Identification, Characterization, and Management of an Emerging Mastitis Pathogen, Lactococcus lactis, subspecies lactis

Project Overview

Project Type: Partnership
Funds awarded in 2011: $14,445.00
Projected End Date: 12/31/2013
Region: Northeast
State: New York
Project Leader:
Dr. Michele Barrett, DVM
Keseca Veterinary Clinic

Annual Reports


  • Animal Products: dairy


  • Animal Production: preventive practices, therapeutics
  • Crop Production: food product quality/safety

    Proposal abstract:

    Mastitis is one of the most costly health concerns affecting dairy farmers in the United States, accounting for approximately 1.8 billion dollars lost by the industry annually. With growing consumer concern about farming practices, as well as pressure for higher milk quality standards, farmers will need to control mastitis in order to sustain their viability in national and international markets. Over the past six months, a new mastitis pathogen has emerged in Western New York, Lactococcus lactis, subspecies lactis, which until now was thought to be nonpathogenic. Without expensive testing, Lactococcus is lumped together with the other Streptococci bacteria. However, unlike Streptococcal infections, cows with Lactococcal mastitis respond poorly to treatment, resulting in higher costs to farmers. Since this pathogen has previously been overlooked, our goal is to establish a simpler and less expensive identification technique by evaluating the API 20 Strep test for its accuracy in identifying Lactococcal mastitis. By doing so, the project will also establish the prevalence of Lactococcal mastitis within the dairy herds served by Keseca Veterinary Clinic. Subsequent herd surveys will assist in risk-factor analysis for infection as a means of proposing practical preventative and control measures for farmers. Finally, all Lactococci isolated from clinical samples will undergo antimicrobial sensitivity testing to create a library for potential future treatment trials. Overall, this project will create a foundation of knowledge about Lactococcal mastitis to aid farmers nationwide in controlling Lactococcus as an emerging pathogen on their farm and a threat to their sustainability in the industry.

    Project objectives from proposal:

    Phase 1: Evaluation of the API Test strip
    All mastitis samples submitted through the Keseca Veterinary Clinic (KVC) mastitis lab are plated on Blood Agar in aerobic conditions at 37 C, and results are read at 24- and 48-hours post inoculation. As part of this project, all clinical samples submitted to Keseca’s mastitis lab that are diagnosed as non-hemolytic, esculin positive, Streptococcus species after 48-hour aerobic culture will be enrolled into this study for further species differentiation. Pure isolates of these eligible Streptococci will be tested using the standard procedures outlined by the manufacturer of the API 20 Strep Test strip, Biomerieux, as follows: Streptococcus samples will be suspended in a saline solution until turbidity levels reach the minimum requirements according to McFarland’s standard. Once this saturation is reached, the first 10 wells of the API 20 Strep strip are filled with this suspension. The remaining suspension is transferred to a GP medium, and then the remaining 10 wells are filled on the strip. The last 11 wells on the plate are then overlaid with mineral oil to provide anaerobic conditions. The strip plate is then incubated at 36+/-2 C for 4 hours. Once incubated for 4 hours, additional reagents are added to the first ten wells based on the specific test, and any color changes to the wells are noted. Color changes are reported as “positive” results, while a lack of color change is considered a “negative” result. The specific combination of test results for all 20 tests are then entered into an online database, and compared with the library of known bacteria. In certain situations, the plate may need to be re-read at 24 hours. Once entered into the database, the most likely identification of species is displayed by the program, along with percentage similarity to “known” bacteria in the library. All isolates will be identified with a unique number, and results of API tests will be recorded in an Excel Spreadsheet.

    These isolates will then be sent to the referral lab at (Quality Milk Production Services-Molecular Lab) where molecular strain typing would be run according to industry standards. At the end of the study period, a statistical comparison of API test results to the “gold standard” of species identification by molecular testing would be used to calculate sensitivity, specificity, and overall accuracy of the API 20 Strep strip in specifically identifying Lactococcus.

    Phase 2: Analysis of Risk Factors and Environmental Sampling
    During the study period, all farms submitting cultures through KVC’s established culture program will be asked to fill out a written herd survey concerning pertinent farm information. The survey will cover specific management concerns, such as types of bedding used, frequency of bedding, bedding method, pre and post teat dip types used, forage types fed, inoculants used (if any), liner types, frequency of liner changes, towel- washcloth or paper used in parlor, manure removal systems, water sources, and teat end score conditions. Analysis of farm records in DairyComp305 for herd demographic data will also be conducted. Individual farm data will be compiled, and the appropriate statistical analysis will be used to identify any strong associations between farm characteristics and Lactococcus mastitis incidence, using enrolled non-infected herds as controls. From this same information, a focused list of environmental areas to sample for culture will be created. Once a list of samples to take is generated, a sampling protocol will be created to maintain consistency of sample collection across farms. One specific person per farm will be responsible for collecting environmental samples, and will be trained by project leader in the established sampling protocol in order to minimize interpersonal sampling bias. Once samples are collected, they will be delivered to the Keseca mastitis lab for aerobic culture in similar fashion as the clinical mastitis samples (37 C). Any samples containing non-hemolytic, esculin positive, Streptococcus will be handled as mastitis samples were in the section above, undergoing API 20 Strep strip testing and molecular strain typing for verification. Results of these tests will be included in the data for API strip accuracy testing. If a farmer wishes to confirm that the strains in environmental samples match the strains found in clinical samples, the owner may opt at that time for further testing at owner’s expense.

    Phase 3: Creation of Antibiotic Sensitivity Database
    All isolates verified by molecular strain typing at the referral lab (QMPS) will undergo antibiotic sensitivity testing via Minimum Inhibitory Concentration (MIC) method at same referral lab according to industry standards. Results of all testing will be combined into a database and analyzed. Descriptive statistics concerning the results of this in vitro testing will be published in the summary of results, and may serve as groundwork for future in vivo drug treatment trials.

    Through this project, Keseca Veterinary Clinic (KVC) aims to create practical management recommendations for those who work in the dairy industry. By doing so, the goal is to increase the productivity and financial sustainability of these farms by minimizing the impact of this pathogen. Distribution of results will need to be aimed both at farmers and industry professionals who consult with farmers concerning milk quality. As a progressive veterinary practice, KVC will be able to disseminate the results of this study on local, regional, and national levels.

    With the primary goal of farm sustainability, farm owners and employees will be the main audience for disseminating results. Locally, a newsletter describing the project’s results and their impact on dairy farms will be distributed to KVC’s nearly 200 client farms, including the two identified in this project. This provides dissemination to farms in five different counties, with word of mouth potentially increasing distribution from there. A farmer-focused PowerPoint presentation will be created summarizing the same material and be presented at KVC’s annual client meeting in March 2012. Regionally, this farmer-oriented presentation will be submitted for presentation at the Northeast Dairy Producers Association, which has members in the dairy industry throughout the Northeastern United States, for presentation at their annual meeting in Syracuse, NY in March 2013.

    As veterinarians, KVC also understands the tremendous impact industry professionals have in helping farmers to create farm-specific protocols, and as such, must be informed of these results. Regionally, KVC will create a second PowerPoint presentation aimed at industry professionals, and submit the presentation to the Northeast Dairy Production Medicine Symposium for their 2013 meeting. Furthermore, KVC intends to share the results of the study with Quality Milk Production Services (QMPS) for dissemination to their extended networks of both farms and professionals throughout the northeast and the nation. Finally, as members of several national/international veterinary organizations, these results can be shared on a much broader scale. Staff veterinarians will submit a summary of the project to the Journal of the American Veterinary Medical Association (distributed to over 80,000 veterinarians nationally), The American Association of Bovine Practitioners (AABP, 6000 members, both US and International), and the newsletter of the National Mastitis Council (NMC, 1500 members throughout 40 countries).

    Furthermore, the PowerPoint presentation, as well as a poster summarizing the project, will be submitted for presentation at the AABP and NMC national conferences. Overall, KVC is poised to distribute the results of this study to farmers and industry professionals alike. Through sharing of written materials to our clients and QMPS, and presentations at regional meetings, KVC can distribute the practical knowledge gained by this study to farmers throughout the Northeast. Through regional and national organizations, scientific papers, PowerPoint presentations and posters KVC will inform industry professionals whose impact extends to a national, and even international scale. Through this process, KVC will be targeting all those who play a role in on-farm decision making, allowing for informed management decisions that will serve to improve farm profitability and sustainability.

    Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.