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<p>Enteric infections caused pathogens such as Salmonella enterica, Clostridium difficile and Porcine Epidemic Diarrhea Virus(PEDV) is a major problem in animal health and food safety. Combined economic cost due to the enteric infections caused byenteric pathogens is several billion US dollars annually.Control of the infections caused by such enteric pathogens requires amultipronged approach of understanding their pathogenesis, tracking the transmission of these pathogens and the developmentof new diagnostic tests. The overall goal of this proposal is to develop an integrative approach to control enteric animal diseasesby developing new animal models to study the pathogenesis, employ genomic methods to track the disease transmissiondynamics and to develop a new generation of diagnostic tests for the accurate detection of these pathogens. Our long term goal is to develop an integrative research tool set for controlling enteric infections by combining traditional animal studies, molecularbiology methods, genomics and bioinformatics.</p><p> Specific objectives of our proposal are:</p><p> Objective 1. Develop a gnotobiotic miniature pig model to study enteric disease pathogenesis: Most studies performed tounderstand the pathogenesis of enteric pathogens have been carried out using in vitro methods or have used animal modelssuch as mouse, hamster or conventional pigs. In this objective, we propose to develop a novel miniature pig model to studyenteric pathogens. We will use Yucatan miniature pigs to develop the new model. The Yucatan miniature pig model we proposeto develop has significant advantages over the conventional pig models. When compared to conventional pigs, Yucatanminiature pigs are preferred for longer-term studies because of their smaller size, stable growth rates, lower feeding costs,smaller housing needs and their docile nature. Also, as opposed to varying genetic background of conventional pigs, Yucatanpigs have been bred to have a uniform genetic background.</p><p> Objective 1A. To study the C. difficile pathogenesis using the miniature pig model. Miniature pigs will be delivered throughcesarean section and will be maintained in sterile isolators, infected in the presence and absence of normal microbiome tounderstand pathogenesis of C. difficile. Host response to the infection will be analyzed by histopathological examination and byassessing immune response. Microbial changes in the gut during infection will be studied by shotgun sequencing of microbiomeand by traditional molecular assays.Objective 1B. To study the effect of dietary changes in controlling C. difficile infection. Since the primary risk factor of C. difficileinfection is the changes in the gut microbiome composition, we will design prebiotic diets that could restore normal microbiomecomposition in pigs. Miniature pigs receiving various prebiotic diets will be then challenged with C. difficile to test protectiveefficacy of these diets against C. difficile infection.</p><p> Objective 2. Study the transmission dynamics and evolution of enteric diseases. Understanding the population dynamicsand transmission mechanisms of enteric pathogens is critical for identifying the genomic changes that lead to the emergence ofmore virulent strains and also for tracking the source of disease outbreaks. In this study, we will perform whole genomesequencing of historic and recent isolates of enteric pathogens that are of animal and environmental origin.</p><p> Objective 2A. Sequence the genomes of historic and recent strains of Salmonella enterica. Historic and recent strains ofSalmonella enterica isolated from animals and environmental sources will be sequenced using Next generation sequencing(NGS) methods. Strains will be collected through the South Dakota ADRDL and through collaborations within and outside theUnited States. Bioinformatics comparison of these genomes against publicly available sequence databases will be performed todevelop analysis pipelines to detect outbreak sources and for identifying genome changes that lead to the emergence of virulentvariants.</p><p> Objective 2B. Sequence genomes of outbreak associated PEDV isolates. NGS based whole genome sequencing of PEDVisolates from the Midwestern US will be performed to identify the causes of PEDV outbreaks since 2013. Comparative genomicanalysis of these strains against other strains from the US, Canada, China and Brazil will be performed to identify mutation hotspots that may contribute to strain variation.</p><p> Objective 3. Develop new a generation diagnostic tests for enteric diseases. Globalization has removed the many of thebarriers that previously prevented the transmission of enteric pathogens between locations within a country and internationally.Modernization of agricultural practices has also changed food processing practices that allow transportation of meat, egg andfresh produce to distant locations. Traditional molecular diagnostic methods such as PCR alone do not have sufficient resolutionpower to track the source of contaminating enteric pathogens in clinical samples and food. By combining traditional diagnosticmethods such as PCR with NGS, we aim to develop new generation of diagnostic tests that are </p><p> (1) culture independent, </p><p> (2)identify the original source of contamination, and </p><p> (3) detect new virulence properties.Objective 3A. Develop NGS based diagnostic assay for the detection of Salmonella enterica. Currently, multiple tests arerequired to identify the serovar, virulence properties and antibiotic resistance profile of Salmonella enterica. We will use NGSbased amplicon sequencing of serovar specific genes, virulence genes and antibiotic resistance genes that can detect theseproperties of Salmonella DNA isolated from animal sources and food samples.Objective 3B. Develop NGS based diagnostic assay for the detection of PEDV. The PEDV genome is about 28 KB in size.Currently used PCR methods could detect only a small region of the PEDV genome and therefore cannot identify genome widevariations in outbreak strains. Direct NGS sequencing of PEDV samples are not cost effective because 98% NGS reads fromdirect sequencing are non-PEDV reads. We will develop a multiplex PCR that can amplify whole genome of PEDV and then useNGS to sequence the amplicons. The new test then can simultaneously detect PEDV from clinical samples and identify newvariants that may contribute to transmission and outbreaks.</p>
<p> We have acquired all the expertise and facilities required for the successful completion of the proposed experiments. Theanimal disease research unit at SDSU has all the necessary facilities to perform the required animal experiments. The Moleculardiagnostics section of ADRDL has purchased an Illumina MiSeq required for NGS experiments. To support the analysis ofsequencing data ADRDL has also purchased a High Performance Computing (HPC) machine with 32 cores, 256 GB memoryand 16 TB of storage capacity. Additional computing capacity is available at the SDSU IT department. These include severalservers loaded with virtualization platforms and parallel processing capacity. Experiments associated with each objective will be performed as per the outline below</p><p> Objective 1. Develop a gnotobiotic miniature pig model to study enteric disease pathogenesis: All the animal experiments will beperformed with the approval of Institutional Animal Care and Use committee (IACUC). Preterm miniature pigs will be obtainedfrom LoneStar laboratory swine farm (http://lonestarswine.com/ ). Following the protocols we have published previously[9, 11],piglets will be delivered under sterile conditions by cesarean section and will be maintained in sterile isolators. Two customprebiotic diets will be designed by the supplementation of the chow diet by 10% inulin and fructooligosaccharide respectively,and will be sourced from Research diets Inc. (http://www.researchdiets.com/ ). These are chosen because they are shown toimprove general gut health by acting as a fermentable fiber source. Groups of three piglets will be transplanted with definedmicrobiome population and will be given different diets. Two weeks post birth, they will be treated with vancomycin and thenchallenged with 103 spores of C. difficile. Microbiome shifts associated between groups will be analyzed using shotgunmicrobiome sequencing. We will follow standard Illumina protocols to perform NGS on MiSeq platform using paired end 300base sequencing chemistry. Host immune response will be assessed using antibacterial qRT-PCR array (Qiagen). Protectiveefficacy of diets will be compared based the reduction of diarrhea, host inflammatory response, and weight loss.</p><p> Objective 2. Study the transmission dynamics and evolution of enteric diseases. We will analyze the disease transmissiondynamics of PEDV and Salmonella enterica using NGS. PEDV positive animal samples from South Dakota and other states inthe US have been already collected and stocked in ADRDL since 2013. We will use standard protocols to isolate total RNA fromthese samples and to convert it to cDNA. Illumina Nextera XT kit will be used for the preparation of sequencing libraries andNGS will be performed on a MiSeq platform. Sequence analysis will be carried out on a BioLinux workstation. Molecular clockanalysis will be used to determine mutation rates. Galaxy suite of programs in BioLinux will be used for determining Singlenucleotide profile (SNP).Sequencing of Salmonella enterica samples will be performed following the NGS protocols described above except that totalgenomic DNA will be used as starting material. SNP analysis and phylogenetic analysis using BEAST software(http://beast.bio.ed.ac.uk/ ) will be used to analyze strain dynamics.</p><p> Objective 3. Develop new generation diagnostic tests for enteric diseases. For developing an improved diagnostic test thatsimultaneously detects an enteric pathogen and identifies the molecular changes in the genome, we will combine traditionalPCR methods with NGS. First targeted genes will be amplified from the samples using an ultra-high multiplex PCR. Degenerateprimers will be used for the amplification several genes in a single reaction tube using ultra-high multiplex PCR. The PCRamplicons will be used for sequencing on Illumina Miseq. For PEDV detection, complete genome of PEDV (28KB) will beamplified. For Salmonella enterica, serovar specific genes, known virulence and antibiotic resistance genes will be amplified andwill be used for strain differentiation by comparison against reference strains of known serotype.</p>