Progress 07/01/08 to 09/30/12
Outputs
OUTPUTS: During 2008-2012, we contributed to meeting the goal of meeting the multistate NE1028 project's #1 and #3 objectives. Specific details of these objectives are reported in the AD 416 for this project. Initially, we focused on establishing a distal teat environment that will function to exclude pathogenic bacterial colonization using probiotics delivered locally, as a teat dip. A secondary goal of our project was to study the response of selected factors in the innate immune system to this microbial flora augmentation. We defined and cultured selected endogenous teat flora, evaluating in vitro efficacy against selected mastitis pathogens. This work helped define the endogenous flora of the teat skin and distal teat canal. Later work responded to an increase in cases of algal mastitis in Maine, and investigated improved diagnostic testing, prevalence in Maine, pasteurization resistance of Maine isolates, and conditions enhancing proliferation of the organism on dairies. An additional major output of 2010-12 was to develop a nested PCR to screen bulk tank filters for prototheca, a colorless algae capable of causing bovine mastitis. This test was developed in our lab, and a statewide prototheca prevalence survey was completed using bulk tank filters from collaborating dairies. The PCR test has been used to screen dairies on a research and clinical basis in our mastitis lab. As well, we have tested pasteurization resistance of Maine prototheca isolates, and evaluated bedding types for prevention of prototheca on farms. Current work focuses on conventional and alternative treatment for prevention and control of prototheca mastitis. Results were reported as presentations to regional dairy producers, state large animal veterinarians, the Maine CDC, and to the multistate research project participants. Yearly presentations have been given to state dairy producers during their regular meetings. Presentations to raw milk producers in Maine (in collaboration with the Maine state veterinarian and with the Maine CDC) have been given during 2010-12. Reports to the state's large animal veterinarians have been given at the state's semiannual Maine Cattle Health Assessment Program meetings. A summary of the effect of pasteurization on Maine prototheca isolates was presented to the Maine CDC during 2011. Presentations were given during 2008, 2009, 2010 and 2011 at the Mastitis Research Workers annual meetings on these topics. A prototheca workshop was given at the National Mastitis Council annual meeting during 2012, and will be given again in 2013. One masters thesis and 4 senior thesis projects have been completed utilizing this project for investigation; an additional masters thesis and senior thesis will be completed this year. PARTICIPANTS: Training and development opportunities for several graduate students, and a number of undergraduate students, were provided by this study. Debbie Bouchard provided assistance in technique development for the probiotic work, and Sarah Barker provided essential support, especially for the development of the PCR technique. The individuals working on the project included several undergraduate students (5 over the life of the project), all of whom were female and several of whom have successfully started veterinary school. As well, the current masters student working on the project is a veterinarian from Nepal. The project has allowed collaborations with mastitis professionals in Canada and Europe, as well as both Idaho and New York state. During the last year, I served as the chair of the Mastitis Research Workers group and led the rewrite and submission of the revised NE 1028 project, which was approved (now NE 1048). Other collaborators on the project included the large animal and the state veterinarians of Maine, and dairy producers in Maine. TARGET AUDIENCES: Target audiences for work over the life of this project included dairy farmers in the Northeast, livestock veterinarians in Maine, members of the Mastitis Research Workers (NE 1028) multistate project, and members of the National Mastitis Council or participants in the NMC meeting. The latter 2 groups include dairy producers, dairy processors, dairy veterinarians and other dairy industry professionals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Development of the nested PCR contributed to a change in knowledge, as an effective means to quickly determine the presence and type of Prototheca in a herd of dairy cattle had not been devised. A change of action has occurred, in that Maine dairy producers are now aware of the test, and many have participated in the screening process. 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 a herd of dairy cattle. In the case of one dairy, the owner is collaborating in changing methods of water delivery, cleaning/sanitizing methods, and repeated testing/culling to keep the disease under control. We expect a change of conditions, in the sense of better and less expensive animal health when this pathogen is cleared from the farm due to rapid detection and prompt action. Projects in the lab to evaluate the pasteurization sensitivity of Maine prototheca isolates have been communicated with the Maine dairy industry, and projects to determine what bedding types might promote prototheca infection have been shared with Maine's large animal vets and the Maine Cattle Health Assurance program. Ongoing projects in the lab will evaluate experimental prototheca treatments. Detection of this organism will allow Extension specialists and veterinarians to counsel producers about changing methods of water delivery, cleaning/sanitizing methods, and repeated testing/culling to keep the disease under control. Rapid detection and prompt action should allow reduction of prototheca mastitis, elevation of milk quality, and improved price per pound of milk. As more information about the state and regional prevalence of protothecal mastitis is developed, the disease should decrease in incidence due to improved hygiene and culling of infected cattle. Work in our lab has shown that Maine prototheca isolates are generally sensitive to standard pasteurization techniques, but small numbers escape pasteurization. As well, work in our lab supports the use of cedar shavings as a bedding source to minimize prototheca transmission on the farm. Current work with this pathogen will assist in finding treatments or other preventive measures. During this project, over 200 dairy farmers and 50 dairy professionals participated in informational events regarding protothecal mastitis. National Mastitis Council workshops given in 2012 and 2013 are expected to encourage discussion and awareness of prototheca on dairy farms, and to help find diagnostic and treatment solutions for dairy producers across the US.
Publications
- No publications reported this period
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: The objectives of the NE 1028 mastitis research multistate project include first, characterization of host mechanisms associated with mastitis susceptibility and resistance; second, characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defense; and third, assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. A primary goal of this study within the NE 1028 project focused on Objective 1: to establish a distal teat environment that will function to exclude pathogenic bacterial colonization using probiotics delivered locally, as a teat dip. A secondary goal of this project was to study the response of selected factors in the innate immune system to this microbial flora augmentation. Modulation of innate immune system component expression in the presence of "normal" flora might provide a non-pharmacologic means of preventing mastitis in cattle. We characterized distal teat flora (streak canal and epithelium) for a subset of our lactating dairy herd, identifying these "normal" flora, and evaluating them for suitability as probiotic teat applications. These findings were discussed in state dairy producer meetings and at the multistate meeting. We also, due to an increased incidence of prototheca mastitis in Maine, focused on the NE 1028's Objective 3, new technologies to advance mastitis control, milk quality and dairy food safety. To help meet this objective, we developed a nested PCR to screen bulk tank filters for prototheca, a colorless algae capable of causing bovine mastitis. This test was validated in our lab. As well, we have conducted a statewide prototheca prevalence survey using bulk tank filters from collaborating dairies. The findings from these studies were discussed at several state dairy producer meetings, and at the multistate project meeting. PARTICIPANTS: The PI has utilized this project as a method to integrate senior capstone students into the activities of the diagnostic lab. Tanya Farrington-Thomason completed her masters thesis working on this project (August, 2011). A new masters student from Nepal, Nirajan Adhikari DVM, has begun work on the project. Currently, 4 undergraduate students have been involved in the project either as work-study students (2) or as senior capstone students (2). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
During 2010-2011, the outcomes for this project included use of a sensitive and specific detection method for prototheca, utilizing bulk tank filters. This method allows screening for pathogenic prototheca species on dairy farms. Where somatic cell counts are elevated, the dairy farmer may choose to followup by conducting individual cow milk testing to find those with protothecal infections. As well, the farmer is advised that scrupulous control of milking hygiene and biosecurity is needed to contain the spread of prototheca mastitis. Additional outcomes include the active participation of 6 undergraduate and 2 master's level students in the project during 2010-11. A bulk tank filter prototheca survey of Maine dairies was completed, including culture and PCR testing. Based on this work, a pasteurization resistance study of the prototheca isolates found in Maine was conducted. We found that several of the Maine isolates were not completely eliminated using standard pasteurization methods, and are reporting this finding to the state CDC at a regional meeting this month. We have also discussed this finding at a Maine dairy veterinary meeting, and with the veterinarians of the participating dairies (bulk tank survey). We are discussing extending our protocols for testing to include dairies in other areas of the country. We are continuing pasteurization studies to evaluate sensitivity in all Maine isolates of prototheca, and to evaluate mechanisms of resistance. We continue to collect and evaluate bulk tank filter and milk samples in order to follow the success of Maine farms in eliminating prototheca from their milking herd. Additionally, our diagnostic lab conducts mastitis screening and diagnosis for a number of dairies in the state, allowing us to report overall mastitis pathogen prevalence and antimicrobial resistance pattern to our dairy producers and practitioners. Of the 9 Maine farms with prototheca, 4 have participated in outreach efforts including repeated testing and on-farm investigation of possible reservoirs of infection. Attending veterinarians have participated in this outreach, and a followup study to evaluate the effectiveness of teat sealants for prevention and control of prototheca intramammary infection in the dry period is planned. Findings from this project have been reported yearly to Maine dairy producers at state-level meetings, and to the Mastitis Research Workers meetings (NE1028 multistate project). Interest from milk processors has focused on possible screening of large units of milk prior to processing.
Publications
- Lichtenwalner, AB; Adhikari, N and Farrington-Thomason, T. Prototheca mastitis in Maine: prevalence and pasteurization studies of a food-borne pathogen. Maine CDC: 2011 Infectious Disease Annual Conference, Augusta ME.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: The objectives of the NE 1028 mastitis research multistate project include first, characterization of host mechanisms associated with mastitis susceptibility and resistance; second, characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defense; and third, assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. A primary goal of our study within the NE 1028 project focuses on Objective 1: to establish a distal teat environment that will function to exclude pathogenic bacterial colonization using probiotics delivered locally, as a teat dip. A secondary goal of our project is to study the response of selected factors in the innate immune system to this microbial flora augmentation. Modulation of innate immune system component expression in the presence of "normal" flora may provide a non-pharmacologic means of preventing mastitis in cattle. Work on this objective, however, has been delayed due to refocusing our project onto the NE 1028's Objective 3, new technologies to advance mastitis control, milk quality and dairy food safety. Therefore, a major output of the last year has been to develop a nested PCR to screen bulk tank filters for prototheca, a colorless algae capable of causing bovine mastitis. This test has been validated in our lab. As well, we have conducted a statewide prototheca prevalence survey using bulk tank filters from collaborating dairies. We have presented 2 talks at the Mastitis Research Workers meeting in Atlanta, GA, and are scheduled to speak at the Maine Dairy Producers workshop in March, 2011. We are working with a local producer to control an outbreak on his dairy. These methods will be submitted as peer reviewed publications, and in industry publications. The PCR test will be a regular diagnostic tool in our mastitis lab. We have also integrated new environmental and milk sample testing strategies for this pathogen. We have begun pasteurization resistance studies using our Maine prototheca isolates. PARTICIPANTS: A new addition to the project was Sarah Barker PhD, who developed the nested PCR> Tanya Farrington is developing her masters project using the Maine prototheca isolates. TARGET AUDIENCES: Our target audiences are the dairy producers, who were contacted via a survey request, and who will be given study results this spring in a producer meeting. An additional benefit is the availability of this assay for other users of our diagnostic lab, including out of state dairies and veterinarians. PROJECT MODIFICATIONS: Due to the emergence of several cases of protothecal mastitis in Maine during 2009-10, we shifted focus of our project onto the NE 1028's Objective 3, new technologies to advance mastitis control, milk quality and dairy food safety. Therefore, a major output of the last year has been to develop a nested PCR to screen bulk tank filters for prototheca, a colorless algae capable of causing bovine mastitis. This test has been validated in our lab. As well, we have conducted a statewide prototheca prevalence survey using bulk tank filters from collaborating dairies.
Impacts
Development of the nested PCR contributed to a change in knowledge, as an effective means to quickly determine the presence and type of Prototheca in a herd of dairy cattle had not been devised. A change of action has occurred, in that Maine dairy producers are now aware of the test, and many have participated in the screening process. In the case of one dairy, the owner is collaborating in changing methods of water delivery, cleaning/sanitizing methods, and repeated testing/culling to keep the disease under control. We expect a change of conditions, in the sense of better and less expensive animal health when this pathogen is cleared from the farm due to rapid detection and prompt action.
Publications
- Lichtenwalner AB and Barker S. The Presence of Pathogenic Prototheca sp. on Dairy Farms in Maine, USA. Mastitis Research Workers conference; Nov. 4-5, Atlanta GA 2010. Barker,S and Lichtenwalner AB. A Nested PCR RFLP Diagnostic Test for the Presence of Pathogenic Prototheca spp. in Dairy Herds. Mastitis Research Workers conference; Nov. 4-5, Atlanta GA 2010.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: In 2008, work on this project generated 2 senior student theses, one masters student project, and 4 producer presentations. Conventional dairy, licensed raw milk dairy, small ruminant dairy and beef producers in Maine have been informed about progress to date on the project. The Mastitis Research Workers multistate meeting was informed on our progress to date in 2008 and 2009. Work on this project is still in process and no publications have as yet been produced. PARTICIPANTS: Technician: Deborah Bouchard Students: Tanya Farrington (Masters candidate in Animal Veterinary Science), Jennifer McIntee, Abby Arena (senior students in Animal Veterinary Science) Students involved in this project were trained in basic microbiology and obtained a working understanding of the concept of probiotics in health maintenance. TARGET AUDIENCES: Dairy producers in Maine: many small or organic producers, including approximately 350 farms in Maine, could benefit from a product that might prevent new mastitis infections. Such an organic product could be rapidly implemented without fear of drug or iodine residues in milk. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In the last year, the primary outcomes from our work on this project have been to establish a group of potential endogenous probiotics. Secondary outcomes are related to technique development and student training. In our initial screening of endogenous flora, we were able to identify a total of 33 teat skin bacterial isolates, and 27 streak milk bacterial isolates. Of these, most were bacillus, arthobacter or coagulase negative staph (CNS) species. Of these, 3 have shown substantial inhibition of known mastitis pathogens in vitro to date. Biofilm testing is underway. Further testing, including in vitro toxicity and efficacy, will be completed prior to tests of in vivo application. Producer groups have indicated interest in this product, especially as an adjunct to organic dairy applications. Resources used to product the outcomes to date include salary support for technician time to train students and culture materials costs.
Publications
- No publications reported this period
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Objectives: The primary objective of this group of projects is the characterization of host mechanisms associated with mastitis susceptibility and resistance. A primary goal of this study is to establish a distal teat environment that will function to exclude pathogenic bacterial colonization. We will evaluate the capacity for a normal flora of the distal teat to reestablish itself between milkings, and the effects of augmenting that flora with suitable probiotics delivered locally, as a teat dip. The general goal is to provide an alternative to the use of teat dips that may be more effective in enhancing local immune responses in the host. A secondary goal of this study is to study the response of selected factors in the innate immune system to this microbial flora augmentation. Modulation of innate immune system component expression in the presence of "normal" flora may provide a non-pharmacologic means of preventing mastitis in cattle. In the first 4 months of this project, are focusing on the primary goal of a local probiotic for distal teat application. We have developed a culture and biofilm assessment protocol to begin evaluations of candidate surface probiotics. Work is underway to select the best candidate from a group of ATCC cultures for trial applications, which are scheduled for spring 2009. A summary of this activity follows: Goals: Characterization of host mechanisms associated with mastitis susceptibility and resistance 1. Characterize normal teat end and distal teat canal flora in healthy dry and lactating dairy cattle a. Collect endogenous nonpathogenic flora for potential probiotic use We are now collecting teat skin and distal teat canal flora in lactating cows for culture and identification. 2. Select exogenous or endogenous microbial nonpathogenic flora for potential probiotic use 3.Evaluate cell adherence and biofilm capacity of potential probiotic a. A biofilm assessment protocol has been developed. b. We are evaluating 3 candidate organisms from ATCC at this time. 4. Evaluate antimicrobial efficacy of potential probiotics in vitro 5. Evaluate antimicrobial efficacy in vivo 6. Evaluate the effect of passaging selected cultures for potential probiotics 7. Test for changes in selected innate immune system components in treated, versus control, cows. One graduate student and one senior student are working on this project, and have each given seminars on the project. In both cases, probiotic utilization and concepts of innate immunity are being explored as learning objectives. PARTICIPANTS: Anne Lichtenwalner DVM PhD has directed student activities, designed the experiments and represented the project at the MRWC meeting in November 2008. Debbie Bouchard BS has provided assistance and direction of assay development, and direct assistance to students in the laboratory setting. Tanya Farrington BS is a Masters candidate who is working with endogenous teat cultures and development of a probiotic suspension for application. Jennifer McIntee is an Animal Veterinary Science senior student who is working with biofilm assay development and assessment. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We are in the 4th month of work on this project. At this time, the main outcome has been to develop laboratory skills in our trainees, and identify a candidate for further testing as a teat dip candidate. We are still in the process of identifying normal flora in our herd. One graduate student and one senior student are working on this project, and have each given seminars on the project. In both cases, probiotic utilization and concepts of innate immunity are being explored as learning objectives. The PI attended the NE1028 meeting and presented the project and its current status to the 2008 Canadian Bovine Mastitis Research Network/Mastitis Research Workers Conference for discussion, and was approved by the MRWC for inclusion in NE1028.
Publications
- No publications reported this period
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