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

2011 Annual Report for ONE11-133

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

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

Summary

During the study period, 4% of the 4,101 clinical mastitis samples submitted by 76 farms to the Keseca Mastitis Lab were identified as non-hemolytic, esculin-positive Streptococci. Of those isolates, 84 were evaluated using the API Strep 20 system, and consequently submitted to Quality Milk Production Services for molecular verification. To date, 18 isolates, representing 5 different farms, have been molecularly identified as Lacotococcus lactis subspecies lactis, an emerging mastitis pathogen. Once testing is complete, statistical accuracy of the API test will be calculated to determine its usefulness in diagnosing Lactococcus infections. The individual cow information collected during the time period will allow analysis of individual cow risk factors for Lactococcus infection. A twelve-page herd survey was created to assess farm level risk factors, and has been completed by the 5 Lactococcus positive herds, as well as 5 Lactococcus negative farms. While the small number of positive farms precludes statistically significant analysis at this time, all positive farms used sand bedding, and thus bedding samples were cultured. Currently, 3 of the 5 positive farms have had Lactococcus identified in bedding samples, and the remaining farms will be tested within the next two weeks. All Lactococcus isolates were tested for antibiotic susceptibility to create reference library, since no data for Lactococcus exists in the literature. While data is still being analyzed, impacts of the study have already been noted- two positive farms have seen reduction in somatic cells levels during the study period due to changing of bedding management and segregation of Lactococcus positive cows from the rest of the herd. With the completion of data collection and analysis in the next few months, conclusions will be presented at the annual client meeting and shared with other industry professionals in hopes of expanding the positive impacts already seen.

Objectives/Performance Targets

The objectives of the study fall into three main categories: evaluation of API test strip to determine its effectiveness as an alternative to expensive molecular diagnostics; evaluation of cow and farm level risk factors of disease to identify sources on the farm and create management strategies; and antibiotic susceptibility testing of isolates to create a data base that may serve as a foundation for future drug treatment trials.

Objective 1: API Strep 20 Test Strip Evaluation
1. Currently, a total of 4,101 clinical mastitis cultures have been evaluated by standard biochemical techniques, and all non-hemolytic, esculin-positive Streptococci isolates were evaluated for inclusion in API testing.
2. Out of those cultures, 84 isolates have been run on API strips, 18 of which were identified by molecular techniques as Lactococcus lactis subspecies lactis. These Lactococcus isolates represent five dairy farms in the region, out of the original sample population of 76 farms.
3. Preliminary sensitivity, specificity, and overall accuracy results have been completed for the API test strip compared to molecular testing. Final results will be ready once all molecular results are reported by reference lab.

Objective 2: Risk Factor Analysis
1. Using farm record programs, data on each of the 84 cows was entered into Excel for risk factor analysis, and included characteristics such as lactation number, production levels, somatic cell levels, reproductive status, pen number, and additional health events.
2. A twelve-page herd was survey created to assess facility, management, nutritional, herd demographics, and other factors that may affect disease presence.
3. Herd survey was completed by the five farms with identified Lactococcus cases and by five farms without an identified case. Survey responses were entered into Excel for evaluation. Due to the small number of farms with identified Lactococcus cases, statistical analysis was not significant at this time.
4. Sand bedding samples were taken from three of the five farms with identified clinical Lactococcus cases were submitted for API testing and molecular verification. Lactococcus lactis subspecies lactis was identified in all three of these samples.

Objective 3: Antibiotic Susceptibility Testing
1. Minimum Inhibitory Concentration (MIC) testing was completed on all 18 clinical isolates and all environmental samples identified as Lactococcus lactis subspecies lactis.
2. MIC results entered into Excel for analysis once data collection is complete.

Accomplishments/Milestones

The first step in the study was to evaluate the API test strip as an accurate, less expensive, and more rapid diagnostic test for Lactococcus infections. While the test has provided faster results than the traditional molecular testing that requires a reference lab, it is clear that interpretation of the test is somewhat subjective, and requires some time to establish a consistent interpretation process. Initially, it was expected that only 40 samples from the mastitis lab would qualify for API testing based on previous experience. However, an unexpected decrease in referral lab fees allowed for a larger number of tests to be run. As a result, 84 isolates have been tested, far exceeding initial expectations. As a result, this has extended the data collection period slightly, which has delayed analysis, but should provide stronger statistical power to our calculations and give more support for study conclusions. Additionally, this means a larger number of dairy farms, our target population, were enrolled in the study process. Additionally, the increased spectrum of isolates may allow evaluation of the API strip for additional groups of bacteria beyond the original focus of the study, Lactococcus lactis. Overall, the small extension of data collection period will give more depth to study results without altering the timeline for dissemination of study conclusions.

As isolates were identified, the cows associated with those samples were entered into the second phase of the process-risk factor analysis. Simultaneously, farms submitting clinical samples were identified as Lactococcus positive or negative. The original plan was to conduct a herd survey and conduct risk factor analysis on a farm-level. While a twelve page survey was created and completed by the ten farms, it became clear that the small number of “positive” farms will severely limit statistically powerful conclusions from this data. The herd data collected was still entered into an Excel file for future analysis, but it did not provide strong indicators to direct environmental sampling as hoped. Therefore, environmental sampling was guided by the fact that all positive farms had sand bedding in common. Despite this change of events, this sampling technique has proven successful in identifying the presence of Lactococcus in bedding samples from positive farms. Additionally, a second Excel file was created to analyze cow-level risk factors since the number of individual cows identified was larger than expected and could provide significant results. While this is a slight change from the original plan of work, it should provide a very detailed analysis regarding cow characteristics and health events that may be associated with disease.

Finally, as both clinical samples and bedding samples were molecularly identified as Lactococcus, antibiotic susceptibility testing was conducted. This testing is still being completed, but has proceeded as planned. Clear trends in susceptibility have been observed, and may provide data useful for further treatment studies either in on-farm studies by veterinarians or by the pharmaceutical industry. As the data collection period comes to a close, the next phase of data interpretation and dissemination of results will achieve the ultimate goals of the study- to assess the API strips accuracy for farmer benefit, and to identify key management procedures to prevent and control Lactococcus mastitis on farms.

Impacts and Contributions/Outcomes

Although data is still being collected and analyzed, this study has already had positive impacts on dairy farmers. Two of the five identified Lactococcus positive herds were struggling with high somatic cell counts prior to the study period. The first farm has reduced their monthly average somatic cell count (SCC) from nearly 400,000 to less than 200,000 by segregating Lactococcus positive cows identified in the study into their own milking group and by improving their sand bedding management following identification in bedding samples. This reduction has resulted in increased quality premiums, and therefore increased income, for this farm. The second farm has also seen improvements in SCC by changing the depth of sand bedding raking so as to prevent Lactococcus in deep bedding from being drawn to bedding surfaces. An unexpected positive impact from farm cooperation in the survey process has been much greater access to herd management information for herd veterinarians beyond what is normally gathered at routine visits. The survey has the potential to improve veterinarian-client communication on a great variety of management topics, and may provide useful in day-to-day veterinary practice. Additionally, there has been unexpected interest from herd nutritionists as to how nutrition may play a role in the pathogenesis and/or prevention of the disease. Furthermore, discussion of the study with all of the herds enrolled on the study has facilitated greater client awareness of general mastitis prevention and control strategies regardless of specific pathogens identified. Additional data analysis will allow for more concrete conclusions for presentation to the remainder of our clients at the annual meeting in March 2012, and provides the opportunity for potential follow-up projects utilizing Quality Milk Production Services concerning testing on additional New York farms or potentially connecting bacterial isolates found in bedding samples to clinical isolates from the same farms.

Collaborators:

Dr. Brenda Moslock Carter, DVM

keseca@rochester.rr.com
Clinic Owner/Collaborator
Keseca Veterinary Clinic
PO Box 267
Geneva, NY 14456
Office Phone: 3157811378
Denise Burnett

keseca@rochester.rr.com
Mastitis Lab Technician
PO Box 267
Geneva, NY 14456
Office Phone: 3157811378
Mary Ellen Charter, LVT

keseca@rochester.rr.com
Mastitis Lab Technician
Keseca Veterinary Clinic
PO Box 267
Geneva, NY 14456
Office Phone: 3157811378