<ol> <li> Develop a regional repertoire of livestock Listeria subtypes. <li> Develop a rapid assay to identify livestock Listeria subtypes. <li> Characterize Listeria on-farm subtypes of livestock origin and compare (differentiate) these subtypes from those of food-borne disease origin. <li> Define the on farm ecology of livestock shedders versus non-shedders to determine if shedders are segregated to certain farms and conditions.
Through culture of on farm samples, develop a repertoire of bovine and ovine Listeria subtypes. Utilizing serotyping, and genetic analyses such as Multilocus enzyme electrophoresis and restriction fragment length polymorphism, the on farm subtypes will be characterized and compared to those of food borne disease origin. These data will be acquired through interactions with farms (initially) in the Pacific Northwest. The data derived from antigenic and genetic analyses will be utilized to develop assays for rapid and accurate identification of Listeria subtypes. An important component of this research is identification of the shedding pattern of cattle and sheep. BSL-1: IBC 9-04-01. Fy00 Program Increase $270,000. 1 New SY added. Previously was 5348-42430-001-00D (05/01).
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 be focusing 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. <p> 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. <p> 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 100%. The current research focus is on farm epidemiology and ecology and genetic analysis through the application of microarray technology. <p> What were the most significant accomplishments this past year? A. Research was done to test the utility of a mixed genome DNA microarray analysis for genetic subtyping of L. monocytogenes strains and to identify the genetic differences that contribute to the virulence differences observed among between subtypes. The research was done as a collaborative project between USDA-ARS-ADRU and Washington State University. A 2000-probe mixed genome DNA microarray was constructed and used to identify and sequence 675 polymorphic (informative) probes for construction of a condensed microarray. A condensed microarray consisting entirely of informative probes is an important next step towards a high throughput subtyping microarray assay. B. Research was done to design an assay that allowed a large number of L. monocytogenes strains to be serotyped rapidly and inexpensively. As part of a collaborative project between USDA-ARS, Albany, CA and USDA-ARS-ADRU, Pullman, WA, the conventional slide agglutination serotyping assay was converted to an ELISA format. ELISA serotyping of 89 of 101 L. monocytogenes isolates agreed with slide agglutination serotyping data, and 100 previously uncharacterized isolates were serotyped unambiguously using the ELISA method. This ELISA assay greatly reduces the time and expense of serotyping L. monocytogenes strains and this will facilitate serotype determination by research and public health labs. Research was done to investigate whether human listeriosis cases are associated with the same subtypes of L. monocytogenes found in raw milk by comparing L. monocytogenes subtypes isolated from area bulk milk tanks with human isolates obtained from the Washington State Department of Health. As part of a collaborative project between USDA-ARS-ADRU, Washington State University, and Washington State Department of Health, 80 L. monocytogenes strains obtained from human and animal sources were subtyped using serotyping, pulsed-field gel electrophoresis and microarray analysis. Although several human isolates were genetically similar to strains isolated from bulk milk strains, no identical matches were found. Although these results suggest that L. monocytogenes strains found on farm are not a major source of human disease, the scope of this experiment was limited and we plan to continue to compare farm and human subtypes obtained from the Pacific Northwest. C. None D. None <p>Describe the major accomplishments over the life of the project, including their predicted or actual impact. Although this project represents a new CRIS within our Unit (2000), the microarray technology has the potential to be very useful for epidemiological studies in both public health and research laboratories. The current subtyping techniques provide rather limited genetic information, require expensive subtyping equipment (PFGE and ribotyping) or have suboptimal reproducibility (PCR-based technologies). The microarray equipment needed to subtype isolates is also expensive, however, many laboratories have or are in the process of obtaining such equipment since it can be used for many different purposes. Therefore, if a microarray was developed that enabled the rapid and reproducible subtyping of L. monocytogenes isolates it would potentially benefit many and results would be comparable between laboratories. Additionally, because L. monocytogenes subtypes differ in virulence and microarray subtyping identifies the genetic regions that differ between subtypes, this research will lead to better understanding of virulence mechanisms unique to epidemic strains. <p> What do you expect to accomplish, year by year, over the next 3 years?<br>
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.
<p>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. <p> 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. <p> 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). "Multiple serotype and lineage-specific differences among Listeria strains". Journal Highlights section of ASM News, April 2003. v. 69(4) p. 193.