with test day milk production (TD-milk), test day somatic cell score (TD-SCS), and udder related conformation traits. Herds were visited up to 6 times over an 18 month period and a composite milk sample was collected from all milking cows at each visit, and the milk samples cultured. Heritabilities of E+C, ENV, and CONT were low. E+C was phenotypically and genotypically correlated with ENV and CONT. ENV had independent phenotypic and genetic correlations with CONT. ENV had a high genetic correlation with TD-SCS. CONT had a high genetic correlation with TD-milk. Selection for decreased TD-SCS would increase the genetic resistance of cows to infection by ENV. In contrast, selection for increased milk production would increase the genetic predisposition of cows to infection by CONT. Phenotypic correlations of E+C, ENV, and CONT were close to zero with the conformation traits. ENV had significant genetic correlations with conformation traits related to capacity, rump, mammary system, fore udder, and rear udder. CONT was not significantly correlated with the conformation traits. 2. The objective of a second study was to describe cow and quarter-level factors associated with the process of drying-off, and to evaluate their impact on new intramammary infections (IMI) during the dry period. Data from 300 cows in five research herds were collected. Overall, 11.1% of quarters developed new IMI in the dry period. The majority of new IMI were caused by environmental streptococci and coliform organisms (34.4% and 29.8%, respectively). Quarters that had a cracked teat-end were more likely to develop new infections than those without cracks (14.8% and 9.8%, respectively). Quarters that closed, through formation of a keratin plug early in the dry period, were significantly less likely to develop a new infection, compared to quarters that did not close (9.7% and 14%, respectively). At the end of six weeks of the dry period, 23.4% of quarters were still classified as open. When the level of milk production on the day prior to drying-off was greater than or equal to 21 Kg., quarters were 1.8 times less likely to close, as compared to when milk production on the day before drying-off was less than 21 kg. 3. The Bacillus Calmette Guérin (BCG) induced/purified protein derivative (PPD)-elicited tuberculin skin test is a reliable measure of cell-mediated immune response (CMIR), specifically delayed-type hypersensitivity (DTH); however, its use in livestock may confound diagnosis of Mycobacterium tuberculosis. Therefore, various alternative antigen/adjuvant combinations were evaluated as inducers of DTH and compared to the BCG/PPD test system. Specifically, 30 non-lactating cows (5/treatment) were sensitized on day 0 with mycobacteria (BCG, M. tuberculosis or M. phlei cell wall extract- MCWE), and ovalbumin (OVA) emulsified in Freunds complete adjuvant (FCA), non-ulcerative Freunds adjuvant (NUFA), complete NUFA or MCWE. On day 21, cows were injected intradermally with various test antigens including PPD tuberculin, phlein, and OVA. Phosphate buffered saline was included as the negative control and the T-cell mitogen phytohemagglutinin (PHA) was also administered. Double skin-fold thickness was evaluated before and at 6, 24 and 48 hours post-injection. Skin biopsies were taken at 24 and 48 hours to assess oedema, necrosis and inflammatory cell infiltration. BCG/PPD and M. phlei/phlein treatments when given with a Freunds adjuvant induced equivalent DTH with peak reactions at 24 - 48 hours after antigen injection. Cows receiving NUFA had fewer injection site granulomas than FCA or CNUFA treatments. The change in skin thickness response to PHA peaked at 6 hours. Only cows receiving mycobacteria in NUFA had skin response to OVA which peaked 6 - 24 hours post-injection. Only sites tested with PPD or phlein had significantly higher lymphocyte infiltration than control, whereas neutrophils were significantly higher at PHA test sites and eosinophils predominated at the PHA test sites. Macrophages were significantly more numerous at the PPD and/or phlein test sites in treatment groups that received killed mycobacteria in a Freunds adjuvant and/or with BCG, and at the PHA test sites in all treatment groups. It was concluded that BCG/PPD and M. phlei/phlein given with a Freunds adjuvant induced similar DTH, but that cross reaction to PPD was evident following induction of DTH using M. phlei, hence this protocol does not alleviate the problem of artificial induction of tuberculin test sensitivity when measuring CMIR of cattle. Iowa: Data from a collaborative study (Guelph, PEI, NY, IA) evaluating cow and quarter factors associated with new dry period IMI and the role of barrier teat sealants in teat closure and/or IMI prevention was completed and is currently being analyzed at PEI. Louisiana: A mycoplasma control program has been initiated in Louisiana in response to recent herd outbreaks of mycoplasam mastitis. All dairies in the state are being screened monthly for mycoplasma as well as other major mastitis pathogens. Thus far, incidence of mycoplama is about 1-2% of tested dairies. In a separate study, dairy heifers are being cultured prepartum to determine incidence of coagulase negative staphylococci and monitored through their first lactation to determine effects of CNS on milk production and SCC. Various mastitis pathogens are being screened by a disc diffusion assay for potential resistance to selected teat disenfectants. Michigan: Continuing field study on the effects of J5 hyperimmunization involving two herds in Michigan. We are also determining mechanisms of neutrophil function in relation to endocrine changes associated with parturition. New York: In 2004, we completed a study on host resistance of dairy cows to E.coli mastitis and the mechanisms of E.coli vaccination on the host resistance. The data will be analyzed. We also started on the development of bovine specific cytokine assays. Ohio: Milk production at dry-off was a significant risk factor for both a cow and a quarter being infected with environmental pathogens at calving. For every 5-kg increase in milk yield at dry-off above 12.5 kg, the odds of a cow having an environmental IMI at calving increased by 77%. Cows fed selenized yeast had higher concentrations of selenium in serum and milk and their newborn calves had higher serum selenium than those fed sodium seleate. The increased concentrations of selenium did not translate into differences in neutrophil function or clinical responses following an intramammary infusion of endotoxin. Prince Edward Island, Canada: The University of PEI continues to contribute to this objective with emphasis on identifying risk factors for mastitis in dry cows. A large OrbeSeal field study and work external teat sealants has been the area of concentration in the last year. Tennessee: A reproducible Strep. uberis challenge model was developed in lactating dairy cows during early lactation. Beta-defensin genes were detected in bovine mammary gland epithelial cells. The association of CXCR2 SNP genotypes with subclinical and clinical mastitis was determined. Holsteins expressing genotype GG had decreased subclinical mastitis, while genotype CC cows had increased percentages of subclinical mastitits. In addition, the ability of neutrophils to migrate as well as survive is altered in cattle with a CC genotype and provides initial evidence for a phenotypic association between neutrophil function and a single nucleotide polymorphism in dairy cows previously associated with altered susceptibility to intramammary infections. Washington: Prepartum IMM treatment of heifers did reduce infections at parturition (mostly CNS IMI), but did not lead to improved milk or reproductive performance, nor lowered SCC. Objective 2. Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defense. Guelph, Canada: 1. Previously published reports of animal trials involving phage therapy against bovine S. aureus mastitis have reported treatment failure. We examined two aspects of the phage-bacterial interaction in a model system of raw bovine milk whey: the rate of phage binding to its host cell, and the ability of a bacterial population to survive phage lysis under growth conditions. Whey collected from 24 animals showed a wide variation in the amount of phage binding inhibition. Bovine serum obtained from commercial sources also strongly inhibited phage binding. Overnight incubation of S. aureus with varying amounts of phage in whey showed that the bacteria are more resistant to phage lysis when grown in whey from all animals, as well as in serum. There was no apparent correlation between phage binding inhibition in whey and long-term survival in this system; whey which showed little inhibition of phage binding still promoted resistance of the bacteria to phage lysis in longer-term experiments. The phage themselves were found to be quite stable in whey. Data suggest that binding inhibition and the ability of the bacterium to survive co-culture with phage are two separate phenomena. The role of the protein component of milk whey in this inhibition was established. Treatment of the whey by both heat and proteases removed the inhibitory activity. Protein was also removed from whey by ultrafiltration. Whey ultrafiltrate showed a markedly reduced ability to inhibit phage lysis in both phage binding and long-term growth experiments. Size-exclusion chromatography was conducted on the raw whey in an attempt to isolate the protein component(s) responsible for the phage binding inhibition. The protein fraction which inhibits phage binding appears to be an aggregate or multimer of 1.5 MDa or greater. SDS-PAGE was conducted on this fraction followed by band excision and MALDI-TOF assisted peptide mapping. Peptide mapping revealed that the major proteins present in the inhibitory whey fraction are associated with the milk fat globule membrane (MFGM). 2. Macrorestriction analysis of SmaI digested chromosomal DNA, using pulsed field gel electrophoresis (PFGE) was performed to type and estimate genetic relationships among 288 Staphylococcus aureus isolates recovered from 58 Eastern Canadian dairy herds. Also, a subset of the collection was phage typed and evaluated for sensitivity to 10 antimicrobial compounds. Of 288 isolates recovered, 29 distinct PFGE types were identified. Based on estimates of genetic relationships the PFGE types were assigned to six lineage groups, designated A through F. Of all the isolates, approximately 93% were assigned to lineage groups A, D or F. In 58.6% of herds, only a single PFGE type was recovered while the remainder had two to four types. Of the 212 isolates evaluated for antimicrobial resistance, 24.5% were resistant to one or more antimicrobials. Resistance to penicillin (9.9%) was most common, followed by resistance to sulfadimethoxine (7.5%). Isolates resistant to multiple antibiotics were rare. A total of 63% of isolates responded to phages from groups 1 and 3, and 32.8% could not be typed with any of the phage strains used. The other 4.1% belonged to a variety of phage types. Most of the PFGE lineage group A and F isolates corresponded to phage groups 3 and 1, respectively, and most group D isolates were not typeable. PFGE typing had better discriminatory power than phage typing in defining the relatedness of the S. aureus isolates. Distribution of PFGE types and phage types was independent across regions and within herds. Missouri: Sample collection was completed for a study that will determine the in vitro and in vivo susceptibility of different bovine mammary Staphylococcus aureus strain-types to Pirlimycin HCl. New York: In 2004 we have worked on further defining and understanding the pathogenicity of chronic coliform infections. E.coli organisms associated with chronic and transient infections have been compared with regard to adhesion and invasion characteristics. The two types of strains have been characterized using DNA micro-arrays. S.uberis strains have been collected to identify strains associated with environmental and contagious transmission patterns. Prince Edward Island, Canada: A single PhD project has been designed through the Canadian Bovine Mastitis Research Network to assess epidemiologically the mastitis situation in Canada, which will include analysis of established mastitis databases in the country, but also build on collection and interpretation of clinical mastitis data. Tennessee: All E. coli from cows with mastitis were multridrug resistant and carried a variety of antimicrobial resistance genes. A high prevalence of foodborne pathogens isolated from the dairy farm environment contain antimicrobial resistance genes. Washington: Mycoplasma strains that cause mastitis also can be found at other body sites as determined by chromosomal digest technology. The urogenital system is most often colonized with the same mastitis strain. Biofilm formation by Staphylococcus aureus is associated with strains that cause mastitis as compared to strains that colonize other sites (teat skin, milking unit liners), suggesting that biofilm is a virulence factor for this pathogen. Objective 3. Development and evaluation of techniques for modulation of host responses to mastitis pathogens. Connecticut: Preparturient heifers in 9 herds, in the US and Canada, were enrolled in a study testing the hypothesis that prepartum intramammary therapy would cure existing intramammary infection (IMI), resulting in increased milk production, reduced somatic cell count, and improved reproductive performance. Prepartum intramammary antibiotic therapy did reduce the number of heifer IMI, but this reduction in IMI did not translate into a significant improvement in milk production, SCC, or reproductive performance. Residues persisted until the sixth milking for 10 cows (7.14%) and until the tenth milking for 5 cows (3.67%). The mean interval between treatment and parturition for cows identified with an antibiotic residue was 8.5 d (range = 1 - 18 d). An increase in interval was associated with a decreased risk of antibiotic residues. Guelph, Canada: 1. We evaluated the usefulness of the California Mastitis Test (CMT) for identifying quarters with an intramammary infection (IMI) at calving. A total of 1851 quarters from 463 cows between 1-3 DIM in 16 dairy herds were enrolled. Overall, the sensitivity and specificity of the CMT for detecting major pathogen infections were 58% and 85%, respectively. Cows with a positive CMT were randomly assigned to receive either intramammary cephapirin sodium, or no treatment. There was a tendency towards significance in cure rates for major pathogens, between the 165 treated and the 190 control quarters, especially for environmental streptococcal infections. Overall, as the CMT score increased, cows had decreased milk production. Cows that cured a major pathogen had a lower LS. In conclusion, early antibiotic treatment of CMT positive quarters would appear beneficial, particularly with environmental pathogens. There remains a need for a method to rapidly identify the causative agent of intramammary infections. 2. A 1.0% iodophore teat disinfectant (Full-Bac) was evaluated in comparison to a positive control (Bovadine), a commercially available 1.0% iodophore teat disinfectant. The test product and a positive control were compared in 41 cows with 82 total teats receiving each product at milking, during a 10-wk study period. There were no differences between the test product and positive control in new intramammary infections by Staphylococcus aureus, which averaged 13.4% in each of the two treatment groups. Additionally, no statistical difference was seen between the test product and positive control in new intramammary infections by Streptococcus agalactiae, which averaged 8.5% and 6.1% for the Full-Bac and Bovadine groups, respectively. Teat skin and teat end condition scores were statistically evaluated at wk 1, 5, and 9 of the study period, and no significant differences were observed between the treatment groups. The test teat disinfectant provided similar germicidal activity to that of the positive control teat disinfectant with no adverse effects on teat skin and teat end condition during the warm-season study period. Illinois: Photoperiod length during the dry period (16 h light/d vs 8 h light/d) did not influence somatic cell count or intramammary infection prevalence at calving or during the first 120 d of lactation. Photoperiod length during the dry period (16 h light/d, 8 h light/d, or ambient lighting conditions) did not influence plasma total protein concentration, plasma globulin concentration, or plasma IgG concentration at calving. Nor did photoperiod length impact colostral volume, colostral IgG concentration, or colostral IgG mass. Significant determinants of colostral IgG concentration were colostral volume and the time delay between calving and milking. Iowa: 1) A study evaluating prepartum treatment of 180 heifers (1200 cow dairy; single tube of lactating cow antibiotic (Pirsue, Pfizer)) was completed. Bacteriological, CMT, and milk production data is currently being merged and analyzed with MO trials. 2) A trial evaluating the effects of powder teat dips on teat condition during winter was completed and is being analyzed. 3) Two trials evaluating effect of chlorous acid dips on teat end hyperkeratosis were completed and are being analyzed. 4) A pilot project evaluating the use of the CMT and/or on farm culture systems to diagnose and treat subclinical infections at calving or clinical mastitis was completed and is being analyzed. 5) A high SCC commercial herd prevention/ therapy trial was initiated. Michigan: We are continuing to apply and develop and on-farm bacteriology system for culture of milk to aid in therapeutic decisions. Minnesota: In 2004, we completed cow enrollment for a study evaluating the effect of metaphylactic intramammary infusion of 200 mg Cephapirin Sodium per quarter in primiparous and multiparous dairy cattle at 2-3 weeks prepartum on selected postpartum production parameters. We also completed data collection for a multistate study (MN, NY, Guelph, PEI) comparing duplicate vs single quarter samples for identification of intramammary infections. Members from University of Minnesota, Cornell University, and Valley Agricultural Software collaborated to develop an electronic data transfer system to transfer mastitis event and milk culture data from the farm to the laboratory and back to the farm. Missouri: Two studies were performed to evaluate the efficacy of extended pirlimycin HCl intramammary therapy with or without vaccination with a commercial Staphylococcus aureus mastitis bacterin (Lysigin, Boehringer Ingelheim Vetmedica, Inc.) in the treatment of chronic Staphylococcus aureus intramammary infection. Results show that vaccination with Lysigin and treatment with extended intramammary pirlimycin has no greater efficacy than treatment with extended intramammary pirlimycin HCl alone. New York: Evaluation of long term NY cell count data has resulted in a better understanding of cell count dynamics in a population. The impact of cell count premiums has been quantified. Further evaluation of milk flow characteristics has been worked on. Milk quality on organic farms has been studied. Dynamics of listeria infections on dairy farms and milk has been studied. Ohio: The use of an oil-in-water adjuvant increased the efficacy of an E. coli J5 bacterin over the use of a water soluble adjuvant. However, the use of the oil-in-water adjuvant also increased the frequency of injection site reactions compared with the water soluble adjuvant. An injection site reaction of greater than 1 cm2 was positively associated with efficacy in cows vaccinated with the bacterin containing water soluble adjuvant. Tennessee: A real-time PCR procedure for detection of Staph. aureus, Strep. agalactiae, Strep. uberis, Strep. dysgalactiae subsp dysgalactiae and L. monocytogenes directly from milk was developed. A real-time PCR method based on the dsDNA binding dye SYBR Green to detect C. jejuni, E. coli O157:H7, and Salmonella spp. simultaneously from raw milk and dairy farm environmental samples was developed. Shiga toxin-producing E. coli, L.monocytogenes, C. jejuni and Salmonella spp. were isolated frequently in bovine fecal samples, fecal slurry, water from lagoons, bedding, bird droppings and rats. Washington: Changes in prevalence in intramammary infection, by pathogen type, in herds applying a stringent contagious mastitis control program did not lead to increased risk of non-contagious mastitis as coliform, environmental streptococcal and coagulase negative staphylococcal intramammary infections decreased after adoption of the program WORK IN PROGRESS Objective 1. Characterize host responses or factors that affect resistance of the mammary gland to mastitis. Connecticut: The effect of breed on the extent of negative energy balance, plasma fat soluble vitamin status and rate of new intramammary infection during the transition period will be investigated. Iowa: Publish 2nd dry period mastitis prevention/ teat closure study (Guelph, PEI, NY, IA). Summarize 3 year intense teat observational study on 2 herds. Michigan: Field study on the effects of J5 hyperimmunization. Determining mechanisms of neutrophil function in relation to endocrine function Tennessee: During the next year we will: 1) continue to delineate the importance of ther CXCR2 receptor on neutrophil function and resisatnce to mastitis in dairy cows using the Strep. uberis experimental infection model, 2) delineate the role of Strep. uberis adhesion molecule on bacterial adherence and internalization, 3) determine inhibitory effects of beta-defensins on mastitis and foodborne pathogens. Objective 2. Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defense. Iowa: Publish results of NE-1009 Environmental streptococci project. (IL,IA,NY,MI,VA,WA) Missouri: Completion of a study to determine of the in vitro and in vivo susceptibility of different bovine mammary Staphylococcus aureus strain-types to extended pirlimycin HCl intramammary therapy. Washington: We will test the hypothesis that mycoplasma strains that cause masititis have a unique genetic fingerprint, genetic elements, as compared to isolates that only colonize the extramammary body sites. Ultimately we would like to deterimine the virulence factors associated with strains of mycoplasma that cause mastitis. Objective 3. Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. Connecticut: Factors affecting the peformance of four antibiotic residue screening tests used to detect residues in milk from individual cows will be evaluated using milk from 400 cows in early lactation. Guelph, Canada: Dairy Farmers of Ontario are starting to install time-temperature recorders on all Ontario dairy farms and have begun a water quality testing program, both intended to decrease the bacterial contamination of raw milk. The objectives of this study are to investigate the relationship between bacteria contaminated wash water and elevated Bactoscan bacteria counts in raw milk; and to evaluate the impact of the time-temperature recorders on the bacterial content of milk and the dumping/loss of raw milk on the farm. The Canadian Bovine Mastitis Research Network has submitted a $7.5 million funding proposal to the National Sciences and Engineering Research Council of Canada to support new initiatives in the monitoring, prevention and elimination of bovine mastitis. This strategic network funding request, involving 39 mastitis researchers across Canada under the leadership of Dr. Daniel Scholl, will provide facilitate international collaboration with many of the current members of the NE-1009 project. A project entitled Assessment of the mastitis situation in Canada (H. Barkema, G. Keefe, R. Dingwell, R. Olde Reinkerink, D. Kelton) is underway to describe the herd level patterns of mastitis in dairy herds across Canada and to quantify mastitis pathogens in bulk tank milk and culture clinical mastitis cases. Specifically, the aims of the project are to determine the seasonal period prevalence of mastitis pathogens in bulk milk, synthesize available Canadian mastitis data and identify important information gaps, determination and prioritization pathogens prevalent in clinical mastitis cases, determination of gaps in producer adoption of mastitis management practices, investigate risk factors at the herd level associated with different prevalence levels of Mycoplasma spp., Staphylococcus aureus and Streptococcus agalactiae, and different BTSCC levels, and investigate risk factors at the herd level for the incidence of clinical mastitis and examine and compare risk factors for pathogen-specific incidence of clinical mastitis. Illinois: Continue to investigate the influence of photoperiod on immune responses and mammary gland function, particularly with regard to the prolactin axis. Complete research on use of population pharmacokinetics to determine factors influencing drug residue times after intramammary antibiotic administration. Compare the effects of milking frequency (2x vs 3x/d) on milk concentrations of intramammary antibiotics. Iowa: 1) Complete analysis of commercial herd prepartum heifer treatment trial (IA,MO); 2)Complete analyses of winter teat dip trial using powder dips and trial examining effect of chlorous acid dip on reducing hyperkeratosis; 4) Analyze on farm culture pilot project; 5) Initiate joint project (MN,IA,PEI,Guelph,WI) evaluating on farm culture systems for strategic treatment of clinical and subclinical intramammary infections; 5) Complete commercial herd prevention/ therapy trial. Louisiana: Testing of teat dips for efficacy will continue. Studies on effect of CNS on SCC and production will continue. Evaluation of mastitis pathogens for possible resistance to germicides will continue. Michigan: Apply and develop and on-farm bacteriology system for culture of milk to aid in therapeutic decisions. Minnesota: Final data will be collected and analyzed from the 2004 study evaluating the effect of metaphylactic intramammary infusion of 200 mg Cephapirin Sodium per quarter in primiparous and multiparous dairy cattle at 2-3 weeks prepartum. Data will be analyzed for the multistate study comparing duplicate vs single quarter samples for identification of intramammary infections. Continue development of the electronic data transfer system to transfer culture data from the laboratory to the DHIA LOOP. Herds and cows will be enrolled into a new large multistate study evaluating the use of an on-farm culture system for strategic treatment of clinical mastitis subclinical mastitis in fresh cows. Missouri: A study is in progress to characterize the humoral immune response in milk and serum following vaccination with a commercial Staphylococcus aureus bacterin using an antibody subclass specific ELISA. Also, we will be completing data analysis and preparing publication of a study evaluating prepartum treatment of heifers with pirlimycin HCl in collaboration with Leo Timms (Iowa Station). New York: Publication of vaccine efficacy data will be completed in 2005. Further development of cytokine assays is planned for 2005. Further work on chronic coliform mastitis will take in 2005. Both chronic E.coli and Klebsiella infections will be studied. Cell count dynamics at herd level will be studied in 2005. Milk quality and food safety on organic dairy farms will be studied. Listeria infections in dairy farms will be studied. Prince Edward Island, Canada: A study will commence in 2005 to examine the association between bulk tank milk analysis of raw milk and on farm management practices.