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NE1048: Mastitis Resistance to Enhance Dairy Food Safety

Annual/Termination Reports (SAES-422): [02/08/2013]

Date of Annual Report: 02/08/2013

Report Information:
  • Annual Meeting Dates: 11/06/12 to 11/08/12
  • Period the Report Covers: 10/2011 to 09/2012

    • Participants:
    Brief Summary of Minutes of Annual Meeting:
    1. The 2012 (FY13) annual business meeting of the NE 1048 Multistate research project was called to order at 4:15 pm by vice-chair David Kerr, presiding in the absence of the current chair Anne Lichtenwalner.

    2. An overview of the 2011 business meeting minutes was presented (the complete minutes of the 2011 business meeting were sent by email and available on the project web site). A motion for approval was seconded and the 2011 business meeting minutes were approved by unanimous vote.

    3. Introduction of members and new members. Attending members from 14 stations introduced themselves. Tanya Gressley (Delaware), Pedram Rezamand (Idaho), Jeffery Bewley (Kentucky), Bill Owens (Louisiana), Ron Erskine (Michigan), John Middleton (Missouri), Greg Keefe (Prince Edward Island), Gina Pighetti (Tennessee), David Wilson (Utah State), David Kerr and John Barlow (Vermont), Christina Peterson-Wolfe (Virginia), Larry Fox (Washington), Pamela Ruegg (Wisconsin).

    4. The following new members were approved by unanimous vote: Chris Luby (Saskatchewan), Simon DuFour (replacing Daniel Scholl; Montreal). The Chair will look into having the new members officially added to the Appendix E list of participants.

    5. One individual is listed on our Appendix E list (David Eide) from Univ. Wisconsin  Dept. of Nutritional Sciences. The Chair will verify if this is correct.

    6. Ghent, Belgium was selected for FY 14 meeting that will take place during the summer of 2014. Dr. DeVliegher (University of Ghent) will oversee the local organization.

    7. The project Administrative Advisor, Dr. Cameron Faustman, advised that Federal funds allocated to each experimental station could be used for international travel. He also indicated that there would likely be more funding available in USDA Foundation grant program for FY13. Finally, he advised the group that there is an award at the national level for multistate hatch projects and that we should nominate our group. Dr. Faustman has the last 4 years winning projects. Deadline for the nomination is February 28, 20113. Dr. Kerr asked to receive the document regarding the nomination.

    8. Concern was raised there are members of the project that do not show up and do we, as a group, need to take some action? Discussion was had on this topic. As a result, we are going to go through the email list over the past 3 years and Dr. Kerr is going to send an email to those not participating explaining by-laws. 9. Dr. Gressley and Dr. Luby were nominated to serve as NE1048 secretary. Voting was done and Dr. Gressley was selected and accepted the position. Dr. Kerr is going to send role and job description to Dr. Gressley.

    10. Seeing no other business the meeting was adjourned after a motion and second. Meeting was adjourned at 5:43 pm.


    URL: Copy of minutes

    Accomplishments:
    Objective 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance.

    DELAWARE: Continued evaluating effects of thymol on in vitro measurements of neutrophil kill of S. aureus.

    GEORGIA: Daily feeding of a general immunostimulant supplement to dairy heifers resulted in increased L-selectin and interleukin-8 mRNA expression in blood leukocytes, increased in vitro phagocytic ability of blood neutrophils and monocytes against S. aureus and E. coli, and increased reactive oxygen species (ROS) production by blood neutrophils compared with unsupplemented controls, all of which may enhance immunity in heifers against mastitis. After calving, supplemented heifers exhibited a 3-fold reduction in prevalence of mastitis in early lactation, a 4-fold reduction in SCC, and a 7-lb increase in milk production compared with unsupplemented controls.

    IDAHO: We are investigating the effect of retinol-binding protein (RBP) status (as regulated by dietary retinol and/or protein) on IMI in periparturient Holstein cows. We have demonstrated a strong positive correlation between mRNA expression of TNF-alpha and that of RBP in the liver and adipose tissues. However, a follow up study showed the relationship between RBP and TNF-alpha is not a cause-effect relationship under our experimental condition. We also finished animal phase of our large dairy project entitled Interaction among energy status, retinol-binding protein status and intra-mammary infection in periparturient dairy cows. Laboratory analyses of this project are being performed.

    MICHIGAN: We investigated if increased NEFA concentrations modify vascular inflammatory responses of endothelial cells in vitro by changing the expression of important inflammatory mediators. In particular, the role of n-3 fatty acid content in altering the profile of vasoactive eicosanoids and attenuating endothelial cell inflammatory responses was a central aim of our work.

    NEW YORK: The Cornell team studied the immune response in late gestation and evaluated the impact of an E. coli challenge in late gestation and compared this to the response in mid lactation. Important differences in immune response were observed.

    TENNESSEE: UT has determined that the interleukin-8 receptor, CXCR1 is highly polymorphic. These polymorphisms can be subdivided into groups that are in linkage and travel together. When polymorphisms of individual groups are combined, this forms a haplotype that is more specific than an individual polymorphism. Specific haplotype combinations were significantly associated with the degree of infection and inflammation following experimental challenge with S. uberis. CXCR1 also is expressed by mammary epithelial cells and would be capable of responding to interleukin-8 released in the mammary gland during infection.

    VERMONT: Dermal fibroblasts are being used as a model cell to investigate genetic and epigenetic differences between cows in their innate responses to mastitis causing pathogens. The in vitro production of the IL-8 chemokine in response to E. coli lipopolysaccharide or to a synthetic Gram positive cell wall component (PAM2CSK4) are reflected to some degree by in vivo responses to intramammary challenge with E. coli or S. aureus. Candidate genes responsible for the between-animal differences are being investigated

    VIRGINIA: We are continuing our characterization of T cell responses to dendritic cells presenting S. aureus antigens. In addition to cytokine profiles, we will be evaluating memory cell proliferation and identifying S. aureus specific antigen responses. To date we have shown an increase in IL-17 production from T cells stimulated with irradiated and live Staphylococcus aureus. In addition to cytokine profiles we are evaluating the ability of S. aureus to inhibit dendritic cell apoptosis during intracellular infection.

    WASHINGTON: A project is on ongoing to determine the innate immune response to experimental challenge by M. bovis. Experimental challenge of dairy calves for 3 days with M. bovis through milk replacer feeding, indicated that M. bovis colonization and shedding after challenge was affected by glucocorticoid administration. A higher carriage and shedding rate in Dexamethasone treated calves, as compared to controls, from the respiratory tract was observed. These results are consistent with shedding in dairy cows during an outbreak of mycoplasma mastitis.

    WISCONSIN: UW Milk Quality lab members are currently collecting data for a study that is evaluating pathogen specific effects on reproductive performance and embryo loss. Almost 1600 cows have been enrolled of the approximately 2800 cows for the full study. Data collection will be completed in 2013.

    Objective 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses.

    GEORGIA: Assessment of 118 non-hemolytic staphylococcal isolates from heifers and cows in 2 herds, presumptively identified as coagulase-negative staphylococci (CNS) on blood agar demonstrated that 32.2% of isolates were actually S. aureus based on coagulase production, fermentation of mannitol on mannitol salt agar, and species-level identification based on the API staph test system; 15.2% were other coagulase-positive staphylococci (CPS); and 52.5% were identified correctly as CNS. Thus, a significant proportion of S. aureus and CPS infections may be misdiagnosed as CNS, and additional testing should be performed to correctly identify such staphylococcal isolates.

    KENTUCKY: A MS student in Microbiology at Eastern Kentucky University (Erica Lynch) is conducting a project to assess antimicrobial resistance in pathogens responsible for causing bovine mastitis in Kentucky. This project is a field survey of multiple herds in Kentucky.

    LOUISIANA: The Hill Farm Research Station continues to collect mastitis pathogens and determine their antimicrobial susceptibility to various antibiotics. Studies are underway to compare susceptibility patterns of veterinary isolated to human isolates of the same pathogens.

    An increase in goat milk submission has lead to the isolation and characterization of mastitis pathogens from goats, the majority of which are CNS. Susceptibility studies are underway on these isolates.

    The Mycoplasma mastitis control program continues to be active in Louisiana, quarterly bulk milk samples are cultured for Mycoplasma and routine mastitis pathogens to determine the incidence of these pathogens in Louisiana dairies.

    MISSOURI: The association between mammary inflammation, duration of infection, and coagulase negative staphylococcal (CNS) intramammary infection continues to be a major focus of our research. In-depth genotypic characterization of predominant CNS species using whole genome sequencing is underway to elucidate putative virulence factors involved in mastitis pathogenesis.

    NEW YORK: Full genome sequencing of a number of mastitis E. coli isolates was completed. Differences between transient and persistent isolates were observed and reported.

    TENNESSEE: This past year, two studies were conducted to prove S. uberis adhesion molecule (SUAM) is a relevant virulence factor in vivo. Dairy cows challenged with a SUAM-negative mutant of S. uberis or S. uberis pre-treated with antibodies against SUAM did not develop as severe an infection or inflammation when compared to the wild-type strain or S. uberis receiving antibodies not specific for SUAM. Additionally, several new genes that potentially contribute to S. uberis were identified using a transcriptomic-based approach that identified S. uberis genes activated following intramammary challenge.

    VIRGINIA: Using irradiated and live S. aureus cultures, we are identifying a role for virulence factors in immune suppression, activation, and manipulation. Specifically we are evaluating changes in T cell activation through granzyme production following antigen presentation and stimulation with live or irradiated S. aureus.

    Objective 3. Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety.

    GEORGIA: Use of a horn fly control program on dairy heifers based on a pour-on containing 5% permethrin, which was administered every 2 wk was successful in reducing fly populations on animals and allowing teats to heal, which had been damaged (lesions and scabs) by the horn flies. Such control led to decreases in new intramammary infections caused by S. aureus.

    KENTUCKY: A two-year study to examine the mastitis detection potential of multiple Precision Dairy Farming Technologies (MilkLine P4C individual quarter conductivity, IceRobotics IceQube lying behavior monitors, DVM Systems temperature boluses, SCR Rumination Tags) began in October 2011 at the University of Kentucky. Di Liang has worked on developing a model to examine the economics of mastitis on dairy farms.

    MAINE: Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety.Implementation of a nested PCR for detection and speciation of prototheca from dairy farm samples has contributed to a change in knowledge by allowing sensitive and specific detection of prototheca in herds of dairy cattle. Culling and changing management has been advised to reduce prototheca on affected farms. Studies of pasteurization resistance of Maine isolates, and of bedding types to reduce prototheca growth on farms, have been completed. Results have been communicated to Maine farmers, and at National Mastitis Council workshops.

    MICHIGAN: We have continued outreach efforts to promote better stewardship of antibiotic use on dairy farms. Key contributions included workshops on mastitis bacteriology for dairy producers and veterinary practitioners to help these stakeholders make better therapeutic decisions. Additionally, several presentations were given to veterinary practitioner and dairy producer groups to highlight the interaction between poor therapeutic decisions and residues in meat and milk.

    MISSOURI: In the last 12 months, we have continued to evaluate prevention and treatment strategies for mastitis. We have evaluated a Lysostaphin-fusion protein as a dry cow therapy for treatment of chronic Staphylococcus aureus mastitis in an experimental challenge trial. Data from these studies are currently being analyzed.

    MINNESOTA: The University of Minnesotas College of Veterinary Medicine is developing an on-line curriculum in mastitis and milking system analysis that will provide web-based, self-directed learning systems for veterinarians and other milking system professionals. Thus far, two modules have been delivered to approximately 30 registrants. The courses are offered through the Office of Veterinary Continuing Education of the College of Veterinary Medicine. Collaborator: University of Wisconsin-Madison.

    The University of Minnesota conducted a multi-state multi-herd randomized non-inferiority clinical trial to compare the efficacy of three commonly used dry cow mastitis formulations: penicillin/dihydrostreptomycin, ceftiofur hydrochloride and cephapirin benzathine. Results indicate there was no difference among the three treatments tested regarding quarter-level outcomes (e.g. new infections, cures), or cow- level outcomes in the first 100 DIM, including SCC, milk production (strong trend present), risk for a clinical mastitis event, risk for culling or death, and risk for conception. Collaborators: Iowa State University and University of California-Davis.

    The University of Minnesota reported on a study designed to validate the use of the Minnesota Easy Culture System II Bi-plate and Tri-plate systems to diagnose the presence of classes and species of bacteria in milk samples. Results suggested that readers using the Bi-plate or Tri-plate systems on farms will get the most accurate and useful results when using this diagnostic tool to identify the presence of a Gram positive intramammary infection (IMI). Either tool has moderate ability to identify no growth, Gram negative and Staphylococcus aureus IMI. With the exception of Staphylococcus aureus, readers should refrain from trying to speciate the type of organism cultured using either the Bi-plate or Tri-plate.

    NEW YORK: We evaluated treatment of Gram-negative intramammary infections using a first and third generation cephalosporin with and without corticosteroid. Efficacy of treatment was observed and reported.

    OHIO: The efficacies of two immune-modulating compounds were tested using a Streptococcus uberis intramammary challenge model. Neither the incidence of intramammary infections, incidence of clinical mastitis, shedding rate of bacteria, nor clinical signs of infection were affected by the test products.

    UTAH: Completed study of SPC bacteria counts in pasteurized waste milk for feeding to calves, as pasteurized on commercial farms. Developed season-specific milk handling guidelines.

    Data collection nearly completed for comparison of blood test strips, milk test strips and Fossomatic automated milk analysis for beta-hydroxybutyrate levels in early lactation dairy cattle. Goal is to use DHIA or other meter-collected milk samples for high throughput inexpensive detection of ketotic/negative energy balance cows.

    VERMONT: A case study to demonstrate contagious pathogen (Staphylococcus aureus and Streptococcus agalactiae) transmission dynamics in a herd using automated milking system (robotic milkers) has been completed. Transmission parameters for these 2 contagious pathogens and the impact of control practices in this herd are being evaluated.

    VIRGINIA: In the past year, we have completed a study examining the changes in animal activity and milk component data around the onset of naturally occurring clinical mastitis. In this study, daily animal activity and milk component data were collected from cows with mastitis and compared to healthy cows. Changes in milk components and daily step activity were observed prior to detection of mastitis. A statistical model utilizing all collected data was able to distinguish cows with mastitis from healthy cows 1 d prior to onset of clinical signs. Early detection of disease may lead to reduced economic losses associated with mastitis.

    WASHINGTON: Factors associated with time to clearance of mycoplasma mastitis were studied in 18 herds. Most herds cleared mycoplasma mastitis within 1 month, greater than 50% culled diseased cows preferentially, yet culling was not associated with hastened clearance as herds that culled preferentially cleared the infection at ostensibly the same rate as those that did not cull preferentially. Milking time hygiene practices were practiced near full by 17 of the 18 herds and such practices were not associated with time to clearance. Other known mastitis biosecurity and management practices were not associated with clearance time.

    WISCONSIN: The UW Milk Quality lab completed a study that characterized the differences in in-vitro susceptibility testing of Ceftiofur & Cephapirin as compared to results using the active metabolites of the same compounds (Desfluoceftiofur & Deactylcephaparin). Differences in outcomes for Staphylococci (ceftiofur) and E coli (cephapirin) probably account for some of the lack of association of in vitro susceptibility test results with outcomes of intramammary treatments.

    UW Milk Quality lab members completed an epidemiological study that characterized the occurrence of clinical mastitis on 51 large modern dairy farms in Wisconsin. Results of this study indicate a strong shift to mastitis caused by a variety of opportunistic environmental pathogens. Upon observation of the cases, approximately 25% of the milk samples resulted in no significant growth of bacteria and a further 20% were caused by pathogens for which no currently available antimicrobials are effective. Most cases were mild and the median days in lactation upon occurrence was approximately 130 days. The association between exposure to antimicrobials and occurrence of resistance will be reported.

    Impact Statements:
    1. Various projects have reported beneficial effects of nutritional supplementation on ability of leucocytes to kill mastitis causing pathogens. Responses of T-cells, dendritic cells, and fibroblasts to pathogens have highlighted the importance of these cell types in mastitis defense. Genetic differences in specific genes for mastitis susceptibility are being evaluated, as are mechanisms that affect the host immune response during various stages of lactation and during periods of stress. The detrimental effects of mastitis on reproductive performance have been documented.
    2. Refinements in identification and characterization of antimicrobial resistance of mastitis causing pathogens have been accomplished leading to more accurate diagnosis and more informed treatment decisions. Advanced gene sequencing techniques, such as whole genome sequencing are being applied to enhance identification of virulence factors. One of these factors, S. uberis adhesion molecule (SUAM), enables development of more severe mastitis, and may prove to be an important vaccine component.
    3. Assessment of new technologies to advance mastitis control (Objective 3) have shown the benefits of a fly control program to reduce mastitis, and a new technique for rapid measurement of milk beta-hydroxybutyrate could improve early detection of negative energy balance and ketosis that are risk factors for mastitis. Assessment of a new anti-staphylococcal enzyme is in progress that, if successful, has the potential to reduce the use of currently approved antibiotics.
    4. On farm assessment of a culture-based system to identify pathogens in milk has shown promise in identifying Gram-positive pathogens, and this will lead to more appropriate treatment options. A new approach to early detection of mastitis based on the animals physical activity level and milk components has been developed that may permit early detection, and potentially more successful treatment.
    5. Educational workshops and on-line materials for producer groups and veterinarians have been used to enhance application of proven technologies. Finally, various management practices were examined for their association with clearance of mycoplasma mastitis but no specific association was found.
    6. In the reporting period, member stations have published 45 peer-reviewed publications and 3 book chapters related to the projects activities. Furthermore, 33 abstracts and 18 conference presentations were made at various meetings across the US and abroad.
    7. The annual meeting of the NE1048 project was held in conjunction with the Annual Mastitis Research Workers meeting where a total of 35 abstracts were presented to the 45 attendees. Attendees included those from 15 States, 3 Canadian Provinces, and Belgium.
    Last Modified: 13-Feb-2013
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