Brief Summary of Minutes of Annual Meeting

Objective 1. Characterize host responses or factors that affect resistance of the mammary gland to mastitis. IA, KS, Cornell, Guelph. Overall, 11% of quarters developed new IMI in the dry period. The majority of new IMI were caused by environmental streptococci and coliform organisms (34% and 30%, respectively). The proportions of new IMI were different among herds, parity levels, and season of drying-off. Quarters with cracked teat-ends were more likely to develop new infections than those without cracks (15% and 10%, 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 (10% and 14%, respectively). At the end of the six-week dry period, 23% of quarters were still classified as open. The risk of quarters closing was significantly associated with the level of milk production on the day before drying-off. Management strategies aimed at reducing milk production, improving teat integrity, and enhancing closure of the teat streak canal should be evaluated and implemented. IL. Exposing cows to short days (8 hr light, 16 hr darkness) during the dry period enhanced neutrophil chemotaxis and lymphocyte blastogenesis at calving and was associated with a 50% decrease in new intramammary infections during the dry period and a 50% decrease in clinical mastitis incidence the first 10 days of lactation, compared with long days (16 hr light, 8 hr darkness). However, cows exposed to short days appeared to have a slightly increased local inflammatory response to experimental Streptococcus uberis mastitis. IA. Three trials evaluating a salve and 2 high emollient (>70%) winter teat dips showed no statistical difference in teat end health compared to a commercial non-barrier dip and BMP. LA. Efficacy of 2 iodine teat dips and 2 sodium chloride teat dips was evaluated using NMC protocol B guidelines. A barrier teat sealant was evaluated for use as a dry cow product. MI. Over 310 genes in bovine neutrophils respond to parturition with changed expression. For ~ 60% of these genes, expression levels were dramatically increased, especially the first 12 hr following parturition. Remaining gene expression changes included pronounced down-regulation at parturition or for at least one day postpartum. The majority of these changes suggested parturition induced survival of blood neutrophils. This hypothesis is supported by pronounced neutrophilia observed for ~48 hr around parturition and survival of neutrophils from parturient cows ~24 hr longer in culture than those from mid-lactation cows. Preliminary experiments suggest that steroids of bovine parturition induce this survival with concurrent down-regulation of cellular metabolic processes that could otherwise lead to severe systemic inflammation. Given these findings, we preliminarily conclude that the parturient blood environment induces an adaptive response in bovine neutrophils that may be critically important to overall animal health. NY. The immune response to E.coli mastitis was evaluated in both a field and challenge study. The results indicate a significant increase in IgG2 antibody titer after challenge both in the natural challenge and the experimental challenge. OH. Immunization site reactions were negatively correlated with clinical signs in cows immunized with E. coli J5 and challenged with a heterologous strain of E. coli. TN. A Interleukin-8 receptor single nucleotide polymorphisms are present at defined frequencies and may be linked to mastitis development. B Experimental Strep. uberis IMI induced local production of TNF-alpha, IL-1, and IL-8, which may play a role in the pathogenesis of Strep. uberis mastitis. Other mediators may be involved in initial leukocyte recruitment to the mammary gland since increases in milk somatic cells occurred earlier than adhesion molecule expression or cytokine production. C A cloned calf from a cow susceptible to mastitis exhibited a significantly greater percentage of gd-T cells overall when compared to age-matched controls. These values also were within the upper range expressed by all calves when evaluated on a single day. Further investigation using cloned animals could result in identification of potential genes, immune components, and/or other factors associated with mastitis resistance. Objective 2. Characterization of virulence factors of mammary gland pathogens. NY. S. uberis infections in dairy cows are increasing. In this project we aim to better understand the differences in pathogenesis of S. uberis strains isolated from cows and the environment. First a genetic classification system will be developed, and second further studies into the pathogenesis of S. uberis infections will be undertaken. OH. Culture of E. coli in medium to induce FecA expression resulted in decreased susceptibility of bacteria to phagocytosis. TN. Strep. uberis avoids intracellular bactericidal mechanisms by exploiting preferential intracellular trafficking pathways in bovine mammary epithelial cells. This strategy would allow Strep. uberis to persist intracellularly and may help explain establishment and persistence of chronic intramammary infections observed in lactating and nonlactating dairy cows. WA. In a study of the shedding pattern of Mycoplasma sp. in cows with mastitis, mycoplasma was not recovered in 29% of composite samples, while 54% of the samples had more than 1 million colonies per ml in milk. In 43% of the quarter milk samples mycoplasma was not recovered while in 39% of the samples there were greater than 1 million colonies per ml. On effects of storage and sample handling, freezing and prolonged storage had a negative impact on organism recovery. Objective 3. Development and evaluation of techniques for modulation of host responses to mastitis pathogens. WA, LA, TN, OH, Cornell, Guelph, CT, KY. A greater number of intramammary infections in heifers were cured by treatment during the peripartum period. As a result, treated cows had less IMI at calving, and also less new IMI at calving. IMI were reduced in all herds by treatment, and CNS were the pathogens accounting for the vast majority of treatable IMI. Postpartum milk production was equivalent in treated and control heifers, and milk somatic cell count slightly lower in treated heifers. IL. Antimicrobial susceptibility test results, using the disk diffusion method, were not correlated with clinical or bacteriological outcome of clinical mastitis episodes treated with intramammary cephapirin and/or intravenous oxytetracycline. A DNA amplification method was developed for detection of Mycoplasma species in bulk milk and can detect 1,000 cfu/ml at a similar cost to traditional culture. Intramuscular administration of ceftiofur HCl (2.2 mg/kg q 24 hr for 3 treatments) to cows with experimentally induced coliform mastitis was associated with a more rapid return to normal of milk albumin, lactose, sodium, and chloride concentrations, compared with no treatment. IA. A 27 new teat dip sealant prototypes for dry cows were evaluated for persistency, with 3 prototypes showing 40% increased persistency compared to commercially available products. B Mean % and mean total number of cytokeratin positive cells/ml milk before and 12 hr after endotoxin challenge were 4.18% and 7711, and 40.63% and 2,954,554, respectively. Treatment with steroidal or NSAID did not mitigate these changes. C Treatment with isoflupredone acetate (20 mg IV 1X) following an endotoxin challenge model and clinical signs resulted in statistically similar milk production as healthy untreated controls. KS. A study was conducted that determined the reliability of a documented test procedure to assess teat canal closure during the dry period. Although fairly reliable when a single technician used the procedure, reliability decreased when multiple technicians were employed. These data should be considered for studies employing teat end closure techniques. LA. Various methods of treating teat injuries in lactating cows were described. MI. A The current 3-dose J-5 vaccination regime increases serum and milk anti-coliform IgG1, but is relatively short-lived and does not promote opsonic phagocytosis of E. coli by bovine neutrophils. In contrast, at least 6 vaccinations given monthly throughout periparturition and lactation elicited significant anti-coliform IgG2 responses in serum and milk. IgG2 antibodies also interacted with multiple coliform mastitis pathogens in ELISA assays and recognized a predominant ~ 38 kDa protein in cell membrane proteins. Thus, we may have identified a coliform outer membrane protein that could be used to improve coliform mastitis vaccines, because opsonizing IgG2 antibodies that recognize this protein also promote effective phagocytosis of E. coli by neutrophils. B We continue to evaluate specific antigens of S. aureus cell wall and capsules for specific activity towards staphylococcal organisms. Several subsets of antigens have been evaluated as a prelude to antibiotic treatment. C Experimental infection models of streptococcal mastitis have demonstrated efficacy of intramammary infusions of a nisin preparation. Additionally, safety studies were completed that suggested this product will not affect milk quality or cow health. NY. Chronic coliform mastitis was studied in three large dairy herds. Incidence of cases was evaluated and isolates were obtained. These isolates will be studied in more detailed in the years to come. OH. Efficacy of an oil and water adjuvant was greater than water soluble adjuvant in a polyvalent E. coli bacterin. WA. A study was done to determine the effectiveness of removal of udder and teat hair on new IMI and bacterial counts in milk. Hair removal did not have a significant affect on milk bacterial counts. However, hair removal might be effective in improving milk quality when less stringent pre- and/or post-milking preparation procedures are used. Objective 4. Characterize dairy food safety issues relevant to bovine mastitis. CT. A The objective of this study was to evaluate the effect of antibodies, milk fat, milk protein, mastitis pathogens and SCC in colostrum and transition milk from Holstein heifers on the specificity rates of several commercially available B-Lactam antibiotic residue screening tests. Specificity rates ranged from 0.16 to 0.88 for colostrum and 0.60 to 1.0 for transition milk. Increased milk protein and IgG1 concentrations in milk were associated with increased probability of false positive outcomes for the Charm Cowside, CITE Snap and Penzyme tests. Milk should not be tested for antibiotic residues prior to the sixth milking after parturition to avoid high rates of false positive outcomes. B Fifty multiparous Holstein and Jersey dairy cows at the University of Connecticut dairy herd are currently being enrolled in a study to determine if the combined effects of NEB and ketosis increase the risk of cows developing mastitis during early lactation. C A new formulation of a chlorohexidine teat disinfectant is being evaluated. NY. The genome of S. agalactia isolated from cows and from humans was compared using a number of classification methods including antimicrobial resistance genes, pcr based methods and sequencing. An important overlap was present between human and bovine isolates. Further studies are needed to look at the potential importance of this finding. TN. Rhamnose may be useful in differential isolation media for Shiga toxin-producing E. coli (STEC)O26. Presence of shiga toxin 1 gene sequences may aid in rapid identification of E. coli O26. About 98% (148 of 151) of E. coli O157 strains (largely serotype H7) were stx+ and almost all elaborated Shiga toxins. Acid-resistance (~96% of STEC) and tellurite-resistance (~86% of STEC) were common characteristics.