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<li> Determine alterations in microbial community structure that occur as a result of infection by MAP in the intestines of MAP-shedding and MAP-exposed and non shedding (healthy) animals using well-established terminal RFLP method; <li> Determine the prevalence of Shiga toxin-producing E. coli (STEC)and Salmonella enterica serovar Typhimurium carriage in MAP-infected and shedding, and MAP-exposed and not shedding dairy animals using well-established microbiological procedures and pathogen-specific primers and probes coupled to quantitative real time PCR analyses. </ol> At the conclusion of these studies, we expect to be able to document the magnitude of public health threat of MAP infection in animal populations. These studies will establish a baseline for future analysis and quantitation of the impact of JD on food safety and public health.
NON-TECHNICAL SUMMARY: The understanding of microbial population structure in a pathogen induced environment would enable more rigorous risk analysis for food pathogen carriage. These studies will also provide critical missing information on the specific microbial populations that may be depleted as a consequence of Johne's disease and provide clues on how probiotic approaches may help in controlling this spread of Mycobacteria.
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APPROACH: We propose to study intestinal microbial community structure and function in JD infected and control animals in a prospective study design. Animals culture positive and negative for MAP residing on infected herds and status negative herds will be sampled for fecal culture and microbial community profiling monthly over a one year period. All samples will be analyzed by terminal-restriction fragment length polymorphism analysis for microbial population profiles and cultured for MAP, Salmonella and E. coli O157. Shifts in microbial structure will be correlated with MAP status and carriage of Salmonella and E. coli O157. In addition, when animals are culled from the herd, they will be purchased and euthanized for a detailed histopathology of the intestines and mucosal microbial profiles using fluorescence in situ hybridization methods.
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PROGRESS: 2007/07 TO 2007/12<BR>
OUTPUTS: The project was initiated in July of 2007 and work is underway. No outputs have been recorded for this period yet. <BR> PARTICIPANTS: Dr. Michael Paustian; National Animal Disease Center, Ames, IA Dr. Gary Andersen; Lawrence Berkeley National Laboratories, CA <BR> PROJECT MODIFICATIONS: We have identified collaborations with Dr. Andersen at the Lawrence Berkeley national laboratories to perform our analyses using more robust, unambiguous, and quantitative Phylochips. Phylochips are 16 rDNA based high density microarrays encompassing both eubacteria and archaea. The arrays are expected to provide quantitative microbial identities within each community which would not have been available with the proposed TRFLP protocols. Thus, we propose to continue the work with Phylochips.
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IMPACT: 2007/07 TO 2007/12<BR>
Since initiation of this project, we have identified a Johne's disease infected herd. Forty fecal samples from culture positive (moderate to heavy shedders) and 10 uninfected animals have been collected. Total microbial community DNA has also been extracted from these samples. Of these, twenty fecal DNA samples have been amplified for universal 16s rDNA encompassing eubacterial and archaeal communities in a gradient PCR. All gradient PCR generated amplicons will be poled by sample prior to analysis with a Phylochip.