Tracking and Controlling Zoonotic Pathogens Throughout the Dairy Food System

NON-TECHNICAL SUMMARY: Zoonotic pathogens, those that are transmitted from animals to humans, are the main caused of foodborne disease due to consumption of raw and pasteurized dairy products. Most illnesses are caused by a subset of highly virulence strains, known as epidemic clones. In order to control these zoonotic pathogens we must first be able to track them throughout the dairy system. The purpose of this study is to enhance the safety of raw and pasteurized dairy products by developing methods for tracking and controlling epidemic clones of zoonotic foodborne pathogens throughout the dairy system.

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APPROACH: Objective 1. Serum and fecal samples from Chrohn's, colitis and control patients will be subjected to serological and bacteriological analyses. Data will be analyzed using various statistical procedures and CDC's Epi Info. <P>Objective 2. DNA from human fecal samples from humans that work on dairy farms will be extracted from suspect colonies of STEC and subjected to PCR. Two sets of multiplex PCR will be conducted, one for detecting the genes stx1 and stx2 and eae and a second multiplex for detecting the genes rfbE of O157 and fliC for H7. Positive samples will be identified based on the presence of bands of the expected sizes compared to results with positive control strain E. coli ATCC 43895. <P>Objective 3. Milk samples and milk filters from organic and non-organic dairy farms will be collected and screened for the presence of MAP, STEC and LM. MAP will be recovered and identified as previously described by Jayarao et al., 2004. STEC will be detected using the methods described in part 3b. Epidemic clones of LM will be detected using the muliplex PCR method developed in 3c and confirmed using the SNP method in 3d. <P>Objective 4. Initial and attachment phases of biofilm formation will be assayed using dye binding and microscropy as previously described. Various environmental conditions will be manipulated to determine their effect on biofilm formation. To determine if the outbreak strains co-habitate with other strains LM cells will be tagged with a fluorescent marker and incubated with various other bacteria strains that are common in foods. Epifluorence microscopy will be used to examine the location and movement of LM cells with the multi-species biofilm. Genome sequences of biofilm-forming and non-biofilm-forming LM will be compared to determine if the biofilm-forming strains have unique sequences that may be involved in biofilm formation. Knock-out mutants will then be created to confirm whether these genes are indeed required for biofilm formation.

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PROGRESS: 2007/09 TO 2008/09 <BR>
OUTPUTS: Dr. Cutter co-directed and taught in the PSU Sanitation Shortcourse (SSC) in October, 2008. She introduced the concept of biofilms to 60 participants of the SSC. Participants were food processors from all over the U.S. Dr. Cutter continued to collaborate with Dr. Gylen Uhlech from USDA-ARS Eastern Regional Research Center on biofilm research. Dr. Uhlech has provided information from a similar project with E. coli O157:H7 that is being incorporated into the existing Listeria monocytogenes biofilm project. Dr. Knabel published the results of his molecular subtyping of Listeria monocytogenes in Penn State Research and also presented the results of his subtyping research to the Pennsylvania Secretaries of Agriculture and Health and the Director of the Pennsylvania Emergency Management Agency at the 2008 Food Safety and Security Harvest Tour Round Table Meeting. Dr. Knabel is collaborating with Dr. Wei Zhang at the National Center for Food Safety and Technology to develop a DNA microarray approach for studying gene expression in Listeria monocytogenes in order to better understand how this organism transitions to a long-term survival stage. Dr. Knabel collaborated with Dr. Edward Dudley in the Food Science Department to apply new DNA-sequence-based subtyping technologies developed for L. monocytogenes to also track E. coli O157:H7 in dairy processing plants. Three graduate students worked on this project during this period and information on this project was also disseminated to undergraduate and graduate students taking courses on food microbiology, including Food Microbiology, Fundamentals of Food Science, Epidemiology and Molecular Epidemiology. Two visiting scientists from Italy worked on this project and one technician worked on this project in our laboratories is an African-American. Information from this project was also disseminated to participants in short courses sponsored by the Department of Food Science, including the Penn State Sanitation Short Course and the Food Microbiology Short Course. Target audiences also included food industry associations such as the Eastern Meat Packers Association, the Pennsylvania Association for Food Protection, and the Institute of Food Technologists. Additional audiences included government associations such as the Central Atlantic States Association of Food and Drug Officials and the National Advisory Committee for the Microbiological Criteria for Foods (NACMCF). Farm families in Pennsylvania were also served by the Mycobacterium avium subspecies paratuberculosis as part of this project. Given their high risk for zoonotic foodborne illness, this project specifically targeted infants, the elderly and those individuals with compromised immune systems, such as patients with AIDs and cancer and those on immunosuppressive drug therapy. <BR> PARTICIPANTS: Dr. Stephen J. Knabel, PI, is responsible for Objective 3 and the development of DNA-sequence-based molecular subtyping methods for tracking zoonotic pathogens. Dr. Bhushan Jayarao, PI, is responsible for Objective 1, to determine the sero-prevalence for Mycobacterium avium subspecies paratuberculosis (MAP) in humans exposed to dairy environemnts. Dr. Chirita Debroy, PI, is responsible for Objective 2, and the charactization of all zoonotic agents that are isolated as part of this project. Dr. Catherine Cutter, PI, is responsible for Objective 4, to determine the factors affecting biofilm formation by Listeria monocytogenes (LM) in dairy production and processing environments and to develop intervention strategies for their control. Dr. Edward Dudley will serve as a co-advisor to a M.S. student working on developing a DNA-sequence-based molecular subtyping strategy for tracking E. coli O157:H7. Two visiting scientists from Italy worked on this project and one technician working on this project in our laboratories is an African-American. Dr. Sara Lomonaco, a visiting scientist from the University of Turin, Italy, conducted research on additional virulence genes in L. monocytogenes to confirm the epidemiologic relevance of MVLST. Dr. Wei Zhang at the National Center for Food Safety and Technology is collaborating with Dr. Knabel to develop a DNA microarray approach to study gene expression in L. monocytogenes during the transition to the long-term survival phase. <BR> TARGET AUDIENCES: Target audiences included graduate students working on this project and undergraduate and graduate students taking courses on food microbiology, including Food Microbiology, Fundamentals of Food Science, Epidemiology and Molecular Epidemiology. Target audiences also include participants in short courses sponsored by the Department of Food Science, including the Penn State Sanitation Short Course and the Food Microbiology Short Course. Target audiences also include food industry associations such as the Eastern Meat Packers Association, the Pennsylvania Association for Food Protection, and the Institute of Food Technologists. Target audiences also include government associations such as the Central Atlantic States Association of Food and Drug Officials and the National Advisory Committee for the Microbiological Criteria for Foods (NACMCF). Farm families in Pennsylvania were also served by the Mycobacterium avium subspecies paratuberculosis part of this project. Given their high risk for zoonotic foodborne illness, this project specifically targets infants, the elderly and those individuals with compromised immune systems, such as patients with AIDs and cancer and those on immunosuppressive drug therapy. <BR> <BR>
IMPACT: 2007/09 TO 2008/09<BR>
Dr. Knabel's Visiting Scientist and Ph.D. student analyzed prophages and 6 more virulence genes and 3 more virulence gene regions within L. monocytogenes using prophage sequencing and Multi-Virulence-Locus Sequence Typing (MVLST). The results identified additional single nucleotide polymorphisms (SNPs) in L. monocytogenes and confirmed that prophage sequences and MVLST possess perfect epidemiological relevance. These finding will allow scientists and food processors to use MVLST and prophage sequences and the novel SNPs within these genes. These can then be used to very accurately track the routes of transmission of epidemic clones and outbreak clones of L. monocytogenes within dairy processing plants and then implement effective intervention strategies to prevent contamination of dairy products. Dr. Cutter's Ph.D. student determined that the ability of Listeria monocytogenes to colonize surfaces is influenced by environmental factors such as temperature and medium composition, which can affect the physiological state of the bacteria. Results indicated that initial attachment of L. monocytogenes at 25C was favored when nutrients (TSB) were present, while growth was prevented in its absence. In contrast, at 4C, initial attachment was favored under starvation conditions (saline) and prevented under the presence of nutrients (TSB). This finding suggests an interesting interaction to be studied in further experiments. The data also indicated differences in biofilm formation may result from the initial inoculum, suggesting the need of a population threshold for initial attachment and subsequent biofilm formation. A cocktail of four strains of L. monocytogenes was treated with full strength TSB, saline solution (control), whole milk, skim milk, or irradiated bovine meat purge at different concentrations at two temperatures (4 and 10C). A significant difference was found in the initial attachment of L. monocytogenes treated with TSB regardless of temperature. This finding suggests that TSB is not a good model media for the study of L. monocytogenes biofilm formation, which is a interesting finding since TSB is the most widely used media to study biofilm formation in vitro. We are currently evaluating potential modifications to the biofilm assays where meat purge is used so it can be used in future experiments. Dr. Jayarao conducted a detailed analysis (60 survey questions ranging from dietary habits to work practices) between cases and controls which did not reveal any significant association between dietary habits (raw milk consumption, consumption of meat/vension, source of water) and work practices (manure handling, history of Johne's disease etc).

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PROGRESS: 2006/09/15 TO 2007/09/14<BR>
OUTPUTS: Dr. Cutter co-directed and taught in the PSU Sanitation Shortcourse (SSC) in October, 2007. She introduced the concept of biofilms to 65 participants of the SSC. Participants were food processors from all over the U.S. Dr. Cutter began collaborating with Dr. Gylen Uhlech from USDA-ARS Eastern Regional Research Center on biofilm research. Dr. Uhlech has provided information from a similar project with E. coli O157:H7 that will be incorporated into the existing Listeria monocytogenes biofilm project. Dr. Knabel began working with Dr. Peter Evans (USDA-FSIS) and Dr. Todd Ward (USDA-ARS) on a project involving analysis of USDA-FSIS Listeria monocytogenes isolated from meat plants in the Northeastern U.S. that appear to be epidemic clone II by PFGE analysis. Dr. Knabel's laboratory confirmed that they were epidemic clone II and found that they were the 1998-1999 outbreak clone, using multi-virulence-locus sequence typing and new a prophage PCR method that was recently developed in his laboratory as part of this grant proposal. Dr. Knabel is now collaborating with Dr. Edward Dudley in the Food Science Department to apply these new DNA-sequence-based subtyping technologies to track E. coli O157:H7 in dairy processing plants. Dr. Jayarao solicited the participation of the Crohn's and Colitis Foundation Chapter in Philadelphia to seek individuals of dairy farm origin with Crohn's disease, irritable bowel syndrome, ulcerative colitis and other gastrointestinal conditions. Dr. Jayarao combined a part of this study with an ongoing statewide study on zoonotic diseases in dairy farm operations. Dairy producers who had indicated in their response to survey that could be contacted, were solicited to participate in this study. A total of 112 adult dairy producers consented to participate in the study. The 42 dairy producers comprised of individuals with (cases; n=19) and without (controls; n=23) chronic gastrointestinal conditions. The cases and controls were matched based on geographical location of the farm, sex and age of the participants. <BR> PARTICIPANTS: Dr. Stephen J. Knabel, PI, is responsible for Objective 3 and the development of DNA-sequence-based molecular subtyping methods for tracking zoonotic pathogens. Dr. Bhushan Jayarao, PI, is responsible for Objective 1, to determine the sero-prevalence for Mycobacterium avium subspecies paratuberculosis (MAP) in humans exposed to dairy environemnts. Dr. Chirita Debroy, PI, is responsible for Objective 2, and the charactization of all zoonotic agents that are isolated as part of this project. Dr. Catherine Cutter, PI, is responsible for Objective 4, to determine the factors affecting biofilm formation by Listeria monocytogenes (LM) in dairy production and processing environments and to develop intervention strategies for their control. Dr. Edward Dudley will serve as a co-advisor to a M.S. student working on developing a DNA-sequence-based molecular subtyping strategy for tracking E. coli O157:H7. <BR> TARGET AUDIENCES: Target audiences include graduate students working on this project and undergraduate and graduate students taking courses on food microbiology, including Food Microbiology, Fundamentals of Food Science, Epidemiology and Molecular Epidemiology. Two visiting scientists from Italy are working on this project and one technician working on this project in our laboratories is an African-American. Target audiences also include participants in short courses sponsored by the Department of Food Science, including the Penn State Sanitation Short Course and the Food Microbiology Short Course. Target audiences also include food industry associations such as the Eastern Meat Packers Association, the Pennsylvania Association for Food Protection, and the Institute of Food Technologists. Target audiences also include government associations such as the Central Atlantic States Association of Food and Drug Officials and the National Advisory Committee for the Microbiological Criteria for Foods (NACMCF). Farm families in Pennsylvania were also served by the Mycobacterium avium subspecies paratuberculosis part of this project. Given their high risk for zoonotic foodborne illness, this project specifically targets infants, the elderly and those individuals with compromised immune systems, such as patients with AIDs and cancer and those on immunosuppressive drug therapy.
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IMPACT: 2006/09/15 TO 2007/09/14<BR>
Dr. Cutter identified a doctoral student and assigned this student to the biofilm project. The student spent three months researching the biofilm literature and researched methods for biofilm detection. The student conducted a series of experiments that indicated that some strains of Listeria monocytogenes from foodborne outbreaks are better biofilm formers than others. This information may be used as another means of characterizing the pathogen and may provide insight into how to control the pathogen in food processing establishments. Dr. Knabel identified a Ph.D. student and a M.S. student to work on this project. The M.S. student is co-advised by Dr. Dudley and will work on developing molecular subtyping methods for tracking E. coli O157:H7. The M.S. student will continue our work on developing DNA-sequence-based methods for tracking Listeria monocytogenes. Both students completed their literature reviews this semester and will start research on their projects next semester. Dr. Jayarao conducted a detailed analysis (60 survey questions ranging from dietary habits to work practices) between cases and controls which did not reveal any significant association between dietary habits (raw milk consumption, consumption of meat/vension, source of water) and work practices (manure handling, history of Johne's disease etc). Bulk tank and milk filter samples will be collected starting December 14, 2007, once a week for 6 weeks from all 38 participating dairy herds. All the samples will be examined for the presence of Salmonella, Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Mycobacterial spp. including Mycobacterium avium subsp. paratuberculosis using real time PCR assays developed in Dr. Jayarao's laboratory. Samples positive on real time PCR assay will be cultured for the respective pathogens and provided to other collaborators including Dr. DebRoy (Escherichia coli, Salmonella and Campylobacter) and Dr. Knabel (Listeria monocytogenes and Escherichia coli) for further characterization. E. coli strains will be further characterized in Dr. DebRoy's laboratory. These will be serotyped to determine if they belong to O157:H7 or any other shiga-toxin producing serogroups. The presence of virulence genes such as shiga toxins (Stx), heat-stable toxins (Sta), heat labile toxins (LT) and other virulence attributes such as adherence and effacing gene (eae) and bundle forming pili (bfp) will be determined by PCR.Salmonella strains will be tested for the presence of gene encoding for invasive gene (inv) known to be a virulence factor. Campylobacter isolates will be tested for cytotoxic distending toxin (cdt). Presence or absence of these pathogens in the stool of cases and control will be matched to that in bulk tank milk and milk filters to determine if there is a causal relationship between gastroenteric disease and the occurrence of over and excess of a pathogen