Progress 09/15/06 to 09/14/09

Outputs
OUTPUTS: The results of the comprehensive administered questionnaire survey (32 attributes) revealed that consumption of raw milk, working on a dairy herd with a history of Johne's disease, and well water consumption were not associated with Crohn's disease. Results from molecular subtyping research were shared with graduate students taking an Advanced Food Microbiology course taught by PI Knabel. PIs Jaryarao and Debroy mentored research technicians working on objectives 1 and 2, respectively. PIs Knabel and Cutter mentored two graduate students working on objectives 3 and 4,respectively. Results of research were shared during the Food Microbiology Short Course, which was offered annually during the period of this grant. Results were also shared at the following national scientific meetings: The American Society for Microbiology and the Institute of Food Technologists. PI Knabel used his expertise in tracking and controlling zoonotic foodborne pathogens as part of a subcommittee of the National Advisory Committee for the Microbiological Criteria for Foods that was charged with answering questions pertaining to new advanced technologies that FSIS might consider using for baseline and certification testing for pathogens. PI Cutter distributed her video, "Control of Listeria monocytogenes in Retail Establishments" to extension agents, food industry personnel and government personnel. PI Cutter also developed a pamphlet called "Proper Care and Handling of Venison: From Field to Table," which was made available online at http://pubs.cas.psu.edu/FreePubs/pdfs/uk066.pdf. To have a free paper copy of the pamphlet mailed to their home, a Pennsylvania resident can call 814-865-6713 and ask for publication UK066. PI Knabel worked with scientists at FSIS to further subtype epidemic clone II Listeria monocytogenes isolates that were generated as part of FSIS's various surveillance programs. He is currently working with FSIS scientists to submit a manuscript for publication on this subject. PARTICIPANTS: PD/PI Knabel was responsible for Objective 3, To detect and track MAP, STEC and Listeria monocytogenes throughout the dairy food system, which will then allow development of effective intervention strategies for control. He worked with PIs Debroy and Jayarao on this objective and also collaborated with Dr. Edward Dudley in the Food Science Department to develop a molecular subtyping method for E. coli O157:H7 based on Rearrangement Hot Spot (RHS) elements. Drs. Knabel and Dudley co-advised a M.S. student on this project. PD/PI Knabel also advised a M.S. student who worked on development of novel DNA-sequence-based methods for subtyping Listeria monocytogenes. PD/PI Knabel was also assisted on this project by a visiting scientist from Italy, Dr. Sara Lomonaco and his postdoctoral scholor, Dr. Bindhu Verghese. PI Jayarao was responsible for Objective 1, To determine the sero-prevalence for Mycobacterium avium subspecies paratuberculosis (MAP) in humans exposed to dairy environments. PI Debroy was responsible for Objective 2, To determine the prevalence of Shigatoxin-producing E. coli (STEC) in humans that consume raw milk. PI Cutter was responsible for Objective 4, To determine the factors affecting biofilm formation by Listeria monocytogenes in dairy production and processing environments and develop intervention strategies for their control. PD/PI Knabel collaborated with FSIS scientist Dr. Peter Evans, FDA scientist Dr. Yi Chen and Dr. Sophia Kathariou from the Food Science Department at North Carolina State University on Objective 3 of this project. Two graduate students in the Department of Food Science at Penn State University were funded by this project and two research technicians in the Department of Veterinary and Biomedical Sciences were funded by this project. Three undergraduate technicians in the Department of Food Science were also funded by this research project. TARGET AUDIENCES: The main target audiences for this project were the food processors and retailers that manufacture and sell dairy products and the public that consume them. However, extension programs were used to also disseminate the results of this research to hunters and consumers. Since infants, the elderly and those who are immunocompromised are at highest risk for morbidity and mortality related to a foodborne illness, these "at risk" groups especially benefitted from the results of this research. One of the undergraduate technicians working on this project was a minority student and the other two students were females. As a result of his experience on this project, the minority student accepted an internship at FSIS to work on public policy related to control of Listeria monocytogenes. All of these students had financial needs that were partially met by the monies they were paid as technicians. The results of this research were disseminated to adult learners via various short courses in the Department of Food Science including the Food Microbiology Short Course, The Penn State Sanitation Short Course and the Ice Cream Short Course. They were also disseminated to 9-11 grade students via a new Science U-Camp entitled, "Natural Borne Killers", which introduced these students to the science of tracking and controlling zoonotic foodborne pathogens. This camp provided these high school students with hands on laboratory experience with the microbiology and molecular biology techniques used to track and control foodborne pathogens and ended with a mock trial at the new Law Building at Penn State, where the students had to either defend or prosecute those being sued. This short course made science come alive for many of these students and motivated them to pursue careers in food science/food safety. The results of this research project were disseminated to graduate students in the Food Science Department Graduate course entitled Advanced Food Microbiology. Finally, the results of this research project were shared with general public through two different Penn State publications. The first was entitled "Killers in the Kitchen" which was published in Penn State Research and focused on recent advances in molecular subtyping of Listeria monocytogenes and the second entitle, "Mission - Food Safety", published in the Penn State Agricultural Sciences Magazine, which focused on tracking and controlling various foodborne pathogens throughout the food system. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The findings from objective 1 clearly suggested that that there is considerable difference in the number and type of bacterial species in the feces between cases (those with Crohns Disease) and controls (those without Crohns Disease). The only attribute that had a causal relationship associated with Crohn's disease was consumption of venison meat (OR 1.93; CI 1.15-13.45, χ2 = 0.0391). This result was disseminated to participants in the "Venison 101 Workshop" offerred by PI Cutter, who emphasized proper processing methods so that partipants would destroy the causative agent(s) during processing of venison. This workshop and the pamphlet that accompanied it (see above) explained how to reduce the risk of foodborne illness by properly handling, processing and preparing deer. The molecular subtyping methods developed by PI Knabel will allow food companies and federal agencies to very accurately track subclones of Listeria monocytogenes both between and within dairy processing plants. This novel method will allow the development of a novel DNA-sequence-based system for tracking zoonotic foodborne pathogens throughout the food system. This will allow federal food safety agencies to identify routes of transmission, which will allow food companies to implement more effective intervention strategies that prevent contamination of ready-to-eat food products. This will decrease the burden of foodborne illness and thus enhance public health. It will also greatly reduce the incidence of costly recalls due to this pathogen. Taken together, these advancements will increase the profitability of food companies and restore public confidence in the safety of our food supply.

Publications

• Chen, Y., W. Zhang, and S.J. Knabel. 2007. Multi-Virulence-Locus Sequence Typing identifies single nucleotide polymorphisms that differentiate epidemic clones and outbreak strains of Listeria monocytogenes. J. Clin. Microbiol. 45:835-846.
• Chen, Y. and S.J. Knabel. 2007. Multiplex PCR for simultaneous detection of bacteria of the genus Listeria, Listeria monocytogenes, and major serotypes and epidemic clones and outbreak clones of L. monocytogenes. Appl. Environ. Microbiol. 73:6299-6304.
• Houser, B.A., S.C. Donaldson, R. Padtee, A. Sawant, C. DebRoy, and B.M. Jayarao. 2008. Assessment of phenotypic and genotypic diversity of Escherichia coli shed by healthy lactating dairy cattle. J. Foodborne Pathogen Dis. 5:41-51.
• Liu, K, S.J. Knabel, and E.G. Dudley. 2009. Rearrangement hot spot (rhs) genes are potential markers for multilocus sequence typing of Escherichia coli O157:H7. Appl. Environ. Microbiol. (Accepted).
• Chen, Y. and S.J. Knabel. 2009. Molecular Epidemiology of Foodborne Pathogens. In, Genomics of Foodborne Pathogens, W. Zhang and M. Weidmann (eds.). Springer, N.Y., USA. (In Press).
• NACMCF New Technologies Subcommittee (includes S. J. Knabel). 2009. Response to the questions posed by the Food Safety and Inspection Service regarding determination of the most appropriate technologies to adopt for FSIS routine and baseline microbiological analysis. J. Food Prot. (In Press).

Progress 09/15/07 to 09/14/08

Outputs
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. 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. 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. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
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).

Publications

• Chen, Y. and S. J. Knabel. 2008. Prophages in Listeria monocytogenes contain single nucleotide polymorphisms that differentiate outbreak clones within epidemic clones. J. Clin. Microbiol. 46:1478-1484.
• Chen, Y. and S. J. Knabel. 2008. Strain Typing. In Handbook of Listeria monocytogenes, Dongyou Liu (Ed.). CRC Press. New York. page 208
• Lomonaco, S., Y. Chen and S. J. Knabel. 2008. Analysis of additional virulence genes and gene regions confirms the epidemiological relevance of multi-virulence-locus sequence typing of Listeria monocytogenes. J. Food Prot. 71(12):2559-2566.

Progress 09/15/06 to 09/14/07

Outputs
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. 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. 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.

Impacts
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

Publications

• No publications reported this period