Barlow, John Food Safety from Milking to Marketing for Vermont Artisanal Cheese Maker
National Institute of Food and Agriculture
The objective of this project is to support Vermont's growing artisanal cheese production regional food chain with food safety microbiology, epidemiology and consumer research, and extension activities directly linked to human health concerns. The project addresses food safety research and extension gaps for Vermont's artisanal cheese makers. Food safety pathogen epidemiology and detection research is addressed across the entire on-farm cheese production chain. Research on a novel pathogen detection technology that may be scalable to all size dairy operations. Integration of these detection technologies with an improved understanding of pathogen epidemiology will fill gaps in our current abilities to mitigate food safety risk on dairy farms producing cheese or other raw milk products. Proposed research on consumer beliefs, attitudes and practices brings the project full circle from milk production to marketing and back to production again by providing artisan cheese makers with knowledge on consumer issues.
Barlow, John Mastitis Resistance to Enhance Dairy Food Safety
National Institute of Food and Agriculture
The multi-state project NE-1048 objectives include: Objective 1) Characterization of host mechanisms associated with mastitis susceptibility and resistance; Objective 2) Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses; Objective 3) Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. The Barlow lab will address objective 2 and the sub-objectives i) characterization of pathogen virulence factors, ii) antimicrobial resistance, and iii) molecular epidemiology and diagnostic tools. We will examine potential associations between antimicrobial use practices, carriage of antimicrobial resistance genes and response to antimicrobial therapy among different strain types of Staphylococcus aureus and other Staphylococcus species. We will characterize and evaluate the impact of putative virulence factors in vitro and the examine association of these factors with DNA-based strain types for S. aureus. We will evaluate the use of molecular epidemiologic tools to describe transmission dynamics of contagious mastitis pathogens and antimicrobial resistance. Our work is focused on the epidemiology of staphylococci in emerging sectors of the dairy industry in Vermont including niche dairy farms (e.g. on-farm artisanal cheese producers and organic dairy farms) and dairy farms adopting automated milking technologies. We have completed a pilot study of contagious mastitis pathogen transmission in a herd milking cows in robotic systems. In this study we demonstrated limited Staph. aureus and Strep. agalactiae transmission on a farm using robotic milking. We have also completed a 12 month study of milk quality on 43 organic dairy farms and have established a library of S. aureus isolates from these farms. We have identified >20 different strain types with 90% of the isolates clustering in 3 clonal complexes. We have also determined that specific S. aureus strain types are associated with the ability to form biofilms and carriage of antibiotic resistance genes. We have determined that Staphylococcus chromogenes is an important mastitis pathogen on many dairy farms. This organism might be described as an emerging mastitis pathogen in that it is more recently being identified and recognized as a cause of persistent infections. In collaboration with a scientist at U.C. Berkeley, we have developed and evaluated a strain typing scheme for S. chromogenes. This typing scheme will be used to improve understanding of the epidemiology of this organism on dairy farms. Our goal is to combine molecular and mathematical epidemiological approaches to unravel the complex problems of chronic intramammary infections and antimicrobial resistance emergence and transmission in cattle populations. In the next year we will conduct in vitro studies of virulence for S. aureus and S. chromogenes isolates from our library. Outputs will include peer reviewed publications, presentations at national and local meetings, and contributions to bacterial isolate collections with epidemiological data.
Barlow, John Duration of Immunity to Vaccination of Cattle with Human Ad5FMDV Vacine
Agricultural Research Service/Dept of Agriculture
The goal of this research is to evaluate the duration of immunity and protection from viral challenge following vaccination of Holstein cattle with the newly approved Foot and Mouth Disease Virus vaccine. The first experiments demonstrated that vaccination can provide protection of cattle from live virus challenge up to 6 months following vaccination. The second experiments demonstrated incomplete protection from live-virus challenge at 9 months following vaccination. Additional experiments will be conducted to quantify the role played by specific arms of the immune system in animals that are protected following vaccination. Associated with these experiments, tools (e.g. antibodies for use in assays and assay systems) are being developed to identify and quantify cellular changes in cattle following vaccination or experimental infection. Results of this work will be disseminated to the research community by presentations at international meetings and through submission of manuscripts for publication in peer-reviewed scientific journals.
Barlow, John Determination of Duration of Immunity Conferred by Ad5FMDV Vaccination of Cattle Agricultural Research Service/Dept of Agriculture The goal of this research is to evaluate the duration of immunity and protection from viral challenge following vaccination of Holstein cattle with the newly approved Foot and Mouth Disease Virus vaccine. The first experiments demonstrated that vaccination can provide protection of cattle from live virus challenge up to 6 months following vaccination. The second experiments demonstrated incomplete protection from live-virus challenge at 9 months following vaccination. Additional experiments will be conducted to quantify the role played by specific arms of the immune system in animals that are protected following vaccination. Associated with these experiments, tools (e.g. antibodies for use in assays and assay systems) are being developed to identify and quantify cellular changes in cattle following vaccination or experimental infection. Results of this work will be disseminated to the research community by presentations at international meetings and through submission of manuscripts for publication in peer-reviewed scientific journals.
Barlow, John BREAD: A Modern Approach for the Development of Cattle Vaccines for Critical Bovine Diseases Impacting Smallholder Farmers in Sub-Saharan Africa
National Science Foundation
Improving cattle vaccine assessment technologies will advance understanding of immune system function, accelerate vaccine development efforts and ultimately improve global cattle health. The benefits of improved global cattle health include reduced vulnerability of farmers to agricultural resource losses due to animal disease. An international team of scientists from the United States, Kenya, and Denmark have established a collaborative research program to examine the use of new technologies in immunology with the goal of accelerating vaccine development for two critical cattle diseases that impact smaller holder farmers in sub-Saharan Africa. The objectives of the project are to establish and advance gene typing methods for the major histocompatibility (MHC), also known as bovine leukocyte antigens (BoLA), class I and II genes of cattle, and to establish and advance immune response assays to identity potential antigens for 2 critically important cattle diseases, Foot-and-Mouth disease and East Coast Fever. We have successfully identified methods and initiated typing of Holstein cattle in a University of Vermont Dairy Center of Excellence herd. We have evaluated the genetic types of approximately 120 animals for the MHC class I and MHC Class II DRB3 genes using a series of molecular typing methods. For typing MHC Class I genes we have extended current methods to a potentially more robust system using next-generation gene sequencing technologies. Collaborators have completed synthesis of a panel of synthetic recombinant bovine MHC molecules which will be used as immunologic tools to evaluate T cell immune responses of Holstein cattle following vaccination or natural infection. Experiments have been completed describing the binding affinity of these molecules. These results are expanding our understanding of BoLA gene expression and diversity in Holstein cattle. Results of this work will be disseminated to the research community by presentations at international meetings and through submission of manuscripts for publication in peer-reviewed scientific journals. In the next years we plan to demonstrate the use of a panel of bovine class II MHC-tetramer molecules in studies of Holstein cattle immune responses.
Barlow, John Mastitis Resistance to Enhance Dairy Food Safety
National Institute of Food and Agriculture
Improving cattle vaccine assessment technologies will advance understanding of immune system function, accelerate vaccine development efforts and ultimately improve global cattle health. The benefits of improved global cattle health include reduced vulnerability of farmers to agricultural resource losses due to animal disease. An international team of scientists from the United States, Kenya, and Denmark have established a collaborative research program to examine the use of new technologies in immunology with the goal of accelerating vaccine development for two critical cattle diseases that impact smaller holder farmers in sub-Saharan Africa. The objectives of the project are to establish and advance gene typing methods for the major histocompatibility (MHC), also known as bovine leukocyte antigens (BoLA), class I and II genes of cattle, and to establish and advance immune response assays to identity potential antigens for 2 critically important cattle diseases, Foot-and-Mouth disease and East Coast Fever. We have successfully identified methods and initiated typing of Holstein cattle in a University of Vermont Dairy Center of Excellence herd. We have evaluated the genetic types of approximately 120 animals for the MHC class I and MHC Class II DRB3 genes using a series of molecular typing methods. For typing MHC Class I genes we have extended current methods to a potentially more robust system using next-generation gene sequencing technologies. Collaborators have completed synthesis of a panel of synthetic recombinant bovine MHC molecules which will be used as immunologic tools to evaluate T cell immune responses of Holstein cattle following vaccination or natural infection. Experiments have been completed describing the binding affinity of these molecules. These results are expanding our understanding of BoLA gene expression and diversity in Holstein cattle. Results of this work will be disseminated to the research community by presentations at international meetings and through submission of manuscripts for publication in peer-reviewed scientific journals. In the next years we plan to demonstrate the use of a panel of bovine class II MHC-tetramer molecules in studies of Holstein cattle immune responses.
Barlow, John Determination of Duration of Immunity Conferred by Ad5FMDV Vaccination in Cattle
Agricultural Research Service/Dept of Agriculture
The goal of this research is to evaluate the duration of immunity and protection from viral challenge following vaccination of Holstein cattle with the newly approved Foot and Mouth Disease Virus vaccine. The first experiments demonstrated that vaccination can provide protection of cattle from live virus challenge up to 6 months following vaccination. The second experiments demonstrated incomplete protection from live-virus challenge at 9 months following vaccination. Additional experiments will be conducted to quantify the role played by specific arms of the immune system in animals that are protected following vaccination. Associated with these experiments, tools (e.g. antibodies for use in assays and assay systems) are being developed to identify and quantify cellular changes in cattle following vaccination or experimental infection. Results of this work will be disseminated to the research community by presentations at international meetings and through submission of manuscripts for publication in peer-reviewed scientific journals.
Barlow, John BREAD: A Modern Approach for the Development of Cattle Vaccines for Critical Bovine Diseases Impacting Smallholder Farmers in Sub-Saharan Africa
National Science Foundation
Improving cattle vaccine assessment technologies will advance understanding of immune system function, accelerate vaccine development efforts and ultimately improve global cattle health. The benefits of improved global cattle health include reduced vulnerability of farmers to agricultural resource losses due to animal disease. An international team of scientists from the United States, Kenya, and Denmark have established a collaborative research program to examine the use of new technologies in immunology with the goal of accelerating vaccine development for two critical cattle diseases that impact smaller holder farmers in sub-Saharan Africa. The objectives of the project are to establish and advance gene typing methods for the major histocompatibility (MHC), also known as bovine leukocyte antigens (BoLA), class I and II genes of cattle, and to establish and advance immune response assays to identity potential antigens for 2 critically important cattle diseases, Foot-and-Mouth disease and East Coast Fever. We have successfully identified methods and initiated typing of Holstein cattle in a University of Vermont Dairy Center of Excellence herd. We have evaluated the genetic types of approximately 120 animals for the MHC class I and MHC Class II DRB3 genes using a series of molecular typing methods. For typing MHC Class I genes we have extended current methods to a potentially more robust system using next-generation gene sequencing technologies. Collaborators have completed synthesis of a panel of synthetic recombinant bovine MHC molecules which will be used as immunologic tools to evaluate T cell immune responses of Holstein cattle following vaccination or natural infection. Experiments have been completed describing the binding affinity of these molecules. These results are expanding our understanding of BoLA gene expression and diversity in Holstein cattle. Results of this work will be disseminated to the research community by presentations at international meetings and through submission of manuscripts for publication in peer-reviewed scientific journals. In the next years we plan to demonstrate the use of a panel of bovine class II MHC-tetramer molecules in studies of Holstein cattle immune responses.
Donnelly, Catherine Strategies to detect and mitigate Listeria in artisan cheese facilities
National Institute of Food and Agriculture/Department of Agriculture
Consumer interest in artisan and farmstead cheeses is driving explosive growth of on-farm cheese operations throughout the United States. As many of these enterprises are small to very small establishments, there is a need for focus on assuring the microbiological safety of cheeses produced on farm. During 2010, the U.S. Food and Drug Administration intensified its scrutiny of U.S. cheese makers. In particular, increased regulatory attention was focused on small scale artisan cheese makers and
those producing cheeses from raw milk. Smaller operations are considered high risk due to lack of resources, capital and technical expertise to implement effective control programs. With 38 artisan cheese producers, Vermont boasts the highest number of artisan cheese makers per capita in the United States. In order to allow this industry to grow and prosper, it is essential that the safety of artisan cheeses be assured.
Guo, Mingruo Beneficial and Adverse Effects of Natural Chemicals on Human Health and Food Safety
National Institute of Food and Agriculture/Department of Agriculture
Use of sutures in veterinary medicine is still the most widely practiced method for wound closure and tissue reconstruction. Scarring is a common defect resulted from sutures on topical use and nerve repair (lack of conduction). However, sutures sometimes are not effective on the sites where fluid and air leakage could occur (e.g., blood vessels, urethra and lung tissues). Tissue adhesives could prevent fluid and air leakage effectively and may minimize scare formation after surgery. Tissue
adhesives incorporating proteins such as bovine serum albumin (BSA) and fibrin are commercially available and have been approved by FDA for clinical use. These commercial tissue adhesives are expensive and single dosed. Whey is a byproduct from cheese making, and more than one third of the whey generated by the cheese industry is disposed to the environment every year in the US. Purified whey protein is much cheaper than BSA and fibrin. Based on the chemistry of the major components of whey proteins (ß-lactoglobulin, a-lactalbumin and BSA), whey proteins might be suitable for formulation of tissue adhesives. Whey proteins are abundant, economical, and structurally modifiable. The objectives of this project are to formulate and develop safe, effective, and low cost whey protein based tissue adhesives, and to evaluate the functional properties of the new adhesives in vitro.
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Johnson, Doug Fungal Biofilms and Development of New Anti-Fungal Drugs National Institute of Food and Agriculture "Biofilms caused by Candida albicans and other fungi lead to significant losses in the maple syrup industry by blocking/clogging
maple sap plastic tubing. The studies described herein will allow us to examine the ability of biofilm inhibitors to block biofilms on
this type of tubing and other abiotic surfaces. These studies will also give us insight into the mechanism by which one of the
inhibitors, ETYA, is able to block these biofilms.
Kerr, David Genetic and Epigenetic Contribution to Variation between Animals in Strength of Response to Vaccination
National Institute of Food and Agriculture/Department of Agriculture
The proposed research seeks to explore genetic and epigenetic causes for variation that exists between cows in the strength of their response to vaccination. The long-term goal is to use this knowledge to reduce the prevalence of poor responder animals. If causative gene mutations can be identified then breeding strategies could be employed to identify and potentially eliminate carrier animals. Alternatively, the emerging field of epigenetics predicts that events occurring in utero or early development may affect the ability of genes to be expressed without a change in the genetic code. One epigenetic modification is DNA methylation at “C” residues. This modification could occur in early development due to various environmental challenges and then be passed on from cell to daughter cell as the animal develops. The possibility exists that desired epigenetic modifications can be ensured through management strategies, such as optimal plane of nutrition of the dam or the young calf, or different animal housing conditions, such that overall vaccine response is enhanced. The ultimate goal of the project is to reduce the incidence of animals that develop poor responses to vaccines.
We have conducted a preliminary experiment with in which 19 animals have been vaccinated with an adenovirus vaccine and their antibody responses to this vaccine were determined. Skin samples from these animals were also collected and fibroblast cell cultures established. The cultures are now in storage in liquid nitrogen. The cells will be revived and propagated, and then subsets will be used for analysis or refrozen for later experimentation. The laboratory analysis will determine the strength of response of the cultures to challenge with various viral and bacterial components. Challenge responses will include the ability of the cells to produce immune associated proteins such as cytokines and chemokines. These factors are considered as critical components of the “innate” immune system with functions such as attracting immune cells to the sight of infection, and in producing the animal’s fever response. In parallel we are evaluating the antibody response of these same animals to the initial vaccination. Blood serum samples were taken monthly for 9 months to evaluate the strength of the adaptive antibody response to the vaccine. Cells of the “adaptive” immune response generate the long-term protection provided by vaccines and produce antibodies. Our work seeks to determine if animals that have a highly responsive innate immunity, as determined by the fibroblast testing, also have enhanced antibody responses to the vaccine. The results of this preliminary experiment will focus our future studies.
Kerr, David Mastitis Resistance to Enhance Dairy Food Safety
National Institute of Food and Agriculture/Department of Agriculture
Mastitis is an inflammatory disease resulting from bacterial infection of the mammary gland and ranges in severity from mild to severe. The severe form is very detrimental to dairy animal health and often results in premature removal of cows from the lactating herd. Our long-term goal is to develop biological tests that can be applied to samples obtained from young dairy calves to predict if they will develop into dairy cows with reduced or enhanced susceptibility to develop severe mastitis. Producers could use this information to decide if those calves predicted to be pre-disposed to developing severe mastitis could be sold or raised for beef production rather than being raised as replacements for the dairy herd. We use a small skin biopsy to supply fibroblast cells for our laboratory-based testing of how vigorously an animal’s cells respond to infection. We have published some data indicating that an animal with high responding cells also has a greater severity of response to experimentally induced infection of the mammary gland. We are also examining the causes of the differential responses we observe in the fibroblast cells. Very recently we submitted a manuscript that describes substantial differences between beef (Angus) and dairy (Holstein) breeds of cattle. The cells from the Holstein animals are much more responsive to immune challenge than are cells from the Angus animals. There are likely to be genetic and epigenetic causes of this difference. Epigenetics effects are mediated by modifications of an animal’s DNA during development and these changes have the potential to significantly affect the functioning of immune response genes. DNA methylation is a type of epigenetic modification that primarily involves the addition of a methyl groups to the DNA. Environmental differences during fetal development in utero or during the pre-weaning growth phase are thought to contribute to differences between animals in the amount and location of DNA methylation. Some types of methylation can reduce the strength by which certain genes respond to a given signal. This differential response of genes can thus contribute to differences observed between animals. Interestingly, beef animals are raised quite differently to dairy animals and this may relate to the differences we observe in how their cells responds to infection-type stimulus. Our results have been submitted for publication in peer-reviewed scientific journals and presented in oral form at national and international scientific meetings. A future project will make use of an enhanced laboratory-based testing scheme to rank 100 dairy cows based on their cells response to infection. We will then select the top 6 and bottom 6 animals for an intramammary challenge with E. coli to compare the severity of response.
Kindstedt, Paul Characterization of crystals that enhance the value of artisanal cheeses
National Institute of Food and Agriculture
Artisanal cheese making has become a vibrant and highly visible component of the Vermont dairy industry. Artisanal cheeses must command premium prices in the marketplace in order to be economically sustainable, therefore, they must be readily differentiated from lower cost conventional cheeses through quality attributes that render them more interesting and satisfying, and thus worth paying more for. Crystallization in cheese is a fascinating phenomenon that has the potential to differentiate long-aged artisanal cheeses from lower cost conventional alternatives. Though poorly understood, crystallization also plays a role in cheese quality. By developing an understanding of the factors that control crystal formation, it may be possible to develop new ways to control cheese ripening
The general objectives of this project are to characterize the occurrence of visible crystals in long aged cheeses, identify factors that predispose specific cheeses to specific forms of crystallization, and establish relationships between predisposing factors and traditional practices used in artisanal cheese making. During the past year we have focused on crystals that form at the surface of soft bloomy rind cheeses (i.e., white mold cheese such as Brie and Camembert) and soft washed rind cheeses (i.e., smear ripened cheeses, such as Limburger). Several accomplishments have been achieved so far towards understanding the role of surface crystals in the quality of these cheese families: 1.) We have determined that the crystals that form on the surface of bloomy rind cheese consist of brushite, a form of calcium phosphate; 2.) We have elucidated the mechanism by which brushite forms on the cheese surface, and have linked this crystallization to the characteristic internal softening of this cheese during ripening. These results point to potential new strategies to control the ripening process of bloomy rind cheese; 3.)We have identified two crystals, ikaite and struvite, on the surfaces of washed rind cheeses. These were unexpected discoveries that have opened up new lines of research into the effects of carbon dioxide and ammonia in the atmosphere of the ripening room on interior cheese softening during ripening. These results suggest that the gaseous makeup of the ripening room profoundly influences cheese ripening rate and the shelf life of washed rind cheeses, thus opening the door to new practical strategies to control cheese ripening ; 4.) We have demonstrated that these crystals (ikaite and struvite) are likely associated with gritty mouthfeel, which may be viewed as a positive or negative quality attribute, depending on the specific market. Our results suggest strategies that can be used to accelerate or repress the formation of these crystals in order to tailor the cheese to specific markets; 5.) We have constructed, for the first time, the 3-dimensional molecular structure of ikaite and struvite using single crystal diffractometry. Single crystal diffractometry represents a powerful new analytical approach for conducting cheese crystal research
We presented our research at the Ninth Cheese Symposium in Cork, Ireland in November 2014, the 2015 Annual Meeting of the American Dairy Science Association, and the 2015 Annual Meeting of the Geological Society of America. We also published a journal article in Dairy Science and Technology. The next phase of our research will be to perform an in-depth study of the role of crystal formation during the ripening of washed rind cheeses with the goal of developing specific strategies to improve the control of ripening.
Neher, Deborah Persistence of Enteric Pathogens in Manure-Amended Soils in Northeast U.S. Produce-Growing Environment
Department of Agriculture USDA
The overall objective is to quantify how long the bacterium Escherichia coli (E. coli) will survive in soil after applications of raw dairy manure and/or a spray containing benign forms of the bacteria. Two field sites at the UVM farm were chosen because they represent different soil types. The experiment contained six treatments which were arranged in a randomized complete block and replicated four times. Treatments were 1) application of raw dairy manure, 2) spray a cocktail of three E. coli isolates, 3) combination of raw dairy manure and E. coli, and 4) a control without either manure or E. coli. All treatments combinations were cultivated; treatments 2 and 3 were also implemented without tillage keeping material on the surface. Sampling occurred frequently after treatments were applied and gradually extended to one month apart for a cumulative total of nine months. The experiment follows the same experimental design as the Food and Drug Administration has implemented at other geographic locations including California, Pennsylvania, and Maryland. Results from the first year suggest that soil cultivation affects survival of E. coli more than application of raw manure. Survival of E. coli was greater in cultivated than surface (non-tilled) treatments. We conclude that both soil type and soil management practices affect the level of E. coli persistence in manure-amended and unamended soils in the Northeast U.S. We established a second year of the trial in September 2015 at the same locations but plots were off-set from the previous year so they represent soil receiving the first tillage and manure application in many years. We made two changes in the field experiment for year 2, 1) increased the selectivity of the media for quantifying E. coli populations and planted a winter spinach crop. In spring 2016, we plan to overlay a treatment of applying either dairy or poultry compost and plant a root (radish) and shoot (spinach) crop that are eaten fresh. This will allow us to test the impact of compost amendments on the survival of E. coli and the ability of E. coli to move from soil to plant tissue for human consumption.