NE1009: Mastitis Resistance to Enhance Dairy Food Safety

(Multistate Research Project)

Status: Inactive/Terminating

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SAES-422 Reports

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[01/08/2004] [01/11/2005] [12/06/2005] [02/08/2007] [12/21/2007]

Date of Annual Report: 01/08/2004

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Annual Meeting Dates: 11/07/2003 - 11/07/2003
Period the Report Covers: 11/01/2002 - 11/01/2003

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Objective 1. Characterization of host mechanisms associated with mastitis susceptibility and resistance. Louisiana The Hill Farm Research Station dairy herd of 102 cows was used in a controlled infection trial to determine efficacy of two iodine teat dips for preventing intramammary infections (IMI) with Staphylococcus aureus and Streptococcus agalactiae. Connecticut Efforts include determining the efficacy of teat disinfectants and the effect of extent of negative energy balance on prevalence of pathogens during the periparturient period. Ohio - Plasma and milk concentrations of ascorbic acid and dehydro-L-ascorbic acid were measured before and after 21 Holstein cows were given an intramammary infusion of Escherichia coli. Increased severity of clinical signs was associated with large decreases in concentration of vitamin C in milk from the challenged quarter Iowa 1) Research identified that undifferentiated bovine mammary gland progenitor cells (BMGPC) are resistant to Coliform mastitis organisms while, in contrast, differentiated BMGPC are susceptible to E. coli infections. 2) The uterocalin gene was identified as a potential host protection factor against inflammation 3) Fourteen new dry cow teat dip sealant prototypes were evaluated for persistency of teat end coverage during the early and late dry period 4) A targeted treatment trial was conducted after 2 months of stabilization in a herd who had dipped teats in formaldehyde for 3 milkings. Michigan - Isotype-specific antibody responses and cross-reactivity were profiled following hyperimmunization of steers with J5 Escherichia coli bacterin. Two immunizations increased mean serum IgM and the IgG1antibody profiles above pre-immunization levels but five immunizations were required to detect significant IgG2 Missouri - The influence of S. aureus intramammary infection (IMI) on serum Cu, Zn, and Fe concentrations was studied. Results showed a significant decrease in serum Zn concentrations 24 hours after the final intramammary infusion of S. aureus. Washington - We analyzed the difference of Nramp1 gene expression between clinical mastitis resistant and susceptible cows. Results demonstrate that resistant cows produced more Nramp1 mRNA than susceptible cows. Tennessee 1) A 311 bp segment located within the coding region of exon 3 of the CXCR2 gene was amplified and sequenced. We detected 5 SNPs expressed by both breeds of cattle within our amplified sequence. Breed was shown to have a significant effect on allele and genotype frequencies for four of the five loci. 2) Association between functional activity of neutrophils and single nucleotide polymorphisms of CXCR2 in dairy cows was investigated. Cows exhibiting the CG genotype in SNP3 had lower neutrophil migration after stimulation with zymosan activated sera than cows exhibiting the GG genotype. This coincided with weaker downregulation of CD62 after stimulation with IL8 or phorbol myristate acetate (PMA). Cows exhibiting the AA genotype in SNP5 had higher upregulation of CD18 in conjunction with a greater downregulation of CD62 after stimulation with IL8 compared to genotype AG. Vermont - In order to characterize the response of the mammary epithelial cells to infection, an in vitro model system of mammary epithelial cells in primary culture has been developed and used to quantify changes in gene expression in response to infection. Guelph 1) Factors associated with dry period mastitis was studied. Variables of interest included daily milk production, formation of the teat canal keratin plug, and quarter milk bacteriological culture results. Overall, 11% of quarters developed new IMI in the dry period. Quarters that closed were significantly less likely to develop a new infection. 2) Intramammary Infection at First Calving in Ontario Dairy Heifers: Bacterial culture results from composite milk samples collected after calving indicated that 45% of fresh heifers were culture negative, while 3% were culture positive for Staphylococcus aureus. IMI at calving increased the probability of subsequent positive milk cultures, increased somatic cell count and increased the risk of being culled. 3) In order to facilitate the genotyping of a large population, a rapid PCR screening method, multi-primer target-PCR (MPT-PCR) was developed. Four primers were combined in one PCR reaction to detect the presence of alleles DRB3.2*16 and *23 simultaneously. No associations were found between alleles and clinical mastitis, mastitis at calving or somatic cell score. Objective 2. Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defense. Ohio - The synergistic effects IgG from cows vaccinated with ferric citrate receptor (FecA) and IgG from cows vaccinated with ferric enterobactin receptor (FepA) were measured in an in vitro iron uptake assay. The combination of anti-FecA IgG and anti-FepA IgG reduced 55Fe uptake compared with either anti-FecA or anti-FepA alone. Missouri - A study was conducted to compare the efficacy of three different S. aureus bacterins against non-vaccinates. Results showed no significant differences between groups with regard to IMI rate. Washington - Microbial shedding rates in the milk were studied in cows with mycoplasma mastitis. A bimodal distribution of shedding rates was seen, and some shedding rates were zero for several days, some were low enough that on average, a single cow with mycoplasma mastitis would not be discerned through the herds bulk tank milk sampling. Tennessee Streptococcus uberis isolates were collected and Fingerprinting was performed on these isolates using the following methods: RiboPrinter., pulsed-field gel electrophoresis and randomly amplified DNA fingerprinting. Based on results of the present study, the PFGE method appears to be more discriminatory for detecting differences in strains. 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. Virulence factors of S.uberis that distinguish isolates causing chronic and transient infections are being investigated. Vermont Characterization and manipulation of Bacteriocins, autolysins, and bacteriophage enzymes is being pursued in order to understand their potential role in regulating growth and virulence of the mastitis pathogens S. aureus and S. uberis. Guelph - Staphylococcus aureus Isolated From Intramammary Infections Before and After the Dry Period of Dairy Cattle in Canada was studied using Sensititre.. Very few of the S. aureus studied were resistant to common antibiotics used in dairy herds, even those drugs most often used for dry cow antibiotic therapy. Overall, there was very little change in the resistance pattern of organisms that were cultured from intramammary infections prior to drying off. Objective 3. Development and evaluation of techniques for modulation of host responses to mastitis pathogens. Connecticut Evaluated the effect of intramammary antibiotic therapy at 2 weeks prior to anticipated calving on pathogen prevalence, milk production and reproduction in primiparous cows. Ohio - Antimicrobial susceptibility of mastitis pathogens isolated at calving from first lactation and older cows were compared. MIC90 for penicillin was higher among isolates from older cows. Resistance to tetracycline was more common and MIC90 higher among isolates from first lactation cows than from older cows. Iowa - A study evaluating prepartum treatment of heifers ( single tube of lactating cow antibiotic was completed on a 1200 cow commercial dairy. Michigan - A cDNA microarray resource has been developed with the goal of providing integrated functional genomics resources for cattle. A web-accessible database (http://www.nbfgc.msu.edu) has been established to provide pertinent data related to NBFGC clones, including sequence data, BLAST results, and ontology information. The NBFGC microarray represents the largest cDNA microarray for a livestock species prepared to date and should prove to be a valuable tool in studying genome-wide gene expression in cattle. Missouri - A method was developed for detection of chlorhexidine in raw milk using HPLC. Subsequently, a study was completed to evaluate the elimination kinetics of chlorhexidine in milk following intramammary infusion as a treatment to stop lactation in mastitic mammary quarters of cows. Washington - A study was made to determine the effect of strict control program on. S. aureus IMI. With the program that was based on segregation and on application of hygienic and therapeutic protocols, prompt collection of milk samples at parturition and at 7 and 14 days after parturition, incidence of new IMI was reduced to < 1case/100 cow-months. Tennessee 1) One hundred and three quarters of 68 dairy cows experimentally infected with S. uberis during early lactation were used to evaluate efficacy of extended pirlimycin therapy regimens. Cows were allocated randomly to one of three treatment groups: 2X, 5X, and 8X. Differences in treatment efficacy were observed between the 2X and 8X treatment groups (58% vs. 80%). 2) A study was sone to detect Shiga toxin-producing Escherichia coli O26 using phenotypic and genetic markers. There were few observable differences in carbohydrate fermentation patterns of most E. coli isolates. Fourteen of 21 STEC O26 were rhamnose-negative. Seven STEC O26 and all non-O26 STEC isolates fermented rhamnose. Of the non-O26 strains, 14%, 17% and 0% of STEC O157, STEC O111 and generic E. coli, respectively, were rhamnose-negative. Incorporation of rhamnose in STEC O26 isolation media was reported to be a useful diagnostic. Minnesota - A field study was completed describing the effect of infusion with an internal teat sealant, when used with a long-acting antibiotic (DCT) at dry off. Results showed that quarters infused with an internal teat sealant in addition to DCT were 30 % less likely to develop a new IMI between dry off and 1 to 3 DIM, and 33% less likely to experience a clinical mastitis event. New York - New technologies that are being developed included milking parlor assessment and evaluation of milk flow curves and equipment cleaning quantification. Food safety research includes work on Salmonella and Listeria infections in dairy herds. Vermont - We have previously shown that transgenic production of antibacterial enzymes in the lactating mammary gland is possible using a transgenic mouse model producing lysostaphin to create resistance to Staphylococcus aureus mastitis. In order to extend this work to enhance resistance during the dry period, we have identified and cloned gene promoter regions that are active during mammary gland involution and infection. A transgene using this promoter to direct expression of lysostaphin has been constructed. Guelph 1)Effects of Two Gram-Negative Mastitis Immunization Protocols on Daily Milk Production and Dry Matter Feed Intake Values.-- A total of 198 Holstein cows and heifers from two research herds were enrolled. Each animal was randomly assigned to one of two treatment groups (Standard or Experimental). The Standard Group was immunized according to the label directions of a commercial J5 Escherichia coli .The Experimental Group was immunized at two weeks prior to drying off, at drying off and at three weeks before the expected calving date. There was no significant difference in milk production prior to drying off or on the average daily milk production to 60 days between the two groups 2) As a second objective, the Immunization protocol did not induce antibody to J5 E. coli, likely as a result of the high pre-existing antibody to this organism.

Publications

Louisiana Foret, C. J., W. E. Owens, R. L. Boddie, P. Janowicz. Efficacy of two iodine teat dips during experimental challenge with Staphylococcus aureus and Streptococcus agalactiae. Our Industry Today. (In press). 2003 Norman, C. B., W. E. Owens, R. L. Boddie, N. T. Boddie, and C. H. Ray. Efficacy of prepartum, intramammary lactating cow antibiotics in dairy heifers. National Mastitis Council Annual Meeting Proceedings. Fort Worth, TX. 2003. Norman, C. B., W. E. Owens, R. L. Boddie, and B. F. Jenny. Heifer mastitis is all too common. Hoards Dairymen. p. 381. 2003. Owens, W. E., R. L. Boddie, C. J. Foret, and P. Janowicz. Efficacy of a 0.1% iodine teat dip against Staphylococcus aureus and Streptococcus agalactiae during experimental challenge. Our Industry Today. (Submitted). 2003 Owens, W. E. Mastitis therapy and control: Medical therapy options in encyclopedia of dairy sciences. Roginski, H., J. W. Fuquay, and P. F. Fox (eds.) Academic Press, London. 2002. Owens, W. E. Microbiological procedures for the diagnosis of bovine udder infections in Microbiological procedures for the diagnosis of bovine udder infections, 4th edition, National Mastitis Council. Arlington, VA. 2002, Owens, W. E. The staphylococci in Microbiological procedures for the diagnosis of bovine udder infections. 4th edition, National Mastitis Council. Arlinton, VA. 2002. Connecticut: Book Chapter: Andrew, S. M. 2003. Antibiotic Residues in Animal-Based Foods. In Current Topics in Food Safety in Animal Agriculture, Iowa State University Press, Ames, IA. Abstracts and Proceedings Andrew, S. M., and R. Van Saun. 2003. Guidelines for investigating a mastitis/milk quality problem. Mid Atlantic Consortium 5th Annual In-Service Training Conference. June 11-13, 2003, Wilkes Barre, PA. Andrew, S. M. 2002. Got Biosecurity? Whats New in Residue Avoidance. Pages 36-44 In Proc. Natl. Mastitis Counc. 2002. Syracuse NY. Ohio: Takemura, K., J.S. Hogan, and K.L. Smith. 2003. Effect of immunoglobulin G from cows immunized with ferric citrate receptor (FecA) on iron uptake by Escherichia coli. J. Dairy Sci. 86:133-137. Wise, A.J., J.S. Hogan, K. Takemura, and K.L. Smith. 2003. Opsonic activity of serum and whey from cows immunized with the ferric citrate receptor. J. Dairy Sci. 86:146-151. Aslam, M., J. Hogan, and K. L. Smith. 2003. Development of a PCR-based assay to detect Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella in milk. Food Microbiol. 20:345-350. Hogan, J.S., and K.L. Smith. 2003. Coliform mastitis: review article. Vet. Res. 34:507-519. Iowa: Nilsen-Hamilton M., Q. Liu, J. Ryon, L. Bendickson, P. Lepont and Q. Chang. 2003. Tissue Involution and the Acute Phase Response, Annals of the NY Academy of Sciences, 995:94-108 Timms, L. L. and P. Ruegg. 2002. Chapter 13. Heifer mastitis and production of quality milk. Raising Dairy Replacements. MWPS RDR: 101. Morelli, J., L. Timms, and J. Schnitzler. 2003. Split udder comparison of teat disinfectants on skin toleration during winter and spring. NMC Ann. Proc.:332. Paulsen, D., L.L. Timms, and M.A. Faust. 2003. Evaluation of a salve or two novel winter teat dips to maintain or enhance teat end integrity during winter. NMC Ann. Proc.:385. Wedemeier, S.G., and L.L. Timms. 2003. Controlling Staphylococcus aureus and other mastitis and lowering somatic cell counts in a modern freestall dairy. J. Dairy Sci. 86 (Suppl. 2): 90. Paulsen, D. and L.L. Timms. 2003. Evaluation of a salve or two novel winter teat dips to maintain or enhance teat end integrity during winter. J. Dairy Sci. 86 (Suppl. 2): 14 Michigan: Chaiyotwittayakun, A., J.L. Burton, P.S.D. Weber, K. Kizilkaya, F.F. Cardoso, and R.J. Erskine. 2003. Hyperimmunization of Steers With J5 Escherichia coli Bacterin: Effects on Isotype-Specific Serum Antibody Responses and Cross Reactivity With Heterogeneous Gram-Negative Bacteria. Journal of Dairy Science (in review). Burton, J.L, P.M. Coussens, M. VandeHaar, M. Allen, G.W. Smith, and J.J. Ireland. 2003. Functional Genomics and the Dairy Cow: From Genes to the Farm. Michigan Dairy Review (in press) (see also http://www.msu.edu/user/mdr/) Weber, P.S.D., T. Toelboell, L-C. Chang, J. Durrett-Tirrell, P.M. Saama, G.W. Smith, and J.L. Burton. 2003. Mechanisms of glucocorticoid-induced down regulation of neutrophil L-selectin in cattle: Evidence for effects at the genome level and primarily on blood neutrophils. Journal of Leukocyte Biology (accepted for publication). Madsen, S.A., L-C. Chang, M-C. Hickey, G.J.M. Rosa, P.M. Coussens, and J.L. Burton. 2003. Microarray analysis of gene expression in blood neutrophils of parturient cows. Physiological Genomics (in press) (also see 10.1152/physiolgenomics.00121.2003, at: http://physiolgenomics.physiology.org/papbyrecent.shtml) Suchyta, S.P., S. Sipkovsky, R. Kruska, A. Jeffers, A. McNulty, M.J. Coussens, R.J. Tempelman, R.G. Halgren, P.M. Saama, D.E. Bauman, Y.R. Boisclair, J.L. Burton, R.J. Collier, E.J. DePeters, T.A. Ferris, M.C. Lucy, M.A. McGuire, J.F. Medrano, T.R. Overton, T.P. Smith, G.W. Smith, T.S. Sonstegard, J.N. Spain, D.E. Spiers, J. Yao, and P.M. Coussens. 2003. Development and testing of a high-density cDNA microarray resource for cattle. Physiological Genomics 15:158-164. Erskine RJ, S.A. Wagner, and F.J. DeGraves. 2003. Mastitis therapy and pharmacology. Vet Clinics N Am: Food Animal Practice, 19:109-138. Burton, J.L., and R.J. Erskine. 2003. Immunity and mastitis: Some new ideas for an old disease. Vet Clinics N Am: Food Animal Practice, 19:1-45. Missouri: Refereed Journals Hebert, V. R., J. R. Middleton, E. Tomaszewska, and L. K. Fox. 2003. Methodology for quantifying residues of chlorhexidine in raw dairy milk. J. Agric. Food. Chem. 51(3):567-570. Middleton, J. R., V. R. Hebert, L. K. Fox, E. Tomaszewska, and J. Lakritz. 2003. Elimination kinetics of chlorhexidine in milk following intramammary infusion as a treatment to stop lactation in mastitic mammary gland quarters of cows. J. Am. Vet. Med. Assoc. 222(12):1746-1749. Middleton, J. R., D. Hardin, B. Steevens, R. Randle, and J. W. Tyler. Utility of quarter California mastitis test and quarter milk somatic cell count as screening tests for detecting subclinical intramammary infections in a herd with a high bulk tank somatic cell count. J. Am. Vet. Med. Assoc. In Press. Middleton, J. R., C. D. Luby, L. Viera, J. W. Tyler, and S. Casteel. Influence of Staphylococcus aureus intramammary infection on serum Cu, Fe, and Zn concentrations. J. Dairy Sci. In Press. Abstracts and Proceedings Middleton, J. R. 2003. Recent research on Staphylococcus aureus mastitis. Proceedings of the 111th Annual Meeting of the Missouri Veterinary Medical Association. pp. 333-338. Luby, C. D., L. Viera, J. R. Middleton, J. W. Tyler, and S. Casteel. 2003. Influence of Staphylococcus aureus Intramammary Infection on Serum Copper, Zinc, and Iron Concentrations. Proceedings of the 42nd Annual Meeting of the NMC. pp. 326-327. Middleton, J. R. 2003. Staphylococcus aureus Mastitis: Recent Developments. Proceedings of the 21st Annual American College of Veterinary Internal Medicine Forum. pp. Washington: Biddle, Mary K , Larry K. Fox, and Dale D. Hancock 2003. Patterns of mycoplasma shedding in the milk of dairy cows with intramammary mycoplasma infection. 223:1163-1166 Fox, L.K., G. A. Bohach, S. U. Lee, K.T. Park, and Y.H. Park. 2003. Mastitis: Effect on milk quality, causative agents and control. Kor. J. Vet. Publ Health. 27:69-76. Lee, S. U., Y. H. Park, W. C. Davis, L. K. Fox, and G. A. Bohach. 2003. The properties of human and bovine CD8+CD26+ T Cells induced by a microbial superantigen. Advances in Experimental Medicine and Biology 524:1-4. Fox, LK, Hancock, DD, Mickelson, A, and A. Britten. 2003. Bulk tank milk analysis:Factors associated with appearance of Mycoplasma sp in milk. J. Vet. Med B 50:235-240 Middleton JR, Hebert VR, Fox LK, Tomaszewska E, Lakritz J. 2003. Elimination kinetics of chlorhexidine in milk following intramammary infusion to stop lactation in mastitic mammary gland quarters of cows. J Am Vet Med Assoc. 222(12): 1746-1749 Hebert V.R., J.R. Middleton, E. Tomaszewska, and L.K. Fox. 2003. Methodology for quantifying residues of chlorhexidine in raw dairy milk. J. Ag. and Food Chem. 51:567-570. R. N. Zadoks, W. B. van Leeuwen, D. Kreft, L. K. Fox, H. W. Barkema, Y. H. Schukken, and A. van Belkum. 2002. Comparison of Staphylococcus aureus isolates from bovine and human skin, milking rquipment, and bovine milk by phage typing, pulsed-field gel electrophoresis, and binary typing. J. Clin. Microbiol. 40:3894-3902. Middleton J.R., L.K. Fox, J.M. Gay, J.W. Tyler, and T.E. Besser. 2002. Use of pulsed-field gel electrophoresis for detecting differences in Staphylococcus aureus strain populations between dairy herds with different cattle importation practices. Epidemiol. Infect. 129:387-395. Middleton J.R., and L.K. Fox. 2002. Influence of Staphylococcus aureus strain on mammary quarter milk production. Vet Rec. 150(13):411-412. Middleton J.R., L.K.Fox, J.M. Gay, J.W. Tyler, and T.E. Besser. 2002. Influence of Staphylococcus aureus strain-type on mammary quarter milk somatic cell count and N-acetyl-beta-D-glucosaminidase activity in cattle from eight dairies. J. Dairy Sci. 85:1133-40. Oura, L.Y., L.K. Fox, C.C. Warf, and G. K. Kemp. 2002. Efficacy of two acidified chlorite postmilking teat disinfectants with sodium dodecylbenzene sulfonic acid on prevention of contagious mastitis using an experimental challenge protocol. J. Dairy Sci. 85:252-257 Joo, Y.S., J. S. Moon, L.K. Fox, G.H. Suh, N. H. Kwon, S. H. Kim, and Y. H. Park. 2003. Comparison of natural resistance-associated macrophage protein (NRAMP-1) expression between cows with high and low milk somatic cells counts. Asian-Aust. J. Anim. Sci. 16:1830-1836 Tennessee: Journal Articles Gillespie, B. E., H. Moorehead, H. H. Dowlen, D. L. Johnson, K. C. Lamar, M. J. Lewis, S. J. Ivey, and S. P. Oliver. 2002. Efficacy of extended pirlimycin therapy for treatment of chronic environmental Streptococcus species intramammary infections in lactating dairy cows. Veterinary Therapeutics, 3:(4)373-380. Murinda, S. E., L. T. Nguyen, S. J. Ivey, R. A. Almeida, and S. P. Oliver. 2002. Development of a novel single tube agar-based test system for motility enhancement and immunocapture of Escherichia coli O157:H7 by H7 flagellar-specific antibodies. J. Clin. Microbiol. 40:4685-4690. Oliver, S. P., M. J. Lewis, B. E. Gillespie, H. H. Dowlen E. C. Jaenicke, and R. K. Roberts. 2003. Milk production, milk quality and economic benefit associated with prepartum antibiotic treatment of heifers. J. Dairy Sci. 86:1187-1193. Almeida, R. A., D. A. Luther, R. Nair, and S. P. Oliver. 2003. Binding of host glycosaminoglycans and milk proteins: role in the pathogenesis of Streptococcus uberis mastitis. Vet. Microbiol. 94(2):131-141. Oliver, S. P., R. A. Almeida, B. E. Gillespie, S. J. Ivey, H. Moorehead, P. Lunn, H. H. Dowlen, D. L. Johnson, and K. C. Lamar. 2003. Efficacy of extended pirlimycin therapy for treatment of experimentally-induced Streptococcus uberis intramammary infections in lactating dairy cattle. Veterinary Therapeutics 4(3):299-308. Pangloli, Philipus, Yobouet Dje, S. P. Oliver, A. G. Mathew, D. A. Golden, W. J. Taylor, and F. A. Draughon. 2003. Evaluation of methods for recovery of Salmonella from dairy cattle, poultry and swine farms. J. Food Prot. (Accepted). Rambeaud, M., R. A. Almeida, G. M. Pighetti, and S. P. Oliver. 2003. Dynamics of leukocytes and cytokines during experimentally induced Streptococcus uberis mastitis. Vet. Immunol. Immuopathol. (Accepted). Gillespie, B. E., A. G. Mathew, F. A. Draughon, B. M. Jayarao, and S. P. Oliver. 2003. A PCR-ELISA technique for detection of somatic group-specific Salmonella spp. J. Food Prot. (Accepted). Nam, H. M., S. E. Murinda, L. T. Nguyen, and S. P. Oliver. 2003. Evaluation of universal pre-enrichment broth for isolation of Salmonella spp., Escherichia coli O157:H7 and Listeria monocytogenes from dairy farm environmental samples. Foodborne Pathogens & Disease (Accepted). Book Chapters Oliver, S. P., R. N. Gonzalez, J. S. Hogan, B. M. Jayarao, and W. E. Owens. 2003. Microbiological procedures for the diagnosis of bovine udder infection. 4th Edition, The National Mastitis Council, Inc., Madison, WI. (In press). PROCEEDINGS Gillespie, B. E., R. A. Almeida, S. J. Ivey, H. Moorehead, P. Lunn, H. H. Dowlen, D. L. Johnson, K. C. Lamar, and S. P. Oliver. 2003. Efficacy of extended pirlimycin therapy for treatment of experimentally-induced Streptococcus uberis intramammary infections in lactating dairy cattle. In: Proc. Natl. Mastitis Counc. pp. 342-343. Batson, S. D., S. E. Murinda, L. T. Nguyen, and S. P. Oliver. 2003. Detection of shiga toxin-producing Escherichia coli O26 using phenotypic and genetic markers. In: Proc. Natl. Mastitis Counc. pp. 344-345. Rambeaud, M., R. A. Almeida, G. M. Pighetti, and S. P. Oliver. 2003. Characterizing the pathogenesis of experimentally-induced Streptococcus uberis mastitis. In: Proc. Natl. Mastitis Counc. pp. 346-347. Murinda, S. E., L. T. Nguyen, T. L. Landers, F. A. Draughon, A. G. Mathew, J. S. Hogan, K. L. Smith, D. D. Hancock, and S. P. Oliver. 2003. Multiplex PCR for detection of pathogenic Escherichia coli. In: Proc. Natl. Mastitis Counc. pp. 348349. Gillespie, B. E., and S. P. Oliver. 2003. Comparison of an automated ribotyping system, pulsed-field gel electrophoresis and randomly amplified polymorphic DNA fingerprinting for genotyping Streptococcus uberis. In: Proc. Natl. Mastitis Counc. pp. 350-351. Youngerman, S. M., A. M Saxton, J. L. Edwards, F. N. Schrick, C. J. Davies, S. P. Oliver, and G. M. Pighetti. 2003. Interleukin-8 receptor: A promising candidate gene for mastitis resistance. In: Proc. Natl. Mastitis Counc. pp. 358-359. Pighetti, G. M., J. L. Edwards, F. N. Schrick, A. M. Saxton, C. J. Davies, and S. P. Oliver. 2003. Cloning adult dairy cows: a viable new tool in the fight against mastitis. In: Proc. Natl. Mastitis Counc. pp. 360-361. Oliver, S. P., S. E. Murinda, and R. A. Almeida. 2003. Mastitis control, food safety and milk quality. In: Proc. National Ag in the Classroom Teachers Conference. Oliver, S. P., and F. A. Draughon. 2003. Research and educational programs of The University of Tennessee Food Safety Center of Excellence. In: Proc. National Ag in the Classroom Teachers Conference. Rambeaud, M., R. A. Almeida, G. M. Pighetti, and S. P. Oliver. 2003. Characterizing the pathogenesis of experimentally-induced Streptococcus uberis mastitis. In: Department of Animal Science Annual Report, The University of Tennessee, Knoxville. http://www.agriculture.utk.edu/ansci.annualreports.htm#AnimalHealth. Murinda, S. E., L. T. Nguyen, T. L. Landers, F. A. Draughon, A. G. Mathew, J. S. Hogan, K. L. Smith, D. D. Hancock, and S. P. Oliver. 2003. Multiplex PCR for detection and confirmation of Shiga-toxin producing Escherichia coli. In: Department of Animal Science Annual Report, The University of Tennessee, Knoxville. http://www.agriculture.utk.edu/ansci.annualreports. htm#AnimalHealth. Batson, S. D., S. E. Murinda, L.T. Nguyen, and S. P. Oliver. 2003. Phenotypic and genetic characterization of Escherichia coli O26 isolates from North America using carbohydrate fermentation and genes encoding virulence factors. In: Department of Animal Science Annual Report, The University of Tennessee, Knoxville. http://www.agriculture.utk.edu/ansci. annualreports.htm#AnimalHealth. Oliver, S. P., S. E. Murinda, and R. A. Almeida. 2003. Mastitis control, food safety and quality milk production. In: Department of Animal Science Annual Report, The University of Tennessee, Knoxville. http://www.agriculture.utk.edu/ansci.annualreports.htm#AnimalHealth. Murinda, S. E., L. T. Nguyen, H-M. Nam, R. A. Almeida, and S. P. Oliver. 2003. Detection of Shiga-toxin producing Escherichia coli, Listeria monocytogenes, Campylobacter jejuni and Salmonella spp. from dairy farms. In: Department of Animal Science Annual Report, The University of Tennessee, Knoxville. http://www.agriculture.utk.edu/ansci.annualreports.htm# Animal Health. Youngerman, S.M., S.P. Oliver, A.M. Saxton, J.L. Edwards, F.N. Schrick, C.J. Davies and G.M. Pighetti. 2003. A Novel Candidate Genetic Marker for Mastitis Resistance in Jersey Cattle. In: Department of Animal Science Annual Report, The University of Tennessee, Knoxville. http://animalscience.ag.utk.edu/pdf/Reports/Youngerman_DeptReviewArticle2003.pdf ABSTRACTS Youngerman, S. M., J. L. Edwards, F. N. Schrick, M. van Amstel, H. H. Dowlen, S. P. Oliver, and G. M. Pighetti. 2002. Leukocyte phenotypes expressed by a calf cloned from a mastitis susceptible dairy cow. Abstract 84P in Proc. Conference of Research Workers in Animal Diseases.. Batson, S. D., S. E. Murinda, L.T. Nguyen, and S. P. Oliver. 2002. Phenotypic and genetic characterization of Escherichia coli O26 isolates from North America using carbohydrate fermentation and genes encoding virulence factors. Abstract 79 in Proc. Conference of Research Workers in Animal Diseases. Murinda, S. E., L. T. Nguyen, T. L. Landers, F. A. Draughon, A. G. Mathew, J. S. Hogan, K. L. Smith, D. D. Hancock, and S. P. Oliver. 2002. Multiplex PCR for detection and confirmation of Shiga-toxin producing Escherichia coli. Abstract 53 P in Proc. Conference of Research Workers in Animal Diseases. Almeida, R. A., and S. P. Oliver. 2002. Trafficking of Streptococcus uberis in bovine mammary epithelial cells. Abstract 11P in: Proc. Conference of Research Workers in Animal Diseases. Rambeaud, M., G. M. Pighetti, and S. P. Oliver. 2002. Dynamics of leukocytes and cytokines during experimentally-induced Streptococcus uberis mastitis. Abstract 10P in: Proc. Conference of Research Workers in Animal Diseases. Pighetti, G. M., S. Youngerman, and S. P. Oliver. 2002. A novel and promising candidate gene for mastitis resistance. Abstract 134 in Proc. Conference of Research Workers in Animal Diseases. Jackson, F. R., P. Pangoli, Y. Dje, S. P. Oliver, A. Mathew, D. A. Golden, W. J. Taylor, and F. A. Draughon. 2003. Evaluation of methods for recovery of Salmonella from poultry and swine feed. Abstract PO86 in Intl. Assoc. Food Prot. p.88. Pangoli, P., Y. Dje, W. J. Taylor, D. A. Golden, S. P. Oliver, and F. A. Draughon. 2003. Evaluation of methods for recovery of Salmonella from dairy environmental samples. Abstract PO92 in Intl. Assoc. Food Prot. p.91. Lamar, K. D., P. Pangoli, D. A. Golden, S. P. Oliver, and F. A. Draughon. 2003. Geographic Information System and epidemiological associations among foodborne pathogens at the farm. Abstract PO141 in Intl. Assoc. Food Prot. p.108. Almeida, R. A., and S. P. Oliver. 2003. Development of an experimental Streptococcus uberis intramammary infection model. Abstract submitted to Conference of Research Workers in Animal Diseases. Rambeaud, M, S.M. Youngerman, A.M. Saxton and G.M. Pighetti. 2003. Association between functional activity of neutrophils and single nucleotide polymorphisms of CXCR2 in dairy cows. Abstract submitted to Conference of Research Workers in Animals Diseases. Pighetti, G.M., S.M. Youngerman, A.M. Saxton, and S.P. Oliver. 2003. Identification of Single Nucleotide Polymorphisms Within the Bovine CXCR2 Gene in Holstein and Jersey Cattle. Abstract submitted to Conference of Research Workers in Animals Diseases. Minnesota: Peer-Reviewed Literature Godden, S., P. Rapnicki, S. Stewart, J. Fetrow, A. Johnson, R. Bey, and R. Farnsworth. 2003. Effectiveness of an Internal Teat Seal in the Prevention of New Intramammary Infections During the Dry and Early Lactation Periods in Dairy Cows. J. Dairy Sci. In Press. Godden, S., R. Bey, R. Farnsworth, J. Reneau, and M. LaValle. 2002. Field validation of a milk line sampling device for monitoring milk quality and udder health. J. Dairy Sci. 85:1468-1475. Abstracts Godden, S., P. Rapnicki, S. Stewart, J. Fetrow, A. Johnson, R. Bey, and R. Farnsworth. Effectiveness of an internal teat sealant in the prevention of new intramammary infections during the dry and early lactation periods in dairy cows when used with an intramammary antibiotic. J. Dairy Sci. 86(Suppl. 1): 85-86. Conference Proceedings Godden, S., P. Rapnicki, S. Stewart, A. Johnson, R. Bey, and R. Farnsworth. 2003. 2003. An evaluation of Orbeseal teat seal for prevention of intramammary infections in dry cows. In Proc. Annual Dairy Health Management Update Meeting. Guelph, Ont. May 28-29, 2003. Godden, S., P. Rapnicki, S. Stewart, A. Johnson, R. Bey, and R. Farnsworth. 2003. Effectiveness of an internal teat sealant in the prevention of new intramammary infections during the dry and early lactation periods in dairy cows when used with an intramammary antibiotic. In Proc. Annu. Meet. of the Minnesota Dairy Health Conference. St. Paul, MN. May 20-22, 2003. Pg. 75-97. Godden, S., P. Rapnicki, S. Stewart, A. Johnson, R. Bey, and R. Farnsworth. 2003. Effectiveness of an internal teat sealant in the prevention of new intramammary infections during the dry and early lactation periods in dairy cows when used with an intramammary antibiotic. In Proc. 106th Annual Meeting of the Minnesota Veterinary Medical Association. Bloomington, MN. Feb. 6-8, 2003. Godden, S., P. Rapnicki, S. Stewart, A. Johnson, R. Bey, and R. Farnsworth. 2003. Effectiveness of an internal teat sealant in the prevention of new intramammary infections during the dry and early lactation periods in dairy cows when used with an intramammary antibiotic. In Proc. 42nd Annual Meeting of the National Mastitis Council. Fort Worth, Texas. Jan. 26-29, 2003. Pg. 136 152. New York: Gonzalez R.N., Wilson D.J. 2003. Mycoplasmal mastitis in dairy herds. Vet Clin North Am Food Anim Pract 19:199-221. Grohn Y.T., Rajala_Schultz P.J., Allore H.G., DeLorenzo M.A., Hertl J.A., Galligan D.T. 2003. Optimizing replacement of dairy cows: modeling the effects of diseases. Prev Vet Med 61:27-43. Sauders B.D., Fortes E.D., Morse D.L., Dumas N., Kiehlbauch J.A., Schukken Y.H., Hibbs J.R., Wiedmann M. 2003. Molecular subtyping to detect human listeriosis clusters. Emerg Infect Dis 9:672-980. Schukken Y.H., Wilson D.J., Welcome F., Garrison_Tikofsky L., Gonzalez R.N. 2003. Monitoring udder health and milk quality using somatic cell counts. Vet Res 34:579-596. Schukken Y.H., Grohn Y.T., McDermott B., McDermott J.J. 2003. Analysis of correlated discrete observations: background, examples and solutions. Prev Vet Med 59:223-240. Schukken, Y.H., Grohn, Y.T., McDermott, B., McDermott, J.J., Analysis of correlated discrete observations: background, examples and solutions. Preventive Veterinary Medicine 59 (2003) P. 223-240. Schukken, Y.H., Wilson, D.J., Welcome, F., Garrison-Tikofsky, L., Gonzalez,R., 2003. Monitoring udder health and milk quality using somatic cell counts. Vet. Res. 34:1-18. Tikofsky L.L., Barlow J.W., Santisteban C., Schukken Y.H. 2003. A Comparison of Antimicrobial Susceptibility Patterns for Staphylococcus aureus in Organic and Conventional Dairy Herds. Microb Drug Resist 1: 39-45. Wilson D.J., Gonzalez R.N. 2003. Vaccination strategies for reducing clinical severity of coliform mastitis. Vet Clin North Am Food Anim Pract 19:187-197. Zadoks R.N., Gillespie B.E., Barkema H.W., Sampimon O.C., Oliver S.P., Schukken Y.H. 2003. Clinical, epidemiological and molecular characteristics of Streptococcus uberis infections in dairy herds. Epidemiol Infect 130:335-349. Proceedings Schukken, Y.H. New Developments in the treatment and prevention of mastitis. Recueil Des Conferences 28th Congres. L‘Association Des medecins Veterinaires Practiciens du Qeubec (A.M.V.P.Q) - Laval. Quebec-August 24, (2003) P. 1-19. Schukken Y.H., Zadoks R.N., White L., Dopfer, D.D. 2003. Mastitis: Old Problems, New Approaches, Minnesota Dairy Health Conference, May 20-22, P. 25-41. Schukken, Y.H. 2003. Mastitis Control Strategies Q-Max and beyond. The Northeast Veterinary Dairy Symposium, Cooperstown, NY May 27-29. Schukken, Y. H, van Schaik G., Zadoks, R. N. 2003. Handling of infectious herd problems: role of the individual in herd and population. 95th Annual Conference for Veterinarians, March 14-16. Pg. 104-114. Schukken Y.H., Zadoks R. N., White L., Dopfer D.D. 2003. Molecular and Mathematical Epidemiology of Bovine Mastitis. Society for Veterinary Epidemiology and Preventive Medicine. March-April, Pg. 13-29. Wallace J. A., Schukken Y. H., Welcome F. 2003. Measuring Stimulation‘s Effect with Milk Flow Curves. 95th Annual Conference for Veterinarians, March 14-16, Pg. 228-238. Zadoks Ruth N. 2003. Streptococcus uberis: Contagious or environmental? - A question that also pertains to other mastitis pathogens, in particular Staphylococcus aureus. 95th Annual Conference for Veterinarians, March 14-16, Pg 115-119. Vermont: Refereed Journals Fan, W., K. Plaut, A.J. Bramley, J.W. Barlow, S.A. Mischler, and D.E. Kerr. 2003. Persistency of adenoviral-mediated lysostaphin expression in goat mammary glands. J. Dairy Sci. (in press) Kerr, D.E., and O. Wellnitz. 2003. Transgenic models for animal science research and application. J. Dairy Sci. (in press). Mitra, A., K.S. Hruska, O. Wellnitz, D.E. Kerr, A. Capuco, and R. J. Wall. 2003. Expression of lysostaphin in milk of transgenic mice affects the growth of neonates. Transgenic Research 12:597-605. Carter, E.W., and D.E. Kerr. 2003. Optimization of a DNA-based vaccination in cows using green fluorescent protein and Protein A as a prelude to immunization against staphylococcal mastitis. J. Dairy Sci. 86:1177-1186. Abstracts and Proceedings Wellnitz, O. and D.E. Kerr. 2003. Establishment of a bovine cell-culture system to study the genomic response of mammary epithelial cells to infection with Staphylococcus aureus. J. of Dairy Sci. 86 (Suppl. 1):92. Powell, A.M., N.C. Talbot, K.D. Wells. D.E. Kerr, V.G. Pursel, and R.J. Wall. 2003. Source of fetus determines efficiency of production of nuclear transfer jersey cattle genetically engineered to resist mastitis. Theriogenology 59:281. Guelph: Proceedings: Bassel, L, Kelton, DF, Godkin, MA, Leslie, KE, Lissemore, KD. Risk factors for intramammary infection at first calving in Ontario dairy heifers. Proc 36th Annual AABP Convention . Pg. 177. 2003. Leslie, K.E. and Dingwell, R.T. Background to Dry Cow Therapy: What, Where, Why Is It Still Relevant. 42nd National Mastitis Council Annual Meeting Proceedings. Forth Worth, Texas. P5-17. 2003. Barratt, K., Leslie, K.E., and Bashiri, A. An Evaluation of the PortaSCC Test for Determining Udder Health Status in Dairy Cows at Dry Off. 42nd National Mastitis Council Annual Meeting Proceedings. Forth Worth, Texas. P280-281. 2003. Bassel, L, Kelton, D, Godkin, A., Leslie, K.E., and Lissemore, K. Risk Factors for Intramammary Infection at First Calving in Heifers from Ontario Sentinel Herds. 42nd National Mastitis Council Annual Meeting Proceedings. Forth Worth, Texas. P282-283. 2003. Dingwell, R.T., Leslie, K.E., Schukken, Y.H., Sargeant, J.M., Timms, L. and Shirley, J.E. Evaluation of the California Mastitis Test to Detect Intramammary Infections in Early Lactation. 42nd National Mastitis Council Annual Meeting Proceedings. Forth Worth, Texas. P294-295. 2003. Dingwell, R.T. McLaren, C.J., Osborne, T.M., Petersson, C.S., and Leslie, K.E. Reliability of a Manual Assessment Technique to Determine Teat Canal Closure During the Dry Period. 42nd National Mastitis Council Annual Meeting Proceedings. Forth Worth, Texas. P296-297. 2003. Dingwell, R.T., Timms, L.L., Sargeant, J.M., Kelton, D.F., Schukken, Y.H., and Leslie, K.E. The Association of Teat Canal Closure and Other Risk Factors for New Dry Period Intramammary Infections. 42nd National Mastitis Council Annual Meeting Proceedings. Forth Worth, Texas. P298-299. 2003. Leslie, K.E., Schukken, Y.H., Archambault, M., Dingwell, R.T., Bashiri, A., and Leslie, C.F. Antimicrobial Resistance Patterns of Staphylococcus aureus Isolated from Intramammary Infections Before and After the Dry Period of Dairy Cattle in Canada. 42nd National Mastitis Council Annual Meeting Proceedings. Forth Worth, Texas. P324-325. 2003. Petersson, C.S., Leslie, K.E., Kelton, D.F., and Mallard, B.A. Impact of Two Coliform Mastitis Vaccination Schedules on Immune Response, Milk Yield, Intramammary Infection and Dry Matter Feed Intake in Holstein Dairy Cattle. 42nd National Mastitis Council Annual Meeting Proceedings. Forth Worth, Texas. P356-357. 2003. Scholl, D., Barkema, H., Lesperance, A., Lacasse, P., Leslie, K.E., Kelton, D., and Tomita, G. Canadian Bovine Mastitis Research Network. 42nd National Mastitis Council Annual Meeting Proceedings. Forth Worth, Texas. P370-371. 2003. Wallace, J.A., Stipetic, K., Schukken Y.H., Dingwell, R.T., Baillargeon, P., Bacic, G., and Leslie, K.E. Impact of Intramammary Treatment of CMT-Positive Quarters in Early Postpartum Dairy Cows. 42nd National Mastitis Council Annual Meeting Proceedings. Forth Worth, Texas. P391-392. 2003. Gawrylash, S., Leslie, K.E., Archambault, M., and Bashiri, A. Evaluation of the ColiMast test for Detection of Coliform Mastitis in Dairy Cattle. American Association of Bovine Practitioners Proceedings. Madision, WI. 35:192. Sept 2002. Petersson, C., Leslie, K.E., Kelton, K., and Mallard, B. Impact of two Coliform Mastitis Vaccination Schedules on Milk Yield, Dry Matter Feed Intake and Intramammary Infections of Holstein Dairy Cattle. American Association of Bovine Practitioners Proceedings. Madision, WI. 35:266. Sept 2002. Dingwell, R.T., Duffield, T.F., Leslie, K.E., Keefe, G., DesCoteaux, L., Kelton, D.F., Lissemore, K.D., and Schukken, Y.H. Efficacy of Intramammary Tilmicosin at Drying off and other risk factors for prevention of new intramammary infections during the dry period. American Association of Bovine Practitioners. Madision, WI. 35:269. Sept 2002. Publications: Dingwell, R.T., Leslie, K.E., Duffield, T.F., Schukken, Y.H., DesCoteaux, L., Keefe, G.P., Kelton, D.F., Lissemore, K.D., Shewfelt, W., Dick, P., Bagg, R.: Efficacy of intramammary Tilmicosin and risk factors for cure of Staphylococcus aureus infection in the dry period. Journal of Dairy Science. 86:159-168, 2003. Dingwell, R.T., Duffield, T.F., Leslie, K.E., Keefe, G.P., DesCoteaux, L., Kelton, D.F., Lissemore, K.D., Schukken, Y.H., Dick, P., Bagg, R.: Efficacy of intramammary Tilmicosin administered at drying-off, and other risk factors, for the prevention of new intramammary infections during the dry period. Journal of Dairy Science. 85:3250-3259, 2002. Dingwell RT, KE Leslie, YH Schukken, JM Sargeant, and LL Timms. Evaluation of the California Mastitis Test to Detect an Intramammary Infection With a Major Pathogen in Early Lactation Dairy Cows. Canadian Veterinary Journal 44:407-412, 2003. Dingwell RT, TF Duffield, KE Leslie, GP Keefe, L. DesCoteaux, P. Dick and R. Bagg. Comparative efficacy of a novel intramammary dry cow antibiotic to eliminate subclinical mastitis in the dry period. Bovine Practitioner 37: 98-106, 2003. Dingwell RT, DF Kelton, and KE Leslie. Management of the Dry Cow in Control of Peripartum Disease and Mastitis. Veterinary Clinics of North America: Food Animal Practice. March (19): 235-265. 2003. Wagter, L.C., Mallard, B.A., Wilkie, B.N., Leslie, K.E., Boettcher, P.J., and Dekkers, J.C.M. The Relationship Between Milk Production and Antibody Response to Ovalbumin During the Peripartum Period. J Dairy Sci. 86:169-173. Jan 2003. LeBlanc, S.J., Duffield, T.F., Leslie, K.E., Bateman, K.G., TenHag, J., Walton, J.S. and Johnson, W.H. The Effect of Prepartum injection of Vitamin E on Health in Transition Dairy Cows. J Dairy Sci, 85:1416-1426. 2002. Wallace, J.A., Stipetic, K., Schukken, Y.H., Dingwell, R.T., Baillargeon, P., Bacic, G., and Leslie, K.E. Mastitis Diagnosis and Treatment in Fresh Cows: An evaluation of a diagnostic and treatment protocol for intramammary infections in early postpartum dairy cows. The Bovine Practitioner. Submitted. Oct 2003.

Impact Statements

Greater characterization and understanding of mechanisms associated with mastitis susceptibility and resistance
Further identification and manipulation of virulence factors of mastitis pathogens which greatly enhances host defense
Wide assessment and application of a variety of technologies and modalities advancing udder health control and ensuring food safety

Date of Annual Report: 01/11/2005

Report Information

Annual Meeting Dates: 11/17/2004 - 11/19/2004
Period the Report Covers: 12/01/2003 - 11/01/2004

Participants

Pighetti, Gina (pighetti@utk.edu) - University of Tennessee;
Dingwell, Randy (rdingwell@upei.ca) - University of Prince Edward Island;
Godden, Sandra (godde002@umn.edu) - University of Minnesota;
Timms, Leo (ltimms@iastate.edu) - Iowa State University;
Leslie, Ken (keleslie@ovc.uoguelph.ca) - University of Guelph;
Fox, Larry (fox@vetmed.wsu.edu) - Washington State University;
Morin, Dawn (d-morin@uiuc.edu) - University of Illinois;
Kerr, Kirklyn (kirklyn.kerr@uconn.edu) - University of Connecticut;
Hurley, Walt (wlhurley@uiuc.edu) - University of Illinois;
Owens, Bill (wowens@agcenter.lsu.edu) - University of Louisiana;
Schultz, Bruce (bschultz@vet.k-state.edu) - Kansas State University;
Oliver, Steve (soliver@utk.edu) - University of Tennessee;
Kelton, Dave (dkelton@uoguelph.ca) - University of Guelph;
Jayarao, Bhushan (bmj3@psu.edu) - University of Pennsylvania;
Andrew, Sheila (sheila.andrew@uconn.edu) - University of Connecticut;
Middleton, John (middletonjr@missouri.edu) - University of Missouri;
Barkema, Herman (barkema@upei.ca) - University of Prince Edward Island;
Erskine, Ron (erskine@cvm.msu.edu) - Michigan State University

Brief Summary of Minutes

Minutes Attachment:

Attachment

Accomplishments

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.

Publications

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Impact Statements

The group continued to collaborate (i.e. several multi-state studies were concluded or are in progress) to combine basic and applied science aimed at reducing the incidence and severity of mastitis in dairy cows in order to improve animal health, productivity and milk quality.
Gained a much better understanding of cow-level, quarter-level and management-level risk factors at dry off and during the close-up (transition) period that contribute to new intramammary infections during the dry period. Examples include dry-off strategies, the role of teat sealants, and both nutritional management or photoperiod manipulation to improve immune function. This will lead to more effective management strategies that producers can implement in order to lower the incidence of dr
Continued to improve our understanding of bovine immune/resistance factors and to evaluate vaccination strategies to prevent coliform mastitis. This includes a greater understanding of the relative importance of cow genetics (heritability) vs environment (management) influencing the risk for mastitis.
Continued evaluation of new teat dip formulations to provide producers with reliable information and allow them to choose the best product for reducing mastitis.
Gained a greater understanding of causes of prepartum mastitis in primiparous heifers, as well as concluded a large multi-state study examining the efficacy of prepartum intramammary therapy in primiparous heifers. Preliminary results indicated that while prepartum antibiotic treatment heifers would cure existing intramammary infections, this reduction in IMI did not translate into a significant improvement in milk production, SCC, or reproductive performance.
Continue work to describe molecular typing and distribution plus the antibiotic-susceptibility patterns of common mastitis pathogens such as Staphylococcus aureus and Escherichia coli. Continued work to better understand pathogenicity that differentiate acute from chronic coliform infections.
Continued to investigate the utility of on-farm diagnostic tools such as the California Mastitis Test (CMT) and on-farm culture systems. These tools will allow producers to more effectively diagnose and treat subclinical infections at calving or clinical mastitis cases. Also developed in-lab diagnostic tools such as real-time PCR procedures for detection of common mastitis pathogens in milk.
Initiated a collaborative study to develop an electronic data transfer system to more efficiently and expediently provide producers on a Dairy Herd Improvement (DHIA) testing program with culture results from the mastitis laboratory. This system should allow producers to more efficiently access culture data for the purpose of making timely cow-management and treatment decisions.

Date of Annual Report: 12/06/2005

Report Information

Annual Meeting Dates: 11/02/2005 - 11/04/2005
Period the Report Covers: 11/01/2004 - 11/01/2005

Participants

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Brief Summary of Minutes

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Accomplishments

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.

Publications

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Impact Statements

Greater characterization and understanding of mechanisms associated with mastitis susceptibility and resistance.
Further identification and manipulation of virulence factors of mastitis pathogens which will lead to advances in understanding and managing the host-pathogen interaction.
Wide assessment and application of a variety of technologies and modalities which have led to or will lead to advances in udder health and ensure food safety.

Date of Annual Report: 02/08/2007

Report Information

Annual Meeting Dates: 11/08/2006 - 11/10/2006
Period the Report Covers: 10/01/2005 - 10/01/2006

Participants

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Brief Summary of Minutes

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Accomplishments

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.

Publications

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Impact Statements

Date of Annual Report: 12/21/2007

Report Information

Annual Meeting Dates: 11/07/2007 - 11/09/2007
Period the Report Covers: 10/01/2002 - 09/01/2007

Participants

Herman Barkema - University of Calgary (barkema@ucalgary.ca);
John Barlow - University of Vermont (john.barlow@uvm.edu);
David E. Kerr - University of Vermont (david.kerr@uvm.edu);
A. John Bramley - University of Vermont (john.bramley@uvm.edu);
Daniel Scholl - University of Montreal (daniel.scholl@umontreal.ca);
Gina Pighetti - University of Tenessee (pighetti@utk.edu);
Stephen Oliver - University of Tenessee (soliver@utk.edu);
Bruce Schultz - Kansas State University (bschultz@vet.ksu.edu);
Sheila Andrew - University of Connecticut (Sheila.andrew@uconn.edu);
Pamela Ruegg - University of Wisconsin (PLRuegg@wisc.edu);
Ron Erskine - Michigan State University (erskine@cvm.msu.edu);
Lorraine M. Sordillo - Michigan State University (sordillo@msu.edu) ;
John Middleton- University of Missouri (middletonjr@missouri.edu);
Isis Mullarky - Virginia Polytechnical and State University (mullarky@vt.edu);
Christina S. Petersson-Wolfe - Virginia Polytechnical and State University (cspw@vt.edu);
Kirklyn M. Kerr - University of Connecticut (kirklyn.kerr@uconn.edu);
Ruth Zadoks - Cornell University (rz26@cornell.edu);
Ynte Schukken - Cornell University (yhs2@cornell.edu);
Francis L Welcome -Cornell Cooperative Extension (flw2@cornell.edu);
Sandra Godden - University of Minnesota (godde002@umn.edu);
Lawrence K. Fox -Washington State University (fox@wsu.edu);
Ken Leslie - University of Guelph (keleslie@ovc.uoguelph.ca);
;Steve Nickerson - University of Georgia (scn@uga.edu);
Gary B. Sherman - USDA-CSREES (gsherman@csrees.usda.gov);
Bhushan Jayarao - Pennsylvania State University (bmj3@psu.edu);

Joseph S. Hogan - Ohio State University (hogan.4@osu.edu);

William E. Owens- Louisiana State University (wowens@agctr.lsu.edu)
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Brief Summary of Minutes

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Accomplishments

Over the past 5-years of this project, we have studied three areas focused on identifying new means of detecting, preventing, and treating bovine mastitis. Our specific objectives were: (i) characterization of host mechanisms associated with mastitis susceptibility and resistance, (ii) characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defense, and (iii) assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. Both joint research trials and individual studies have been conducted by NE-1009 researchers and accomplishments include: - Characterization of host mechanisms associated with mastitis susceptibility and resistance led to the identification of genes associated with resistance to mastitis including the CXCR2 gene (TN), Nramp1 gene (WA), and the Uterocalin gene (IA). Ongoing studies continue to evaluate mastitis heritability by identifying susceptibility traits. - Collaborative projects evaluated management tools aimed at decreasing rates of mastitis. Continued work on evaluation of teat dip efficacy and bedding materials and conditioners. - Evaluation of mammary health during the dry period included study of keratin plug formation and development and evaluation of barrier teat sealants such as Orbeseal. - This collaborative project allowed for assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. Evaluation of novel treatment tools including Lysigin vaccine, J5 bacterin hyperimmunization, Pirlimycin therapy, and Heifer prepartum antibiotic treatment. - There was successful development of several novel techniques for identification of pathogens and vaccine efficacy including an ELISA for detection of S. aureus antibody isotypes, Tuberculin tests, Mycoplasma control programs, Microarray development, Pulse field gel electrophoresis for pathogen typing, cDNA microarray, Real-time PCR detection of pathogens in milk, and development of transgenic lysostaphin animal resistant to S. aureus mastitis. - Large collaborative projects evaluated research tools and techniques such as identification of mastitis using single as compared with duplicate aseptic samples and improving animal health and production with 2 as compared to 3 milkings per day. -Several studies focused on host/pathogen interactions including evaluation of nutrients, such as Vitamin C, and positive/negative energy status of the animal on rate of new intrammary infections (IMI). - This project allowed for successful characterization and manipulation of virulence factors of mastitis pathogens for enhancing the host defenses. Bacterial toxins were identified and studied in several species including S. aureus (SEC1), K. pneumoniae (fimbriae), S. aureus biofilms. - Various trials tested vaccines consisting of S. aureus bacterins, S. uberis virulence factors, bacteriosins/bacteriophages, phage therapy, and J5 bacterin. Antibiotic efficacy was evaluated and pathogens were tested for susceptibility and multi-drug resistance. -Innate immunity was studied in response to LPS treatment, BHV-1, and BLV. -In vitro studies were conducted to tease out the mechanisms involved in host resistance to Mastitis. Studies focused on mammary progenitor cells as well as epithelial barrier modification. - Development of new techniques for evaluation of Mastitis on the farm included an in line unit for measurement of SCC (cell sense) and on-farm bacteriology systems. -Improved milk quality through the detection of food pathogens to include, Salmonella, Listeria, and Coxiella burnetti . Improved detection of clorohexidine in milk.

Publications

Peer-Reviewed Literature (2007) Andrews, S.M., K.M. Moyes, A. A. Borm, L. K. Fox, K.E. Leslie, J.S. Hogan, S.P. Oliver,Y. H.Schukken, W. E. Owens, and C. Norman. Fassctors assocated with the risk of antibiotic residues and intramammary therapy. 2007. International Symposium on Mastitis, Ghent, Belgium. In press Bar D, Gröhn YT, Bennett G, González RN, Hertl JA, Schulte HF, Tauer LW, Welcome FL, Schukken YH. Effect of repeated episodes of generic clinical mastitis on milk yield in dairy cows. J Dairy Sci. 2007 Oct;90(10):4643-53. Barlow, J.W. Rauch, B., Welcome, F., Kim, S.G., Dubovi, E., and Schukken, Y.H. 2008. Association between Coxiella burnetii shedding in milk and subclinical mastitis in dairy cattle. Veterinary Research (accepted). Baumgart M, Dogan B, Rishniw M, Weitzman G, Bosworth B, Yantiss R, Orsi RH, Wiedmann M, McDonough P, Kim SG, Berg D, Schukken Y, Scherl E, Simpson KW. Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum. ISME J. 2007 Sep;1(5):403-18. Borm, A. A., L. K. Fox, K. E. Leslie, J. S. Hogan, S. M. Andrew, K. M. Moyes, S. P. Oliver, Y. H. Schukken, D. D. Hancock, C. T. Gaskins, W.E. Owens, and C. Norman. 2006. Effects of prepartum intramammary antibiotic therapy on udder health. J. Dairy Sc,i. 89:2090-2098. Celia, L.K., Nelson, D., and D.E. Kerr. 2008. Characterization of a bacteriophage lysin (Ply700) from Streptococcus uberis. Veterinary Microbiology (in press). Contreras, GA, Guterbock WM and Sears PM. Comparison of Systemic and Intramammary Dry Cow Treatment.. J. Am. Med Assoc. Submitted 2007 Erskine R.J., E.J. VanDyk, P.C. Bartlett, J.L. Burton, M.C. Boyle. Effect of hyperimmunization with an Escherichia coli J5 bacterin in adult lactating dairy cows. J Am Vet Med Assoc 231:1092-1097, 2007. Fox, L.K. 2007. Prevalence, incidence and risk factors of heifer mastitis. Vet. Micro Gehring R and Smith GW. An overview of factors affecting the disposition of intramammary preparations used to treat bovine mastitis. J Vet Pharmacol Ther 29: 237-241, 2006. Gill, J., Sabour, P., Leslie, K., Griffiths, M. Efficacy and pharmacokinetics of bacteriophage therapy in the treatment of subclinical Staphylococcus aureus mastitis in lactating dairy cattle. Antimicrobial Agents and Chemotherapy. Vol 50 (9): 2912-2918. 2006. Gillespie B. E., B.M. Jayarao, J. W. Pankey, and S. P. Oliver. 1998. Subtyping of Streptococcus dysgalactiae and Streptococcus uberis isolated from bovine mammary secretions by DNA fingerprinting. J. Vet Med. [B]-45:585-593. Gilliespie, B. E., B. M. Jayarao, H. H. Dowlen, and S. P. Oliver. 1999. Analysis and frequencies of Bola-DRB3.2 alleles from Jersey cows by PCR-RFLP. J. Dairy Sci. 82:2049-2053. Godden, S., R. Bey, K. Lorch, R. Farnsworth, and P. Rapnicki. 2007. Ability of organic and inorganic bedding materials to promote growth of environmental bacteria. J. Dairy Sci. 2007: Accepted Hogan, J. S., S. L. Wolf, and C. S. Petersson-Wolfe. 2007. Bacterial counts in organic materials used as free-stall bedding following treatment with a commercial conditioner. J. Dairy Sci. 90:1058-1062. Hogan, J. S., S. L. Wolf, and C. S. Petersson-Wolfe. 2007. Bacterial counts in organic materials used as free-stall bedding following treatment with a commercial conditioner. J Dairy Sci. 90(2):1058-1062 Jayarao, B. M., S. C. Donaldson, B. A. Straley, A. A. Sawant, N. V. Hegde, and J. L. Brown.3 2006. A survey of foodborne pathogens in bulk tank milk and raw milk consumption among farm families in Pennsylvania. J Dairy Sci. 89:2451-2458. Lim, G., Leslie, K. Dingwell, R., Timms, L., Kelton, D. Duffield, T. Adherence and Efficacy of an External Teat Sealant to Prevent New Intramammary Infections in the Dry Period. Journal of Dairy Science. Vol 90:1289-1300. 2007. Lim, G., Leslie, K. Dingwell, R., Timms, L., Kelton, D. Herd Management Factors that Affect the Mean Duration and Variation of Adherence of an External Teat Sealant. Journal of Dairy Science. Vol 90: 1301-1309. 2007. Luby CD, Middleton JR, Ma J, Rinehart CL, Bucklin S, Kohler C, Tyler JW. 2007. Characterization of the antibody isotype response in serum and milk of heifers vaccinated with a Staphylococcus aureus bacterin (LysiginTM). J Dairy Res. 74(2):239-246. M. A. Munoz and R. N. Zadoks. 2007. Short Communication: Patterns of fecal shedding of Klebsiella by dairy cows. Journal of Dairy Science 90(3):1220. M. A. Munoz, C. Ahlstrom, B. J. Rauch and R. N. Zadoks. 2006. Fecal shedding of Klebsiella pneumoniae by dairy cows. Journal of Dairy Science 89(9):3425. Middleton JR, Luby CD, Adams DS. 2008. Efficacy of vaccination against staphylococcal mastitis: A review and new data. Vet Microbiol. In Review. Middleton JR, Luby CD. 2008. Escherichia coli mastitis in cattle being treated for Staphylococcus aureus mastitis. Vet Rec. In Press. Molecular Epidemiology of two Klebsiella pneumoniae outbreaks on a dairy farm in New York State. J. Clinical Microbiology. Oct 10; [Epub ahead of print] Munoz, M. A., F. L. Welcome, Y. H. Schukken, and R. N. Zadoks. 2007. Oliver, S. P., B. E. Gillespie, and B. M. Jayarao. 1998. Detection of new and persistent Streptococcus uberis and Streptococcus dysgalactiae intramammary infections by polymerase chain reaction-based DNA fingerprinting. FEMS Microbiol. 160:69-73. Oliver, S. P., B. M. Jayarao, B. M. and R. A. Almeida. 2005. Foodborne pathogens in milk and the dairy farm environment: food safety and public health implications. Foodborne Pathog Dis. 2:115-129. Owens, W. E., and W. Allen Nipper.Development of a Mycoplasma Mastitis Control Program in Louisiana. 2008. Journal of the Professional Animal Scientist. In press Park YH, SU Lee, WA Ferens, S Samuels, WC Davis, LK Fox, JS Ahn, KS Seo, BS Chang, SY Hwang, and GA Bohach (2006) Unique features of bovine lymphocytes exposed to a staphylococcal superantigen. Journal of Veterinary Science 7:233-239. Paulin-Curlee, G. G., S. Singer, S. Sreevatsan, R. R. Isaacson, J. Reneau, D. Foster, and R. Bey. 2007. Genetic diversity of mastitis-associated Klebsiella pneumoniae in dairy cows. J. Dairy Sci. 90:3681-3689. Petersson-Wolfe, C. S., K. E. Leslie, T. Osborne, B. W. McBride, R. Bagg, G. Vessie, P. Dick, and T. F. Duffield. 2007. Effect of monensin delivery method on dry matter intake, body condition score, and metabolic parameters in transition dairy cows. J Dairy Sci. 90(4):1870-1879. Petersson-Wolfe, C. S., S. L. Wolf, and J. S. Hogan. 2007. In vitro growth of enterococci of bovine origin in bovine mammary secretions from various stages of lactation. J Dairy Sci. 90(9):4226-4231. Petersson-Wolfe, C.S., S. Adams, S. L. Wolf, and J. S. Hogan. 2007. Genomic typing of enterococci isolated from bovine mammary glands and environmental sources. J. Dairy Sci. In press. Pol M, and P. L. Ruegg. 2007. Relationship between antimicrobial usage and antimicrobial susceptibility of Gram-positive mastitis pathogens. J Dairy Sci 90:262-273. Pol M, and P. L. Ruegg. 2007. Treatment practices and quantification of antimicrobial usage in conventional and organic dairy farms in Wisconsin. J Dairy Sci 90:249-261. Powell, A.M., D.E. Kerr, D. Guthrie, and R.J. Wall. 2007. Lactation induction as a predictor of post-parturition transgene expression in bovine milk. Journal of Dairy Research 74:247-254. Quesnell RR, Erickson JE, and Schultz BD. Apical electrolyte concentration modulates barrier function and tight junction protein localization in bovine mammary epithelium. Am J Physiol 292: C305-318, 2007. Quesnell RR, Han X, and Schultz BD. Glucocorticoids stimulate ENaC upregulation in bovine mammary epithelium. Am J Physiol 292: C1739-1745, 2007. R. N. Zadoks and J. L. Watts. 2007. Species identification of CNS: Genotyping as the Gold standard. Veterinay Microbiology. Accepted conditional on revisions. Rezamand, P., S. A. Andrew, K. M. Moyes, and R. M. Clark. 2007. Effects of energy status, breed and plasma metabolites on new intramammary infections in periparturient Holstein and Jersey cows during the transition period. J. Dairy Sci. 90:5097-5107. Sanford, C.J., Keefe, G.P., Dohoo, I.R., Leslie, K.E. Assessment of antimicrobial transfer from treated to untreated mammary gland quarters by use of high-pressure liquid chromatography for detection of cloxacillin in milk samples from nonlactating dairy cows. American Journal of Veterinary Research. Vol 67 (7): 1140-1144. 2006. Schmitt-van de Leemput, E., and R. N. Zadoks. 2007. Genotypic and Phenotypic Macrolide and Lincosamide Resistance of Streptococcus uberis from Cases Of Clinical Mastitis. Journal of Dairy Science. Journal of Dairy Science 90(11):5089 Srinivasan V, Gillespie BE, Lewis MJ, Nguyen LT, Headrick SI, Schukken YH, Oliver SP. Phenotypic and genotypic antimicrobial resistance patterns of Escherichia coli isolated from dairy cows with mastitis. Vet Microbiol. 2007 Oct 6;124(3-4):319-28. Stewart, S.C., P. Rapnicki, J. R. Lewis, and M. Perala. 2007. Detection of low frequency external electronic identification devices using commercial panel readers. J. Dairy Sci. 90:4478-4482. Straley, B. A., S. C. Donaldson, N. V. Hedge, A. A. Sawant, V. Srinivasan, S. P. Oliver, and B. M. Jayarao. 2006. Public health significance of antimicrobial-resistant gram-negative bacteria in raw bulk tank milk. Foodborne Pathog Dis. 3:222-233. Wang, L., and B. M. Jayarao. 2001. Genotypic and phenotypic characterization of Pseudomonas fluorescens isolated from bulk tank milk. J Dairy Sci.84:1421-1429. Weiss, W. P., and J. S. Hogan. 2007. Effects of dietary vitamin C on neutrophil function and responses to intramammary infusion of lipopolysaccharide in periparturient dairy cows. J. Dairy Sci. 90:731-739. Wilson DJ, Grohn YT, Bennett GJ, González RN, Schukken YH, Spatz J. Comparison of J5 vaccinates and controls for incidence, etiologic agent, clinical severity, and survival in the herd following naturally occurring cases of clinical mastitis. J Dairy Sci. 2007 Sep;90(9):4282-8. Wilson DJ, Mallard BA, Burton JL, Schukken YH, Gröhn YT. Milk and serum J5-specific antibody responses, milk production change, and clinical effects following intramammary Escherichia coli challenge for J5 vaccinate and control cows. Clin Vaccine Immunol. 2007 Jun;14(6):693-9. Zecconi, A., L Calvinho, and L. Fox. 2006. Staphylococcus aureus intramammary infections. Bulletin of the IDF, No. 408, Brussels, Belgium Abstracts (2007) Apparao, M.D., L. Oliveira, C. Hulland, and P.L. Ruegg. Effect of intramammary treatment with Pirlimycin hydrochloride on antibiotic sensitivity of Gram-positive subclinical mastitis pathogens. J Dairy Sci 90 (supp 1): 10. Church, G. T., L.K. Fox, J.M. Gay, and C. T. Gaskins. 2007. New intramammary infections during the dry period: The effect of short (30 days) vs. long (45 or 60 days) dry periods. A preliminary report. J. Dairy Sci. 90(Suppl. 1): 468. Contreras GA, Neuder LM and Sears PM. Use of Systemic Antibiotic therapy in Prepartum heifers to Reduce the Incidences of Intramammary Infections. Submitted for the 47th Annual Meeting of theNatl Mast Council. Jan 2008 Dingwell, R., S. Godden, D. Kelton, K. Leslie, and Y. Schukken. 2007. Misclassification of udder health status due to culture of single versus duplicate quarter milk samples. Proc. Annu. Meet. of the National Mastitis Council. Jan. 21-24, 2007. San Antonio, TX. Pg. 258-259. Kerr, D.E., M. Latshaw, R. Pareek, J. Zheng, and J.P. Bond. 2007. Comparison of genomic responses to acute LPS challenge of bovine mammary epithelial cells in vitro, and bovine and murine mammary glands in vivo. 8th International Veterinary Immunology Symposium, Ouro Preto, Brazil. Lago A., Godden S.M., Bey R., Leslie K., Dingwell R., Ruegg P. (2007). Persistence of Subclinical Intramammary Infections in Fresh Cows. In: Proceedings of the 46th Annual Meeting of the National Mastitis Council, San Antonio, TX, pp 246-247. Lago A., Godden S.M., Bey R., Leslie K., Dingwell R., Ruegg P. (2007). Prevalence and Etiology of Subclinical Intramammary Infections in Fresh Cows. In: Proceedings of the 46th Annual Meeting of the National Mastitis Council, San Antonio, TX, pp 252-253. Lago A., Godden S.M., Bey R., Leslie K., Dingwell R., Ruegg P. (2006). Preliminary Validation of an On-Farm Culture System. In: Abstracts of the 2006 American Dairy Science Association Annual Meeting, Minneapolis, MN, pp 199. Punyapornwithaya, Veerasak, L. K. Fox, D. D. Hancock and J.M. Gay. 2007. The recurrence of mycoplasma mastitis investigated by bulk tank analysis. J. Dairy Sci. 90 (Suppl. 1):470. Quesnell RR and Schultz BD. Progesterone increases metabolic rate and progesterone withdrawal enhances epithelial integrity of mammary epithelium. FASEB J 21: A1424, 2007. Waechter-Mead L, Toerber SE, Han X, Schultz BD, and Gehring R. An in vitro model using BME-UV monolayers and Ussing flux chambers to study the movement of drugs across mammary epithelial cell membranes. Phi Zeta Research Day, Manhattan, KS, 2007, p. 3. [also presented in poster form on February 27, 2007] Conference Proceedings (2007) Andrew, S.M. K. M. Moyes, A.A. Borm, L.K. Fox, K.E. Leslie, J.S. Hogan, S.P. Oliver, Y.H. Schukken, W.E. Owens and C. Norman. 2007. Factors associated with the risk of antibiotic residues and intramammary pathogen presence in milk from heifers administered prepartum intramammary antibiotic therapy. Congress on Heifer Mastitis. Gent, Belgium, June 24-26. Apparao, M.D., L. Oliveira, C. Hulland, and P.L. Ruegg. Effect of intramammary treatment with Pirlimycin hydrochloride on antibiotic sensitivity of Gram-positive subclinical mastitis pathogens. J Dairy Sci 90 (supp 1): 10. Contreras, GA, Guterbock WM and Sears PM. 2007 Comparison of Systemic and Intra mammary Dry Cow Treatment. Proc. 46th Annual Meeting of the Natl Mast Council. Pg 290. Contreras, GA, Guterbock WM and Sears PM. 2007. Comparison of Systemic and Intra mammary Dry Cow Treatment. Meeting of the 39th Annual Convention of the Am Assoc of Bovine Practitioners. Pg 131 Fox, L.K. 2007. Recent thoughts on heifer mastitis and its control. Proceedings of the NMC 2007 Regional Meeting. Visalia, CA, pages 36-42. Fox, L.K. 2007. Whats up with Mycoplasma Mastitis. Proceedings of the NMC 2007 Regional Meeting. Visalia, CA, pages 18-29. Lago A., Godden S.M., Bey R., Leslie K., Dingwell R., Ruegg P. (2007). Prevalence, Etiology and Self Cure Rates of Subclinical Mastitis in Fresh Cows. In: Proceedings of the 40th Annual Convention of the American Association of Bovine Practitioners, Vancouver, BC, pp 250. Lago A., Godden S.M., Bey R., Leslie K., Dingwell R., Ruegg P. (2007). Subclinical Mastitis in Fresh Cows. In: Proceedings of the 2007 Minnesota Dairy Health Conference, St Paul, MN, pp 111. Lago A., Godden S.M., Bey R., Leslie K., Dingwell R., Ruegg P. (2006). Validation of the Minnesota Easy Culture System II: Results from On farm Bi-plate Culture versus Standard Laboratory Culture. In: Proceedings of the 39th Annual Convention of the American Association of Bovine Practitioners, St Paul, MN, pp 250. Lago A., Godden S.M., Bey R., Leslie K., Dingwell R., Ruegg P. (2006). Validation of an On-Farm Culture System. In: Proceedings of the 2006 Minnesota Dairy Health Conference, St Paul, MN, pp 111. Lago A., Godden S.M., Bey R., Leslie K., Dingwell R., Ruegg P. (2006). Validation of an On-Farm Culture System. In: Proceedings of the 45th Annual Meeting of the National Mastitis Council, Tampa, FL, pp 290-291. Middleton JR, Bader GR, Corbett RB. 2007. Use of pulsed-field gel electrophoresis to evaluate the success of treatment of Staphylococcus aureus mastitis. Proceedings of the 46th Annual Meeting of the National Mastitis Council. San Antonio, TX. 21-24 January 2007. Pages 202-203. Middleton JR. 2007. Efficacy of vaccination against staphylococci in heifers: A review and new data. Proceedings of the Heifer Mastitis Conference. Ghent, Belgium. June 24-26, 2007. Pages 112-113. Munoz, M. A., F. L. Welcome, Y. H. Schukken, and R. N. Zadoks. 2006. What we have learned about Klebsiella Mastitis. Fall Dairy Conference, ProDairy, Auburn, NY, USA. RJ Erskine. Mastitis Therapy and Pharmacology. Seminar presented at the Czech Dairy Management Symposium, October, 2007 Vetrny Jenikov, Czech Republic. RJ Erskine. Mastitis Therapy and Pharmacology. Seminar presented at the Slovakian Dairy Management Symposium, October, 2007, Nitra, Slovakia. RJ Erskine. Mastitis Therapy. Seminar presented at the Dairy Certificate Program, The Pennsylvania University, August, 2007, State College, Pennsylvania. RJ Erskine. New thoughts on J5 bacterins. Seminar presented at the Annual Meeting of the Minnesota Milk Producers, December 2007, St. Cloud, Minnesota RJ Erskine. Mastitis Therapy I and II, Mammary Immunology and Vaccination, and Antimicrobial Resistance in Mastitis Pathogens. Seminar series presented to the Ontario Veterinary Medical Association, January, 2007, Toronto, Ontario, RJ Erskine. The Little Things that Make Quality Milk. Seminar presented at the Great Lakes Dairy Conference, February, 2007, Frankenmuth, Michigan. RJ Erskine. The Little Things that Make Quality Milk. Seminar presented at the Annual Meeting of Field Representatives of the Dairy Farmers of America Cooperative, September 2007, Battle Creek, Michigan. SA Wagner and Erskine RJ. Mastitis Therapy Short Course. Presented at the Annual Meeting of National Mastitis Council, January, 2007, San Antonio, Texas. Schmitt-v. d. Leemput, E. and R. N. Zadoks. 2006. Macrolide and lincosamide resistance in Streptococcus uberis. NMC 45th Annual Meeting Proceedings, Tampa, FL, USA. Schmitt-v. d. Leemput, E. and R. N. Zadoks. 2006. Optimizing detection of macrolide resistance in S. uberis with the agar disc diffusion test by comparing phenotypic to genotypic resistance profiles. World Buiatrics Conference, Nice, France. Zadoks, R. N. 2007. Species identification of CNS - Genotyping as the Gold standard. Pre-Conference Symposium on Coagulase Negatives Staphylococci (CNS) in bovine mastitis. Heifer Mastitis Conference, Gent, Belgium. Zadoks, R. N. and M. A. Munoz. 2007. The emergence of Klebsiella as a major mastitis organism. NMC 46th Annual Meeting Proceedings, San Antonia, TX, USA. Zadoks, R. N., C. Hulland, N. Belomestnykh and P. Ruegg. 2006. Phenotypic and genotypic identification of Staphylococcus spp. from bovine milk. NMC 45th Annual Meeting Proceedings, Tampa, FL, USA. Poster Presentations (2007) Al-Bataineh M, Schultz BD, Gehring R. An in-vitro model using BME-UV monolayers and Ussing-like flux chambers to study drug movement across the mammary epithelium. Presented at: 15th Bienniel Symposium of the American Academy of Veterinary Pharmacology and Therapeutics, Pacific Grove, CA, USA, May 20-24, 2007. Contreras, GA, Guterbock WM and Sears PM. Comparison of Systemic and Intra mammary Dry Cow Treatment. Meeting of the 39th Annual Convention of the Am Assoc of Bovine Practitioners. 2007 Contreras, GA, Guterbock WM and Sears PM. Comparison of Systemic and Intra mammary Dry Cow Treatment. Proc. 46th Annual Meeting of the Natl Mast Council. 2007 Dingwell, R., Godden, S., Kelton, D., Leslie, K., Schukken, Y., Hand, K. Misclassification of Udder Health Status Due to Culture of Single Versus Duplicate Quarter Milk Samples. San Antonio, TX, 2007. Dingwell, R., Miltenburg, C., and Leslie, K. Evaluation of the Method of Infusion of OrbeSeal at Drying Off on the Persistency and Clearance of the Product after Calving in Dairy Cattle. National Mastitis Council, 2007. Herlekar, D., S. C. Donaldson, N. V. Hegde, A. A. Gurjar, S. N. Kallur, B. A. Houser, A. Kaucher, M. Sidhu, S. Cooduvalli and B. Jayarao. 2007. Association between Somatic Cell Counts in Milk of Cows Infected with BHV1, BHV2, and BHV4 Viruses. ASM 107th General Meeting: May 21 - 26, 2007, Toronto, Canada. Houser, B. A., N. V. Hedge, S. C. Donaldson, and B. M. Jayarao. 2007. Development of a Bioreporter System for the Detection of PCBs and Aromatic Hydrocarbons in Milk. ASM 107th General Meeting: May 21 - 26, 2007, Toronto, Canada. Wark, W., and I. K. Mullarky. 2007. Understanding the immune response of epithelial cells during staphylococcal infection. Proc. 2007 Deans' Forum on Health, Food, and Nutrition, Blacksburg, VA. Oral Presentations (2007) Godden, S. Ability of bedding materials to support growth of environmental mastitis pathogens. NE-1009 Mastitis Research Workers Annual Conference. Minneapolis, MN. Nov. 8-10, 2006. Godden, S., A. Lago, R. Bey, K. Leslie, P. Ruegg, R. Dingwell. 2007. Use of on-farm culture systems in mastitis control programs. National Mastitis Council Annu. Regional Meeting. Visalia, CA. May 22-23, 2007. Godden, S., A. Lago, R. Bey, K. Leslie, P. Ruegg, R. Dingwell. 2007. Use of on-farm culture systems in mastitis control programs. Fort Dodge Dairy Summit. Yosemite, CA. Oct. 13, 2007. Lago A., Godden S.M., Bey R., Leslie K., Dingwell R., Ruegg P. (2007). Current On-Farm Culture Research Projects and Preliminary Results. In: Proceedings of the On Farm Culturing Workshop, Madison, WI. Book Chapters (2007) Middleton JR. Mammary Gland Surgeries: Teat Amputation, Mastectomy. In: 5-Minute Veterinary Consult: Ruminant. Blackwell Press. Ames, Iowa. In Press. Middleton JR. Mastitis: Sheep and Goats. In: 5-Minute Veterinary Consult: Ruminant. Lippincott, Williams and Wilkins. Blackwell Press. Ames, Iowa. In Press.

Impact Statements

Greater characterization and understanding of mechanisms associated with mastitis susceptibility and resistance
Further identification and manipulation of virulence factors of mastitis pathogens which greatly enhances host defense
Wide assessment and application of a variety of technologies and modalities advancing udder health control and ensuring food safety
The group continued to collaborate (i.e. several multi-state studies were concluded or are in progress) to combine basic and applied science aimed at reducing the incidence and severity of mastitis in dairy cows in order to improve animal health, productivity and milk quality.
Gained a much better understanding of cow-level, quarter-level and management-level risk factors at dry off and during the close-up (transition) period that contribute to new intramammary infections during the dry period. Examples include dry-off strategies, the role of teat sealants, and both nutritional management or photoperiod manipulation to improve immune function.
Continued to improve our understanding of bovine immune/resistance factors and to evaluate vaccination strategies to prevent coliform mastitis. This includes a greater understanding of the relative importance of cow genetics (heritability) vs environment (management) influencing the risk for mastitis.
Continued evaluation of new teat dip formulations to provide producers with reliable information and allow them to choose the best product for reducing mastitis.
Gained a greater understanding of causes of prepartum mastitis in primiparous heifers, as well as concluded a large multi-state study examining the efficacy of prepartum intramammary therapy in primiparous heifers. Preliminary results indicated that while prepartum antibiotic treatment heifers would cure existing intramammary infections, this reduction in IMI did not translate into a significant improvement in milk production, SCC, or reproductive performance.
Continue work to describe molecular typing and distribution plus the antibiotic-susceptibility patterns of common mastitis pathogens such as Staphylococcus aureus and Escherichia coli. Continued work to better understand pathogenicity that differentiate acute from chronic coliform infections.
Continued to investigate the utility of on-farm diagnostic tools such as the California Mastitis Test (CMT) and on-farm culture systems. These tools will allow producers to more effectively diagnose and treat subclinical infections at calving or clinical mastitis cases. Also developed in-lab diagnostic tools such as real-time PCR procedures for detection of common mastitis pathogens in milk.
Initiated a collaborative study to develop an electronic data transfer system to more efficiently and expediently provide producers on a Dairy Herd Improvement (DHIA) testing program with culture results from the mastitis laboratory. This system should allow producers to more efficiently access culture data for the purpose of making timely cow-management and treatment decisions.