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

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

Summary

In order to characterize the emerging bovine mastitis pathogen, Lactococcus lactis subspecies lactis, a three phase study design was implemented by the Keseca Veterinary Clinic (KVC) Milk Quality Laboratory. Data collection was completed in March 2012. The first phase evaluated the API Strep 20 test system’s ability to identify Lactococcus lactis ssp. lactis in clinical mastitis samples. Out of the 5,261 clinical samples submitted by dairy farms in the Finger Lakes region of New York, 4.2% were classified as non-hemolytic, esculin-positive Streptococci and evaluated for purity and culture density. Isolates meeting the criteria were run on the API Strep 20 system and submitted to Quality Milk Production Services (QMPS) at Cornell University for molecular identification. Data collected in 2012 allowed identification of an additional six Lactococcus isolates from clinical mastitis samples, yielding 24 total clinical Lactoccous isolates for the study period out of the 79 tested. In the second phase, farms both with and without Lactococcus isolates completed a herd survey to identify farm risk factors for disease. While the number of farms with Lactococcus was too low to statistically evaluate farm- level risk factors, it was observed that all five positive farms used sand as their bedding material. Therefore, sampling of sand bedding was completed in 2012 and Lactococcus was identified in the bedding on all five positive farms. Cow level risk factors were also evaluated. Finally, in the third phase, antibiotic sensitivity testing was run on all Lactococcus isolates, both from clinical mastitis samples and bedding samples. Data analysis has now provided one of the most extensive libraries of antibiotic sensitivity of Lactococcus isolates from bovine clinical mastitis. Analysis of data from all three phases of testing began in April 2012 and has been completed. An overview of the research project and preliminary results was shared with approximately 75 dairy farmers at the annual client meeting in March 2012. Results are currently being written up for submission to industry publications and upcoming veterinary conferences. Results will also be shared with clients at a meeting in March 2013, with an emphasis on identifying and controlling the disease-adding a local, practical impact for the dairy industry of the Finger Lakes.

Objectives/Performance Targets

The main objective of our project is to characterize Lactococcus lactis subspecies lactis isolated from bovine mastitis samples in order to provide dairy farmers and veterinarians with practical knowledge to prevent, identify, and control the disease. In order meet this main objective, our project was divided into three phases. In the first phase, the study evaluated the API Strep 20 system as a more economical test for identifying the disease than current alternative testing options. The second phase included using the results obtained in the first phase to identify potential farm-level, cow-level, and environmental risk factors for this particular pathogen. Finally, the third phase included antibiotic sensitivity testing of Lactococcus isolates from the first two phases to inform veterinarians on more successful treatment recommendations.

Objective 1: API Strep 20 Test Strip Evaluation (Phase One)
1. Approximately 5,261 clinical isolates were evaluated in KVC laboratory by standard biochemical techniques during the study period, and all non-hemolytic, esculin-positive Streptococci isolates were evaluated for inclusion in API testing.
2. Out of those isolates, 79 were run on API strips, 24 of which were identified by molecular techniques at QMPS- Cornell University as Lactococcus lactis subspecies lactis. These Lactococcus isolates represent five dairy farms in the region, out of the original sample population of 77 farms.
3. For data analysis, six clinical isolates were excluded after API testing due to concern over contamination of the sample or inability of molecular testing to identify sample to the genus. Therefore, only 73 isolates from this phase were included in data analysis and submitted to Phase Three (see below).
4. Data analysis for this phase of the study was completed this year:
a. Sensitivity, Specificity, Positive Predictive Value, and Negative Predictive Value of the API Strep 20 system for identification of Lactococcus were calculated.
b. These statistics were also calculated for the additional genuses of bacteria identified in our sample population, including Aerococcus, Enterococcus, and other minor Streptococcus species
c. Biochemical variation in Lactococcus isolates was also analyzed by evaluating the variation in isolate results for the 20 individual tests included in the API Strep20 system

Objective 2: Risk Factor Analysis (Phase Two)
1. Data collection completed for cow-level risk factor assessment using on-farm records systems. Information such as lactation number, production level, somatic cell levels, reproductive status, pen number, and additional health events were recorded for every additional cow entered into Phase One.
2. Statistical analysis of cow-level risk factors was completed. A researcher at Cornell University is currently being consulted to verify analysis.
3. Farm-level risk factor analysis was not significant at this time. However, descriptive data of the five positive farms was collected for reporting purposes.
4. Sand bedding samples were analyzed from the final two farms with identified clinical Lactococcus cases. The first three positive farms were sampled and analyzed in 2011.
5. Molecular testing completed on bedding samples. Lactococcus was detected in bedding samples from all five positive farms.
6. Veterinarians discussed bedding sample results with the five farms sampled. Discussion included potential improvements in management of sand bedding for prevention of disease.
7. All Lactococcus isolates from sand bedding cultures of Phase Two entered into Phase Three.

Objective 3: Antibiotic Sensitivity Testing (Phase Three)
1. Minimum Inhibitory Concentration (MIC) testing was completed at QMPS on all 24 clinical isolates and all environmental samples identified as Lactococcus lactis subspecies lactis.
2. Antibiotic sensitivity results have been summarized and compared to sensitivity results found in the literature.
3. Antibiotic sensitivities of clinical isolates from each positive farm were also compared directly to the sensitivities of the environmental samples from that same farm to determine if isolates within farms had specific profiles.

Accomplishments/Milestones

As reported in the 2011 annual report, an overestimate of testing expenses left us with a budgetary excess for reference lab testing. As a result, our data collection period extended through March 2012 and allowed expansion of our data set to a total of 79 clinical mastitis sample isolates. Data collection and testing at reference lab was completed by April 2012. The extended data collection period not only allowed us to collect twice as many samples as originally planned for the first phase of our study, but also permitted the identification of six additional clinical Lactococcus isolates. Of the 79 clinical isolates that were run through the first phase of this process (API system testing), six were excluded from data analysis due to concern over purity of culture or inconclusive molecular results. Once these isolates were identified, data analysis could begin. Unfortunately, the extended time period did not add any additional “positive” farms to our data set. However, it did allow us to finish sampling the sand bedding from the remaining 2 “positive farms”, both of which yielded Lactococcus isolates for evaluation. While a causative relationship cannot be proven by our data, the fact that Lactococcus can be found in the bedding of all the farms in which clinical isolates were found at least points to Lactococcus’ ability to thrive in sand environments. Finally, those additional clinical and environmental isolates were submitted for antibiotic susceptibility. Between the 24 clinical isolates and 7 environmental isolates submitted for MIC testing, this library of antibiotic susceptibility appears to be one of the most extensive identified in the literature.

In March 2012, our research was shared with approximately 75 farmers at the annual client meeting. Basic information regarding Lactococcus was covered and was supplemented with preliminary findings from our study. Initially, we had hoped to present finalized data at this meeting, but the extended data collection period did not leave enough time for analysis prior to the meeting. Furthermore, as data analysis began, it became clear that the sheer amount of data collected during the study period was going to require more extensive analysis than originally planned. Due to twice the number of clinical isolates than expected, we were able to evaluate API strip accuracy not only for Lactococcus, but also for several other species of bacteria identified in our sample population. Additionally, we were able to summarize the variability in biochemical and metabolic properties of the Lactococcus isolates by evaluating the variation in individual tests included in the API Strep 20 system. The larger data set also allowed for more thorough characterization of antibiotic sensitivity data, and allowed a more powerful evaluation of the comparison between clinical mastitis isolates and the environmental isolates from the farms where they reside. Now that the data has been analyzed and an extensive literature survey has been conducted, we are in the process of writing up our results for submission to veterinary and industry journals. Again, the depth of our data may require that our findings be written up in smaller, more manageable pieces than in one long summary (e.g one paper just on the API system and a second one focusing only on the antibiotic sensitivity analysis). While we are still in the process of writing our own paper, we have been able to collaborate with QMPS on their paper detailing their molecular testing method for Lactococcus, which is has already been submitted for publication.

Looking forward, we are transitioning into the data dissemination period with some very promising observations and conclusions. Due to the long lead time needed for applying to present at scientific conferences, our timeline did not afford us the opportunity to apply for presenting at the Spring 2013 veterinary and dairy conferences as originally planned. However, we are on track to apply for presenting our findings at the Spring 2014 conferences when they decide their agendas at the Spring 2013 meetings and also plan to apply for presentation at the Fall 2013 American Association of Bovine Practitioner’s conference. Furthermore, we plan to submit our scientific papers for veterinary journal publication in Spring 2013, and will present our findings at our 2013 client meeting as previously discussed.

Impacts and Contributions/Outcomes

As detailed in the previous sections, this year’s major outcome has been analysis of our large data set. Based on the literature survey completed as part of our research, it appears that our findings will certainly expand the knowledge about Lactococcus as a bovine mastitis pathogen. Our detailed characterization of basic biochemical and metabolic properties of Lactococcus understanding will also aid those in basic laboratory settings where elaborate testing methods may not be available. As reported in 2011, the study has already had a positive impact on two of the five identified Lactococcus positive herds were struggling with high somatic cell counts. Due to the identification of Lactococcus in bedding samples, these farms have made changes to their bedding management to help control new infections in the herd. This reduction has resulted in increased quality premiums, and therefore increased income.

Although the farm-level risk factor analysis was not as rewarding as we hoped, the survey created for this part of the research has been modified to include sections on breeding and vaccine protocols for everyday use by veterinarians who are looking to evaluate on-farm management protocols. This survey has proven useful in maintaining VCPR status (veterinarian-client-patient relationship) between veterinarians and farmers, especially on farms that only use a veterinarian sporadically. It has already served its purpose in stimulating communication between farmers and vets, leading to a more productive working relationship. Also interesting to note was that following the presentation of preliminary results at the client meeting in March 2012, several clients (dairy farmers) brought up discussions about milk quality at subsequent herd health visits. Thus, discussion of this specific project has served to also stimulate general milk quality control discussions with our farmers in terms of general mastitis prevention and control strategies regardless of specific pathogens identified.

Finally, our antibiotic sensitivity results have helped inform our treatment decisions on cows identified with mastitis caused by non-hemolytic, esculin-positive Streptococci, including Lactococcus. By doing so, we believe we have reduced unnecessary and ineffective treatments thereby reducing the cost of mastitis to the dairy producer and supporting more judicious use of antibiotic on the farm.

Collaborators: