Rapid Detection and Identification of Zoonotic Pathogens
Principal Investigator: John Dunn
Abstract: Zoonotic pathogens including those transmitted by insect vectors are some of the most deadly of all infectious diseases known to mankind. In the past, wars and natural disasters were the main catalysts that promoted epidemics of these ancient afflictions, which are normally transmitted by fleas, lice and ticks. Many of these diseases remain endemic in various regions of the world and therefore pose serious threats to U.S. armed forces troops and civilians who might enter endemic disease zones. A number of these agents have been further weaponized and are widely recognized as being the most significant biothreat agents. Study of disease agents and development of rapid means for their detection take on added importance in light of the use of anthrax for a bioterror attack on the U.S.A. The aim of this proposal is to modify a novel DNA-based methodology we have developed for profiling genomic DNAs to permit rapid, cultivation-free differential detection and identification of biothreat infectious agents in their natural environments, including intermediate infected hosts, and clinical specimens from humans or infected animals. We plan to use Borrelia burgdorferi, the arthropod-borne etiologic agent of Lyme disease and Yersinia pestis, the etiologic agent of plague, as our principle test agents to work through the systems. We will begin with B. burgdorferi since although it has a complicated life-cycle involving both arthropod and animal intermediates, it is easy to grow and we have extensive experience in working with it in different complex environments including ticks, rodents and human samples. Thus it gives us the opportunity of detecting this pathogen in a variety of complex environments. We also have significant experience with characterizing and identifying subtle changes in the genome of Y. pestis utilizing genomic signature tags. We will use these methods as the foundation of new, high-throughput sequence-based systems to detect zoonotic and/or vector-borne biothreat agents such as Yersinia pestis, Francisella tularensis, Rickettsia ricketsii and other human pathogens such as Ehrlichia and Babesia species. This technology can ultimately be adapted as a sensitive method to detect specific DNA signature sequences from both known and unknown pathogens in a wide variety of complex environments and since it is PCR-based it has the advantage that only minimal quantities of starting material are needed for analysis.
Funding Period: 2003-09-15 - 2010-02-28
more information: NIH RePORT
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Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA 99352, USA
Proteomics 5:1446-53. 2005....
- Proteome analysis of Borrelia burgdorferi response to environmental changeThomas E Angel
Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington, United States of America
PLoS ONE 5:e13800. 2010..burgdorferi adapts to and is able to survive in a wide variety of environmental conditions and lay a foundation for planned in situ studies of B. burgdorferi isolated from the tick midgut and infected animals...
- Differential binding of Escherichia coli McrA protein to DNA sequences that contain the dinucleotide m5CpGElizabeth A Mulligan
Department of Molecular Genetics and Microbiology, Genomics Core Facility, Stony Brook University, Stony Brook, NY, USA
Nucleic Acids Res 38:1997-2005. 2010..These results provide the first systematic analysis of McrA's in vitro binding specificity...
- Fast, adaptive evolution at a bacterial host-resistance locus: the PFam54 gene array in Borrelia burgdorferiEwa Wywial
Department of Biology, The Graduate Center of the City University of New York, 365 Fifth Avenue, New York, NY 10016, USA
Gene 445:26-37. 2009....
- Genetic vaccines for anthrax based on recombinant adeno-associated virus vectorsTe Hui Liu
Joseph Stokes Jr Research Institute, Children s Hospital of Philadelphia, Abramson Research Center, Philadelphia, Pennsylvania, USA
Mol Ther 17:373-9. 2009..The finding of robust neutralizing antibody responses after a single injection of these rAAV1-based vectors supports their further development as candidate anthrax vaccines...
- Rapid detection and identification of a pathogen's DNA using Phi29 DNA polymeraseYun Xu
Department of Medicine, T 16 Room 027, State University of New York at Stony Brook, Stony Brook, NY 11794 8154, USA
Biochem Biophys Res Commun 375:522-5. 2008..This simple technology can ultimately be adapted as a sensitive method to detect specific DNA from both known and unknown pathogens in a wide variety of complex environments...
- Cloning, purification and initial characterization of E. coli McrA, a putative 5-methylcytosine-specific nucleaseElizabeth A Mulligan
Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA
Protein Expr Purif 62:98-103. 2008....
- Characterization of clinically-attenuated Burkholderia mallei by whole genome sequencing: candidate strain for exclusion from Select Agent listsSteven E Schutzer
Department of Medicine, University of Medicine and Dentistry New Jersey Medical School, Newark, New Jersey, United States of America
PLoS ONE 3:e2058. 2008..Whole genomic sequencing (WGS) is an apt approach to characterize newly discovered or poorly understood microbial pathogens...
- Paired-end genomic signature tags: a method for the functional analysis of genomes and epigenomesJohn J Dunn
Biology Department, Brookhaven National Laboratory, Upton, NY 11973 5000, USA
Genet Eng (N Y) 28:159-73. 2007..This additional feature could permit multiplexing of the data by simultaneous sequencing of several pooled libraries if each used a different linker sequence during diTAG formation (Figure 4)...
- Enzymatic activity of the SARS coronavirus main proteinase dimerVito Graziano
Biology Department, Brookhaven National Laboratory, Upton, NY 11973, USA
FEBS Lett 580:2577-83. 2006..Enzyme activity from the SARS CoV main proteinase dimer could readily be detected at low pM concentrations. The enzyme exhibited a high K(m), and is unusually sensitive to ionic strength and reducing agents...
- Use of single-point genome signature tags as a universal tagging method for microbial genome surveysDaniel Van Der Lelie
Brookhaven National Laboratory, Biology Department, Building 463, Upton, NY 11973, USA
Appl Environ Microbiol 72:2092-101. 2006..This concept was further used successfully to identify the individual members of a defined microbial community...