Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Listeria monocytogenes is a Gram-positive bacterial pathogen capable of causing significant morbidity and mortality in humans. Listeriosis is primarily a food-borne disease that significantly impacts specific risk groups including pregnant women and their fetuses, neonates, and people who are immunosuppressed. L. monocytogenes is capable of surviving and replicating under a wide range of environmental conditions and this, as well as its widespread distribution, make it particularly hard to eradicate from food processing plants. Although high resolution and reproducible molecular epidemiological methods exist for subtyping L. monocytogenes isolates, these methods are labor intensive and provide little information about the virulence potential of the isolate. The identification of a subtyping technique
that is accurate, discriminatory, informative and rapid would greatly aid in subtyping capabilities. Additionally, because FDA maintains a zero tolerance policy for the presence of L. monocytogenes in some food products, identification of genetic markers involved in strain virulence may allow the relative risk of contaminating L. monocytogenes strains to be assessed. The ability to accurately identify strains that are most likely to produce human illness would be of interest to food processors and may result in more widespread testing of products for Listeria contamination. In order to determine if a particular subtyping method is effectively able to differentiate virulent isolates from less virulent environmental isolates one must first be able to assess the virulence potential of a particular L. monocytogenes strain. Isolates known to be associated with human clinical cases are known to be virulent. However, isolates found in the environment may also be virulent given the right host
and dose. Therefore, it is important to develop an assay that is able to predict the virulence potential of environmental strains. In order to assess the role subtype-specific genes play in virulence, in vitro and in vivo assays should be developed that allow the correlation of genotype (genetic subtype) with phenotype (i.e. ability to survive in the environment and cause disease). We will be focusing on developing methods to precisely and reliably identify genetic subtypes that are virulent, developing phenotypic assays to assess the virulence potential of subtypes, and then translate knowledge from these projects into development of a high throughput subtyping microarray for epidemiological investigations. Our research concerning Listeria monocytogenes addresses the following elements of our National Program in Animal Production, Product Value and Safety (108 Food Safety): development and (validation) of diagnostic tools especially microarrays for detection and differentiation
under Section 1.2.1, use of genomic data 1.2.5 (Omics); derivation and use of fundamental data on strain virulence and pathogenicity under Section1.2.9 Pathogenicity; and development and validation of detection technologies for their use in ensuring food security under Section 1.2.9 (Food Security) in the 2006-2010 NP 108 Action Plan. The research also addresses Agency Performance Measure 3.1.2: Develop and transfer to Federal agencies and the private sector systems that rapidly and accurately detect, identify, and differentiate the most critical and economically important food-borne microbial pathogens. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2006) Construct and test virulence gene microarray. Compare resolution with PFGE and subtype grouping clinical background of strains. Test transgenic mouse in vivo assay. If successful, use model to phenotype L. monocytogenes strains. Increase subtyping resolution of suspension by designing and testing
additional subtype-specific probes. Year 2 (FY 2007) Use sequence analysis of polymorphic probes to identify sequences/SNPs that characterize epidemic strains. Use in vivo and in vitro models to characterize mutated (and complemented) L. monocytogenes strains. Use probe genotypic and phenotypic data to identify and design additional information probes to be included in suspension array. Year 3 (FY 2008) Use whole genome microarrays to identify additional virulence genes. Assessment of subtyping resolution and reproducibility using large panel of strain. Technology transfer. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Construct and test condensed subtyping microarray. Milestone Fully Met 2. Combine rapid template amplification with condensed array and test rapid subtyping system. Milestone Substantially Met 3b List the milestones that you expect to
address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The entire project is scheduled to be completed during FY 2005 and a new project will be developed to undergo OSQR review with subsequent implementation beginning FY 2006. OSQR review of new project expected in FY 2006. Milestones for FY 2006 & FY 2007 are projected pending project plan approval. Year 1 (FY 2006) Construct and test virulence gene microarray. Compare resolution with PFGE and subtype grouping clinical background of strains. -Previous subtyping microarrays weve tested have had resolution similar to PFGE (the current gold standard for subtyping), however, not included all genes known to be involved in virulence were included on the array. This virulence gene array includes all informative probes identified in previous genotyping array experiments as well as probes that represent the virulence genes. This array will be used
both for genotyping and gene expression experiments to investigate genetic differences between epidemic and environmental strains. This information will allow us to identify genetic variation that characterizes virulent strains of Listeria monocytogenes. Test transgenic mouse in vivo assay. If successful, use model to phenotype L. monocytogenes strains. -Our previous oral infection mouse model demonstrated that epidemic strains are generally more invasive than environmental strains. Although this model is useful, transgenic mice that express a human intestinal receptor for Listeria monocytogenes have recently been obtained from Dr. Mark Lecuit at the Pasteur Institute as part of a collaborative project. We plan to investigate the utility of this model for characterization of Listeria monocytogenes strain virulence. Increase subtyping resolution of suspension by designing and testing additional subtype-specific probes. -Conventional microarrays have been useful for genetic subtyping
and gene discovery, however, the technique is technically difficult and low throughput. We have designed a high throughput and more user-friendly subtyping assay using suspension microarrays. The resolution of this assay will be increased by the design and testing of additional probes. Year 2 (FY 2007) Use sequence analysis of polymorphic probes to identify sequences/SNPs that characterize epidemic strains. Use probe genotypic and phenotypic data to identify and design additional information probes to be included in suspension array. -Genes identified as characteristic of epidemic strains using via microarray subtyping will be sequenced to identify the exact nucleotide bases that characterize epidemic strains. This sequence information will be used to design probes for PCR and suspension arrays subtyping. Use in vivo and in vitro models to characterize mutated (and complemented) L. monocytogenes strains. -Genes that have been newly identified as important for strain virulence will be
mutated and the affect of the mutation on strain growth and virulence will be assessed using in vitro and in vivo models. Year 3 (FY 2008) Use whole genome microarrays to identify additional virulence genes. - Whole genome expression microarrays will be used to identify new virulence genes that are expressed under various biologically relevant stress conditions (i.e. environmental conditions that mimic those that occur during infection of the host). This information will add to our understanding of which genes are essential for survival within the host and how these genes differ between strains. Assessment of subtyping resolution and reproducibility using large panel of strains. -By year three, we should have a good idea of which genes are important to include in a subtyping array in order to have high resolution subtyping that is informative in regard to the virulence potential of a strains. We will also have a high throughput assay format designed and tested. At this point, the
assay will be validated by testing a large panel of strains that have been previously characterized for virulence using in vitro and in vivo models. Assay resolution, reproducibility, speed and cost can then be accurately assessed. 4a What was the single most significant accomplishment this past year? Construction and testing of a subtyping microarray to allow rapid, accurate and discriminatory subtyping of L. monocytogenes isolates for the purpose of molecular epidemiology and gene discovery. To investigate genetic regions that characterize subtypes, a 2000-probe mixed genome DNA microarray was constructed and used to identify 675 polymorphic (informative) probes for sequence analysis and construction of a condensed microarray. Fifty-five L. monocytogenes strains were genetically characterized using the condensed array including strains. Microarray subtyping had resolution equal to or better than other established methods (pulsed-field gel electrophoesis, ribotyping and multilocus
sequence typing), and correctly grouped eleven out of twelve isolates that had an epidemiological link. Twenty-two probes were identified that distinguish subtypes including several potential markers that were distinct for epidemic strains. These results show that a relatively simple microarray had resolution comparable to, or exceeding, existing methods and correctly identified strains associated with food poisoning outbreaks. Importantly, this method also identified the genetic regions that differ between strains and thus identified numerous genetic markers that are currently being used in epidemiological and pathogenesis studies. 4b List other significant accomplishments, if any. Identification of the sources of Listeria monocytogenes contamination of food processing remains a necessary goal. Work was carried out at the Animal Disease Research Unit in collaboration with Washington State University. It was demonstrated the numerous subtypes (57) of L. monocytogenes located on
two contaminated Dairy farms and showed that one of the subtypes present has been involved in multiple epidemics worldwide. Knowledge from this work contributes to better management of food processing by clearly identifying this source of L. monocytogenes. An understanding of the environmental requirements for growth and persistence of food borne bacterium such as L. monocytogenes is not fully understood. Scientists within the Animal Disease Research Unit conducted work examining this issue. Through construction of a genetic mutant mouse (uvrA mutant - deficient in ability to repair acid-induced DNA damage and adaptation to low pH) it was shown that uvrA is needed for optimal growth and survival of L. monoctyogenes. Description of the genetic basis for virulence of L. monocytogenes aids in management and control strategies for this important bacterium. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A regional
repertoire of human and livestock Listeria subtypes for subtype comparison studies has been developed. A collection of over 1500 L. monocytogenes strains has been assembled including a large number of regional farm isolates. Approximately 775 raw milk samples from 474 Pacific Northwest herds were tested at multiple time points and isolates obtained from the milk samples were serotyped, genetically characterized, and evaluated for unique biological properties. A vast majority of L. monocytogenes isolates obtained from the milk and farm samples were found to be clinically significant serotypes, which persist, in the dairy environment for an extended period of time. This work was an important step in defining the on-farm ecology of L. monocytogenes and accessing the contribution of farm contamination as a source in human listeriosis. The applicability of microarray analysis to L. monocytogenes subtyping and gene discovery has been demonstrated. Results showed that a relatively simple
microarray had resolution comparable to, or exceeding, existing methods and correctly identified strains associated with food poisoning outbreaks. Importantly, this method also identified the genetic regions that differ between strains and thus identified numerous genetic markers that are currently being used in epidemiological and pathogenesis studies. These microarray data also allowed development of an inexpensive, rapid, and user-friendly PCR assay for serotyping isolates that is currently being used by other Listeria researchers. A novel intragastric inoculation mouse model differentiates epidemic strains from environmental strains has been developed. The ability to accurately assess the disease-causing potential of an isolate is essential for defining the genes that control strain virulence. Development of this model is a critical step towards understanding why some strains cause epidemics whereas others do not. Eighty L. monocytogenes isolates were screened for biofilm
formation to determine if there is a robust relationship between biofilm formation, phylogenic division (taxonomic group), and persistence in the environment. Statistically significant differences were detected between phylogenetic divisions. Increased biofilm formation was observed in Division II strains (serotypes 1/2a and 1/2c), which are not normally associated with food-borne outbreaks. The results indicated that persistent strains are more capable of forming biofilms, but did not support a consistent relationship between enhanced biofilm formation and strain virulence. Data from this study allowed us to identify several high biofilm-forming strains that are suitable for gene-mutation studies using a gene believed to be involved in biofilm formation and these strains have been shared with the scientific community for further biofilm studies. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to
the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The PCR serotyping assay is currently used in other research laboratories.
Impacts
(N/A)
Publications
- Borucki, M.K., Reynolds, J.O., Gay, C., Mcelwain, K., Kim, S.H., Knowles Jr, D.P., Hu, J. 2004. Dairy farm reservoir of Listeria monocytogenes sporadic and epidemic strains. Journal of Food Protection. 67(11):2496- 2499.
- Borucki, M.K., Kim, S.H., Call, D.R., Smole, S.C., Pagotto, F. 2004. SELECTIVE DISCRIMINATION OF LISTERIA MONOCYTOGENES EPIDEMIC STRAINS USING A MIXED-GENOME DNA MICROARRAY AS COMPARED TO PULSED-FIELD GEL ELECTROPHORESIS, RIBOTYPING AND MULTI-LOCUS SEQUENCE TYPING. Journal of Clinical Microbiology. 42(11):5270-5276.
- Borucki, M.K., Reynolds, J.O., Call, D., Ward, T.J., Page, B.T., Kadushin, J. 2005. Suspension Microarray with Dendrimer Signal Amplification Allows Direct and High-Throughput Subtyping of Listeria monocytogenes from Genomic DNA. Journal of Clinical Microbiology. 43(7):3255-3259.
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Progress 10/01/03 to 09/30/04
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Listeria monocytogenes is a bacterium capable of causing serious disease in humans and animals. L. monocytogenes infection of cattle and sheep can lead to disease of the central nervous system and death. Human listeriosis is a potentially fatal food borne disease often associated with the consumption of contaminated dairy products. Because L. monocytogenes is widely distributed in the environment, a major problem associated with the food safety issue is the source(s) of L. monocytogenes in food borne illness. The role of domestic animals in dairy product contamination is not clearly understood. The focus of our research program is to elucidate the sources and significance of dairy farm contamination as related to human and animal disease. A majority of human outbreaks are caused by just 3 of the 13
L. monocytogenes serotypes, therefore we will focus on identifying the prevalence of these serotypes on the dairy farm and developing methods to precisely and reliably identify genetic subtypes that are especially virulent. Approximately 2500 cases of human listeriosis occur annually in the U.S. resulting in about 250 deaths. Therefore, although human listeriosis is a relatively rare food borne disease it is significant due to its high mortality rate. Additionally this bacterium causes disease in the central nervous system of cattle and sheep. Outbreaks of listeriosis have been traced to pasteurized milk, cheese, coleslaw, and meat products. However, the role of subclinical infections of animals in the distribution of listeriosis in animals and humans is unknown. Clearly, it is important to define all the contributing sources of the organism to the food chain and human and animal health problems. The aim of this project is to understand the role and significance of farm-associated
subtypes in veterinary and human listeriosis. In order to understand the significance of particular subtypes, one must be able to accurately determine strain subtype using a high resolution and genetically informative subtyping assay. Therefore, the project has four objectives: (i) develop a regional repertoire of human and livestock Listeria subtypes for subtype comparision studies; (ii) construct and test a DNA microarray to allow rapid, accurate and discriminatory subtyping of L. monocytogenes isolates for the purpose of on farm molecular epidemiology and gene discovery; (iii) characterize on-farm subtypes of livestock origin and compare these subtypes to those of food- borne disease origin. L. monocytogenes subtypes are genetically characterized using pulsed-field gel electrophoresis (the current gold standard) and microarray analysis (a new technology that allows identification of genetic regions that differ between subtypes). (iv) Develop in vitro and in vivo virulence models
for L. monocytogenes infection that allow the phenotypic characterization of putative virulence genes identified by microarray analysis. Our research concerning Listeria monocytogenes addresses the following elements of our National Program in Animal Production, Product Value and Safety: 103 Animal Diseases 80% and NP108 Food Safety (20%). In particular this research addresses epidemiology of disease, microbial genomics, and pathogen detection. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY 2003) Construct and test 2000 spot microarray. Compare resolution with PFGE. Use 2000 spot microarray to identify potential virulence-associated genes. Test guinea pig in vivo assay. If successful, use model to phenotype L. monocytogenes strains. Year 2 (FY 2004) Construct and test 6000 spot microarray. Compare resolution with PFGE. Use 6000 spot microarray to identify potential virulence-associated genes. Use genes identified as virulence associated to design
PCR primers for subtyping. Use in vivo model to characterize mutated (and complemented) L. monocytogenes strains. Year 3 (FY 2005) Construct and test condensed subtyping microarray. Combine rapid template amplification with condensed array and test rapid subtyping system. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004 and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. Milestones scheduled to be addressed in FY2004: The milestones listed for this year were: 1) Construct and test 6000 spot microarray. Compare resolution with PFGE. 2) Use 6000 spot microarray to identify potential virulence-associated genes. Use genes identified as virulence associated to design PCR primers for subtyping. 3) Use in vivo model to characterize mutated (and complemented) L. monocytogenes strains. Due to insight gained from previous data from
our 2000 spot array and preliminary in vivo testing, our goals changed slightly: Towards milestones 1&2) The 2000 probe mixed genome microarray was constructed and tested this last year, allowed identification of 675 polymorphic probes, and laid the groundwork for future studies involving epidemic strain identification. We have used this information to construct a 620-probe "condensed" microarray consisting solely of informative probes. We will test this array and compare the results of this subtyping method with those obtained with pulsed-field gel electrophoesis subtyping (PFGE; the current gold standard) and multilocus sequence analysis subtyping (MLST, a new subtyping technique that is currently being developed by a number of labs). In addition, we plan to use data gathered from this condensed array to identify the 100 most informative probes so we can construct and test a more high through-put bead microarray format for subtyping. 2) We are currently testing a newly described in
vivo mouse model to identify virulence differences between L. monocytogenes strains. Once a model is established that reliably differentiates between clinical and nonclinical isolates, this model will be used to evaluate the virulence potential of genes identified by microarray analysis to be present only in epidemic strains. Progress: 1) We have used the condensed array to compare microarray subtyping resolution to PFGE, ribotyping, and MLST and for identification of genes that characterize epidemic strains. A publication describing this work has been accepted (Borucki et al., Journal of Clinical Microbiology). We are in the initial stages of testing a high throughput bead (suspension) microarray. 2) A murine oral infection model has been developed and tested (Kim et al., 2004). Data indicate that epidemic strains as a group are more invasive than environmental strains when assessed using this model. B. List the milestones that you expect to address over the next 3 years (FY 2005,
2006, & 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Year 3 (FY2005) We plan to develop and test a rapid, reliable, and efficient suspension microarray subtyping assay and to characterize virulence genes exclusive to epidemic strains using in vivo and in vitro (mouse oral infection) models. Planar microarrays (genomic comparison and expression) will also be used for identification of subtype specific genes. Gene knock out and mutant phenotyping experiments will be used to assess role of subtype specific genes. Year 4 (FY2006) We plan to validate a high through put subtyping suspension array using a large panel of well-characterized strains and ready the technology for transfer. Genes identified as important for strain virulence using gene knock out experiments and phenotypic characterization will be included as probes in the subytping array. High through put microarray subtyping will also be used to investigate the on-farm ecology
of L. monocytogenes, define the risk of the on-farm sources for food-borne outbreaks and formulate recommendations for control. Year 5 (FY2007) We anticipate that the identification (via planar microarray gene discovery) and phenotypic characterization (gene knock out and testing using in vitro and in vivo models) of virulence-associated genes will be a lengthy process. It is likely that a combination of genes (presence and absence, or polymorphisms), phage insertions/gene disruptions, and gene expression differences all contribute to strain phenotype. Therefore we expect the experiments planned for FY 2005-2006 to continue in 2007. 4. What were the most significant accomplishments this past year? A. In order to determine if a particular subtyping method is effectively able to differentiate virulent isolates from less virulent "environmental" isolates one must first be able to access the virulence potential of a particular L. monocytogenes strain, therefore, it is important to
develop an assay that is able to predict the virulence potential of environmental/nonclinical strains. Six human epidemic strains and six environmental strains were assayed for invasiveness using an intragastric inoculation A/J mouse model (Kim et al., 2004) in a project preformed by the Listeriosis program (ARS Animal Disease Research Unit) and Washington State University. Variation in strain invasiveness was observed and epidemic strains were significantly more invasive than environmental strains. This is the first study demonstrating that a biologically relevant in vivo model can be used to assess L. monocytogenes strain virulence which is a critical step towards understanding why some strains cause epidemics whereas others do not. B. In order to identify genomic regions that differ between L. monocytogenes strains, 2000-probe mixed genome DNA microarray was constructed and used to identify 675 polymorphic (informative) probes for sequence analysis and construction of a condensed
microarray. Fifty-five L. monocytogenes strains were genetically characterized by the ARS Animal Disease Research Unit Listeria lab using the condensed array including strains that had previously been subtyped previously by Dr. Frank Pagotta (Health Canada) and Dr. Sandra Smole (MA Department of Public Health) using pulsed-field gel electrophoresis (PFGE), ribotyping, and multilocus sequence typing (MLST). Microarray subtyping had higher resolution than the PFGE, ribotyping and MLST (using 8 housekeeping genes) and correctly grouped eleven out of twelve isolates that had an epidemiological link, and a majority of epidemic strains were grouped together within phylogenetic Division I. Discriminant function analysis allowed identification of 26 features from the mixed-genome array that distinguish serotypes and subtypes including several potential markers that were distinct for the epidemic cluster. C. Significant activities that support special target populations. None D. Progress
Report None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A regional repertoire of human and livestock Listeria subtypes for subtype comparision studies has been developed. A collection of over 1200 L. monocytogenes strains has been assembled including a large number of regional farm isolates. Approximately 775 raw milk samples from 474 Pacific Northwest herds were tested at multiple timepoints and isolates obtained from the milk samples were serotyped, genetically characterized, and evaluated for unique biological properties. A vast majority of L. monocytogenes isolates obtained from the milk and farm samples were found to be clinically significant serotypes which persist in the dairy environment for an extended period of time. Research was done to investigate whether human listeriosis cases are associated with the same subtypes of L. monocytogenes found in raw milk and area dairy farms by comparing farm-associated
subtypes to human isolates. Several human isolates were genetically similar or identical to farm and milk strains and it was also determined that a strain responsible for a large 1985 epidemic has a current, local on-farm reservoir and continues to cause sporadic human disease. This work was an important step in defining the on-farm ecology of L. monocytogenes and accessing the contribution of farm contamination as a source in human listeriosis. The applicability of microarray analysis to L. monocytogenes subtyping and gene discovery has been demonstrated. Results showed that a relatively simple microarray had resolution comparable to, or exceeding, existing methods and correctly identified strains associated with food poisoning outbreaks. Importantly, this method also identified the genetic regions that differ between strains and thus identified numerous genetic markers that are currently being used in epidemiological and pathogenesis studies. This microarray data also allowed the
development of PCR primers for serotype identification so that strains can be serotyped quickly and inexpensively. A novel intragastric inoculation mouse model differentiates epidemic strains from environmental strains has been developed. The ability to accurately assess the disease-causing potential of an isolate is essential for defining the genes that control strain virulence. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Genetic sequences were deposited in GenBank, making them available to the global research community free of charge. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. Suszkiw, J. 2003. New Tool for Fighting Listeria on Tap. ARS News Service.
Aegerter, M. 2004. A Quick Test for a Killer. Washington State Magazine.
Impacts
(N/A)
Publications
- Borucki, M.K., Peppin, J.D., White, D., Loge, F., Call, D.R. Variation in biofilm formation among strains of Listeria monocytogenes. Applied Env. Microbio. 2003. v. 69. p. 7336-7342.
- Kim, S.H., Bakko, M., Knowles Jr, D.P., Borucki, M.K. 2004. Oral inoculation of a/j mice detects invasiveness differences between listeria monocytogenes epidemic and environmental strains. Infection and Immunity. 72(7):4318-4321.
- Ward, T.J., Gorski, L.A., Borucki, M.K., Mandrell, R.E., Hutchins, J., Pupedis, K. 2004. Intraspecific phylogeny and lineage group identification based on the Prfa virulence gene cluster of Listeria monocytogenes. Journal of Bacteriology. 2004. 186(15):4994-5002.
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Progress 10/01/02 to 09/30/03
Outputs
1. What major problem or issue is being resolved and how are you resolving it? Listeria monocytogenes is a bacterium capable of causing serious disease in humans and animals. L. monocytogenes infection of cattle and sheep can lead to disease of the central nervous system and death. Human listeriosis is a potentially fatal food borne disease often associated with the consumption of contaminated dairy products. Because L. monocytogenes is widely distributed in the environment, a major problem associated with the food safety issue is the source(s) of L. monocytogenes in food borne illness. The role of domestic animals in dairy product contamination is not clearly understood. The focus of our research program is to elucidate the sources and significance of dairy farm contamination as related to human and animal disease. A majority of human outbreaks are caused by just 3 of the 13 L. monocytogenes serotypes, therefore we will focus on identifying the prevalence of these
serotypes on the dairy farm and developing methods to precisely and reliably identify genetic subtypes that are especially virulent. 2. How serious is the problem? Why does it matter? Approximately 2500 cases of human listeriosis occur annually in the U.S. resulting in about 250 deaths. Therefore, although human listeriosis is a relatively rare food borne disease it is significant due to its high mortality rate. Additionally this bacterium causes disease in the central nervous system of cattle and sheep. Outbreaks of listeriosis have been traced to pasteurized milk, cheese, coleslaw, and meat products. However, the role of subclinical infections of animals in the distribution of listeriosis in animals and humans is unknown. Clearly, it is important to define all the contributing sources of the organism to the food chain and human and animal health problems. 3. How does it relate to the National Program(s) and National Program Component(s) to which it has been assigned? Our research
concerning Listeria monocytogenes addresses the following elements of our National Program in Animal Production, Product Value and Safety: 103 Animal Health 70% and 108 Food Safety 30%. The current research focus is on farm epidemiology and ecology and genetic analysis through the application of microarray technology. 4. What were the most significant accomplishments this past year? A. Although it is widely recognized that L. monocytogenes serotypes vary in virulence potential, many researchers do not serotype isolates due to the cost and technical difficulty of the assay, therefore data from our microarray subtyping experiments and other published studies was used to design PCR primers for serotyping L. monocytogenes strains. Four sets of PCR serotyping primers were designed and used to serotype a panel of 120 strains of L. monocytogenes as part of a collaborative project between USDA-ARS-ADRU and Washington State University. All four serotyping primer sets showed greater than 95%
sensitivity and specificity when compared to conventional agglutination serotyping. This method of serotyping is more rapid, inexpensive, and easier to interpret than the conventional agglutination method and will allow both research and public health labs to easily serotype isolates. B. Biofilm formation allows bacterial strains to persist in the food processing environment increasing the likelihood of product contamination, therefore research was done to identify the relationship between L. monocytogenes subtype and the ability to form biofilms. As part of a collaborative project between USDA-ARS-ADRU and Washington State University, 80 strains were tested using a modified microtiter plate biofilm assay and biofilm formation was verified using a carbohydrate binding stain and scanning electron microsopy. We found that biofilm formation correlated with phylogenetic division and strain persistence, and scanning electron microscopy clearly showed that high biofilm forming strains form
a dense, three-dimensional structure on stainless steel whereas low biofilm forming strains do not. This study allowed us to identify several high biofilm-forming strains that are suitable for gene- mutation studies using a gene believed to be involved in biofilm formation that was previously identified by our microarray analysis. Research was done to identify the source(s)of bovine listeriosis cases on two Pacific Northwest dairy farms using pulsed-field gel electrophoresis subtyping. As part of a collaborative project between USDA-ARS-ADRU and Washington State University, approximately 600 L. monocytogenes strains were isolated from environmental samples collected from the two infected dairy farms and subtyped. Two L. monocytogenes strains were identified in the feces of two nondiseased cows on Farm "A" that were genetically similar to the clinical isolate obtained from the Farm "A" bovine listeriosis case, and interestingly, these two fecal isolates were identical to the bovine
listeriosis strain from Farm "B" (located 50 miles away from Farm "A"). These findings suggest that closely related subtypes may vary in virulence potential (a hypothesis that will be tested using an animal model) and that virulent subtypes may be transported between farms via infected food, water or animals. C. None D. None 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Although our Listeria program is relatively new, we successfully designed and tested a mixed genome Listeria subtyping microarray in FY2002. We also identified the serotypes and genetic subtypes of L. moncytogenes on Pacific Northwest dairy farms using used pulsed field gel electrophoresis and microarray and compared them to human isolates obtained from the Washington State Department of Health. Our Listeria program has become much more comprehensive and multifaceted during FY2003. In particular, we are currently testing a condensed mixed genome
microarray for L. monocytogenes strain subtyping and virulence gene discovery. Unlike our previous mixed genome microarrays, all probes on this condensed microarray were previously identified as polymorphic (informative) using a 2000-probe "screening microarray" and all probes have been sequenced and characterized. This microarray will not only lead to the development of new subtyping tools that are rapid (as with the PCR primers described above) and informative, but it will increase our understanding of L. monocytogenes biology, ecology and epidemiology. For example, a glycosyltransferase gene identified by microarray as serotype-specific is currently being used in a gene knock-out (mutation) experiment. Gylcosyltransferase (GLT) is part of biofilm synthesis and we plan to characterize the effect of GLT knock-out on biofilm formation using biofilm assays developed by Washington State University. We are also testing a newly-described mouse model that more accurately simulates human
infection because the animals are orally infected rather than injected with L. monocytogenes as with previous models. Preliminary results are promising, and we expect to use this model to further characterize potential virulence-associated genes identified by microarray analysis. 6. What do you expect to accomplish, year by year, over the next 3 years? FY2004: 1) We plan to continue testing the reproducibility and resolution of our L. monocytogenes microarray. The 2000 probe mixed genome microarray constructed and tested this last year identified 675 polymorphic probes and laid the groundwork for future studies involving epidemic strain identification. We have used this information to construct a 620-probe "condensed" microarray consisting solely of informative probes. We are now in the process of testing this array and comparing the results of this subtyping method with those obtained with pulsed-field gel electrophoesis subtyping (the current gold standard) and multilocus sequence
analysis subtyping (a new subtyping technique that is currently being developed by a number of labs). In addition, we plan to use data gathered from this condensed array to identify the 100 most informative probes so we can construct and test a more high through-put bead microarray format for subtyping. 2) We are currently testing a newly described in vivo mouse model to identify virulence differences between L. monocytogenes strains. Once a model is established that reliably differentiates between clinical and non-clinical isolates, this model will be used to evaluate the virulence potential of genes identified by microarray analysis to be present only in epidemic strains. FY2005: We plan to finish optimization and testing of a rapid, reliable, and efficient microarray subtyping assay and to characterize virulence genes exclusive to epidemic strains using the in vivo model. FY2006: We will use microarray subtyping to investigate the on-farm ecology of L. monocytogenes, define the
risk of the on-farm sources for food-borne outbreaks and formulate recommendations for control. 7. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? No technologies have been transferred. If the microarray assay proves useful the technology may be transferable within the next 2 years. 8. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: This does not replace your peer-reviewed publications listed below). "New tool on tap for fighting listeria". DVM News Magazine. July 2003. p. 2F.
Impacts
(N/A)
Publications
- Muraoka, W.T., Gay, C., Knowles, D., Borucki, M.K. Prevalence and types of Listeria monocytogenes in dairy herds of the Pacific Northwest. Journal of Food Protection. 2003. v.66 (8) pp. 1413-1419.
- Peppin, J.D., Borucki, M.K., White, D., Loge, F.P., Call, D.C. Investigation of Intraspecies Variation in Biofilm Formation Among Listeria monocytogenes Isolates. American Society of Microbiology. 2003.
- Call, D.R., Borucki, M.K., Loge, F.J. Detection of bacterial pathogens in environmental samples using DNA microarrays. Journal of Microbiological Methods. 2003. v. 53. 235-243.
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