MA, Kent State University, 2024, College of Arts and Sciences / Department of Anthropology

Genotyping animals is necessary for various field-based applications that require precise knowledge of the sampled individuals. Though feces are considered a low-quality source of host DNA, molecular techniques are increasingly prioritizing its usage for field-based noninvasive projects. Here, we describe a reproducible workflow to genotype individuals using a whole-genome sequencing approach with the portable, high throughput MinION MK1B and the BWA-GATK variant calling pipeline. After filtering, only 4 of the original 5,394 SNPs passed the filtering criteria, leading to an unsuccessful attempt to generate an informative multiloci SNP panel to confidently and accurately differentiate animals. In the filtered SNPs, 5 samples were entirely void of genotyping data. The majority of SNPs exhibited allelic dropout and a lack of called heterozygote genotypes, leading to the presumable false genotypes of the sampled individuals. On average, approximately 97% of the genome remained unsequenced, with only about one read covering each base in the mapped regions. Despite the limitations of employing a whole-genome sequencing approach to differentiate individuals with the MinION using feces, for species lacking known variants, this strategy may be an effective way to initially identify SNPs for subsequent resequencing and genotyping. Future studies are necessary to validate the authenticity of the identified SNPs and to assess their ability to discriminate individuals effectively with enrichment and targeted sequencing techniques.

Committee: Rafaela Takeshita (Advisor); Sangeet Lamichhaney (Committee Member); Richard Meindl (Committee Member); Anthony Tosi (Committee Member) Subjects: Bioinformatics; Genetics

Doctor of Philosophy, The Ohio State University, 2024, Plant Pathology

Often overshadowed by staple crops, vegetables contain many essential vitamins and minerals and play a key role in global food and nutritional security. However, vegetable production is threatened by a variety of diseases, including bacterial wilt and bacterial spot. My dissertation research utilizes genomic surveys to elucidate the diversity of the bacteria causing these diseases, both globally and locally. Bacterial wilt disease is endemic within the country of Cambodia, causing significant yield losses for Cambodian growers. However, the diversity of the Ralstonia solanacearum Species Complex (RSSC), the causal agent responsible for bacterial wilt disease, has not been defined in Cambodia. Therefore, we conducted a bacterial wilt survey within Cambodia, collecting RSSC isolates from four distinct host plants (tomato, hot pepper, long bean, and bitter gourd) over three locations, for a total of 24 RSSC isolates. We found that all 24 of the Cambodian RSSC isolates belong to phylotype I and are classified as Ralstonia pseudosolanacearum. Through disease progress assays on susceptible hosts, we observed that variation in the Cambodian isolate's ability to cause consistent wilt was dependent on the method of inoculation. Additionally, the Cambodian R. pseudosolanacearum isolates exhibited a wide range of phylogenomic diversity. When comparing the core and accessory genome and the Type III effector profile of the Cambodian isolates, we found that the R. pseudosolanacearum accessory genome better reflected the host of isolation and host range of the isolates compared to the core genome. Altogether, this research provides a glimpse into the RSSC diversity present within Cambodia and insight into R. pseudosolanacearum host range. Bacterial spot disease affects tomato and pepper production worldwide and is caused by a species complex of Xanthomonas bacteria: X. hortorum pv. gardneri, X. euvesicatoria pv. euvesicatoria, X. euvesicatoria pv. perforans, and X. vesicatoria. We (open full item for complete abstract)

Committee: Jonathan Jacobs (Advisor); Mary Rodriguez (Committee Member); Francesca Hand (Committee Member); Sally Miller (Advisor) Subjects: Plant Pathology

Doctor of Philosophy, Case Western Reserve University, 2023, Genetics

Whole genome sequencing (WGS) is an ever expanding tool in the field of genetics, and is widely used to characterize human genetic variation. There are multiple large-scale sequencing studies being conducted today worldwide, like All of Us, Three Million African Genomes, and GenomeAsia 100k. The addition of these diverse datasets alone can be transformative to our understanding of genetics, but the increase in the diversity of populations sampled also has the potential to reveal additional and novel information relevant to health and disease. Specifically, whole genome sequence (WGS) analyses of DNA from human whole blood may be able to capture genetic variation in other species that can affect both individual and public health. The research detailed in this dissertation aims to illustrate the utility of human WGS data for infectious disease, by showing that the malaria causing parasite Plasmodium can be sensitively detected from unmapped reads (UMRs) from WGS data. Malaria has a significant global health burden, and elimination of the disease has been a goal since the 1950s. Recently, there have been roadblocks in the progress of malaria elimination that can only be resolved through additional research efforts. Development of this detection methodology could be the tool required to better define the parasite population, identify problematic populations, and solve the roadblocks limiting elimination success.

Committee: Thomas LaFramboise Ph.D. (Committee Chair); Scott Williams Ph.D. (Advisor); Peter Zimmerman Ph.D. (Advisor); Dana Crawford Ph.D. (Committee Member); Arlene Dent MD, Ph.D. (Committee Member) Subjects: Biomedical Research; Epidemiology; Genetics; Parasitology

BS, Kent State University, 2020, College of Arts and Sciences / Department of Biological Sciences

As strains of Staphylococcus aureus gain resistance to antibiotics, the risk of bidirectional transmission of resistant strains between humans and animals increases. Interactions with domestic animals and livestock may lead to human colonization, but there is scant research on whether urban wildlife do the same. The objective of this study is to examine the genomes of 20 S. aureus isolates collected from wild rodents in Boston, Massachusetts and determine their genetic relationship to human and animal strains. A total of 168 bacterial isolates collected from 45 Brown rats (Rattus norvegicus) were analyzed. Standard methods were used for bacterial culture and diagnostics. MecA (methicillin resistance) and PVL (Panton-Valentine Leukocidin) genes were detected using polymerase chain reaction. All S. aureus isolates were spa typed and tested for antibiotic susceptibility. Non-S. aureus bacterial isolates were identified via VITEK 2 analysis. Whole genomes of 20 S. aureus isolates were sequenced and mapped to reference genome. Assembled genomes were interrogated for known antibiotic resistance genes. Our results indicate that wild rats in Boston, MA are indeed carriers of S. aureus, and that wild rodents may play a role in S. aureus ecology in urban areas. spa typing revealed strain types distinct from typical hospital and community-associated strains, but susceptibility results are consistent with human infections. Further research to characterize lineages of S. aureus strains is needed in order to minimize the risk of Staphylococcal infection from city rodents.

Committee: Tara Smith Ph.D (Advisor); Helen Piontkivska Ph.D (Committee Member); Elda Hegmann Ph.D (Committee Member); Christopher Woolverton Ph.D (Committee Member) Subjects: Microbiology

Doctor of Philosophy, Case Western Reserve University, 2020, Epidemiology and Biostatistics

Hypertension (HTN) or elevated blood pressure (BP) affects 1 in 3 adults in the US. Across ethnicities, BP levels have been consistently higher for African Americans (AA) with an earlier onset of HTN. Many studies have investigated racial differences in HTN, especially genetic factors contributing to disease progression. While genome-wide association studies (GWAS) have identified over 900 loci associated with BP variation, these variants together only explain a small portion of the heritability. Several studies have shown that rare variants could explain a portion of the “missing heritability”. Linkage analysis of families is a promising approach for detecting genetic signals because it is insensitive to allelic heterogeneity and facilitates the discovery of missing heritability due to rare variants. This dissertation includes two of the first studies to leverage linkage evidence from family-based studies and search for BP-associated rare variants in trans-ethnic whole genome sequencing (WGS) data. Because only a small amount of GWAS variants fall within linkage regions, combining linkage and association analyses would yield a powerful and robust approach for detecting rare variants. Given the increasing availability of WGS data, efficient approaches are needed to interrogate a large number of genetic variants involved in disease etiology. Although directly searching the whole genome using window-based or gene-based approaches are commonly implemented, these methods may suffer from statistical power lost due to the large number of statistical tests. In contrast, confining to genomic regions with linkage evidence helps to reduce the multiple testing burden. Furthermore, because variant annotation is independent from linkage information, it can be incorporated into rare variant association analysis. By leveraging linkage evidence from European American (EA) families, SLX4 was shown to be associated with pulse pressure in EA. Linkage evidence in AA families led to the (open full item for complete abstract)

Committee: Scott Williams (Committee Chair); Xiaofeng Zhu (Advisor); Fredrick Schumacher (Committee Member); Jing Li (Committee Member) Subjects: Biostatistics; Epidemiology; Genetics

Master of Public Health, The Ohio State University, 2019, Public Health

Multidrug-resistant (MDR) Salmonella enterica is a threat to public health, in part due to a broad spectrum of resistance to antimicrobials critically important for treating infections. Salmonella Dublin is a bovine adapted S. enterica serotype. Human cases tend to manifest in a more invasive manner and the isolates have high prevalence of antimicrobial resistance (AMR). The aims of this study were to genetically characterize AMR features in MDR S. Dublin isolates from cattle in Ohio, assess the capacity of whole-genome sequencing (WGS) to predict AMR phenotypes, and characterize the genetic relatedness of the strains. Twenty-four S. Dublin isolates recovered from samples submitted to the Ohio Department of Agriculture by veterinarians from sick cattle were selected for WGS with Illumina MiSeq. Genotypic analysis of AMR determinants and plasmid replicons in each assembly was performed through Center for Genomic Epidemiology (https://cge.cbs.dtu.dk/services/) and nucleotide BLAST analyses. Cohen's kappa coefficient of agreement between phenotypic and genotypic AMR was calculated. The webtool CSIPhylogeny 1.4 was used for phylogenetic reconstruction to determine S. Dublin relatedness. Average genotype and phenotype agreement for characterization of AMR was 94.8%. Identification of a resistance gene had a mean 88.9% sensitivity and 84.2% specificity for the identification of the corresponding resistance phenotype. The majority of resistance genes were located on plasmid incompatibility group IncA/C2. IncFII(S) and IncX1 replicon groups were also present. WGS was able to identify chromosomal point mutations conferring resistance to quinolones and fluoroquinolones. All isolates were multilocus sequence type (MLST) ST-10 and showed close clustering in single nucleotide polymorphism (SNP) phylogenetic analyses. Results indicate that WGS is a robust method to accurately detect comprehensive AMR determinants in MDR S. Dublin isolates from bovine sources. WGS could also simul (open full item for complete abstract)

Committee: Gregory Habing DVM, MS, PhD (Advisor); Thomas Wittum MS, PhD (Committee Member); Jiyoung Lee MS, PhD (Committee Member) Subjects: Public Health

PhD, University of Cincinnati, 2018, Medicine: Immunology

Juvenile idiopathic arthritis (JIA) is a complex disease characterized by inflammation of the joints and surrounding tissues that manifests before the age of 16 and persists for at least six weeks. It is a heterogeneous disease categorized into seven subtypes and has a prevalence of approximately 1 in 1,000 children. Two subtypes, oligoarticular and rheumatoid factor-negative (RF⁻) polyarticular JIA (poly RF⁻), are the most common and are primarily differentiated by the number of involved joints during the first six months of disease. JIA has a strong genetic component, which has been elucidated by association studies that interrogate common variation in case-control populations. These studies have identified more than 30 JIA susceptibility loci. Yet, a significant proportion of the genetic heritability for JIA remains unaccounted for, as such studies have included limited sample sizes, heterogeneous patient cohorts, only interrogated candidate regions or common variants, or have not adequately considered additive or epigenetic effects. The overarching hypothesis for the work described in this dissertation is that both common variants and de novo mutations (DNMs) associated with oligoarticular and poly RF⁻ JIA contribute to disease pathogenesis. This hypothesis was tested using genome-wide arrays and whole exome sequencing (WES), followed by bioinformatic analyses. Common variants and DNMs among oligoarticular and poly RF⁻ JIA patients identified novel susceptibility loci and highlighted biological pathways of potential significance to disease pathogenesis. First, evidence of common variation association was found for nine loci (P < 1x10⁻⁶): PRR9_LOR, ILDR1_CD86, WDFY4, PTH1R, RNF215, AHI1_LINC00271, JAK1, LINC00951, and HBP1. Of these, PRR9_LOR, ILDR1_CD86, RNF215, LINC00951, and HBP1 are reported as autoimmune disease susceptibility loci for the first time, while WDFY4, PTH1R, AHI1_LINC00271, and JAK1 have been associated in other autoimmune diseases. Single nucle (open full item for complete abstract)

Committee: Susan Thompson Ph.D. (Committee Chair); Alexei Grom M.D. (Committee Member); John Harley M.D. Ph.D. (Committee Member); Jonathan Katz Ph.D. (Committee Member); William Nichols Ph.D. (Committee Member); William Ridgway M.D. (Committee Member) Subjects: Immunology

Doctor of Philosophy, The Ohio State University, 2018, Plant Pathology

Salmonellosis cases caused by Salmonella enterica through pre-harvest contamination of fresh produce represent a risk to human health worldwide; however, little is known about the interactions between Salmonella, phytopathogens, environment, and the plant host contributing to this food safety issue. Furthermore, the control of Salmonella from “farm to fork” is challenging due to the development of resistance mechanisms towards current control methods and restrictions on use of antimicrobials imposed by regulatory agencies. We investigated the effects of specific environmental conditions on the persistence and dissemination of Salmonella enterica subsp. enterica serotype Typhimurium (S. Typhimurium) following artificial contamination of `Tiny Tim' tomato plants. We found that higher temperatures (30°C day/25°C night) reduced the persistence of S. Typhimurium in the phyllosphere compared to lower temperatures (20°C day/15°C night) when plants were sprayed on the leaves with a S. Typhimurium -contaminated solution. Wounding cotyledons with contaminated tools increased S. Typhimurium persistence and internalization in planta compared to spray inoculation. Low relative humidity enhanced the dissemination of Salmonella into non-inoculated plant tissues. S. Typhimurium was detected in the root systems for at least 98 days-post inoculation. Further, we showed that splice-grafting (`Celebrity' with 'MaxiFort') is a major risk for the internalization and long-term survival of S. Typhimurium inside the tomato plant. S. Typhimurium was detected in the root system for over 137 days if at least 5 x 10^3 colony-forming units were introduced during grafting. The survival of S. Typhimurium in tomato foliage was also affected by the presence of phytopathogens, the genotype of S. Typhimurium and tomato variety used. We found that rfbV, involved in O antigen synthesis, might be essential for S. Typhimurium persistence in inoculated tomato plants and especially in `Tiny Tim' plants (open full item for complete abstract)

Committee: Gireesh Rajashekara (Advisor); Sally Miller (Advisor); Laurence Madden (Committee Member); Christopher Taylor (Committee Member); Corey Nislow (Committee Member) Subjects: Agriculture; Bioinformatics; Biology; Environmental Health; Molecular Biology; Plant Pathology; Public Health

Doctor of Philosophy, The Ohio State University, 2017, Food Science and Technology

Fresh produce is low in calories and high in the fibers, essential vitamins and minerals. Consumption of fresh fruits and vegetables helps prevention of cardiovascular diseases, cancer, diabetes and improves gastrointestinal health. Thus, fruit and vegetables have been gaining popularity among consumers and the increased consumption has been endorsed by many health organizations. On the contrary, fresh produce has continuously been associated with foodborne diseases outbreaks. It is technically challenging to decontaminate fresh produce due to the lack of microbial kill-step that effectively eliminate pathogens without causing product quality deterioration. Currently, chlorine wash is the most-used antimicrobial treatment of postharvest fresh produce. Gaseous ozone and bacteriophages have become popular as natural and environmental-friendly alternative decontamination technologies. Bacteriophages can be applied as alternative to chlorine spray or used as a final rinse before packing. Gaseous ozone was previously found very effective when combined with vacuum cooling to inactivate Escherichia coli O157:H7 on baby spinach. The objectives of this research were: (i) to evaluate the suitability of a single lytic bacteriophage, Escherichia phage OSYSP, as a fresh produce decontaminant; (ii) to assess the efficacy of bacteriophage OSYSP and gaseous ozone against E. coli O157:H7 on spinach leaves; and (iii) to develop a combination treatment involving bacteriophage and gaseous ozone to eliminate E. coli O157:H7 on spinach leaves. To justify using Escherichia phage OSYSP in fresh produce decontamination and other applications, the phage characteristics and genomic makeup were investigated (Chapters 2-4). The method to determine phage titer was optimized for maximum phage recovery and plaque clarity during enumeration. Stability of the phage at different incubation temperatures was investigated. The titer of a phage preparation did not change considerably during storage (open full item for complete abstract)

Committee: Ahmed E. Yousef (Advisor); V.M. Balasubramaniam (Committee Member); Farnaz Maleky (Committee Member); Jiyoung Lee (Committee Member) Subjects: Food Science

Doctor of Philosophy, The Ohio State University, 2017, Comparative and Veterinary Medicine

There are four avian mycoplasma species (spp.) that are pathogenic to poultry; Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), Mycoplasma iowae (MI), and Mycoplasma meleagridis (MM). Avian mycoplasma caused infections represent significant economic burden on commercial poultry industry in the form of decreased egg and meat production efficiency, decreased market value of breeder flocks, carcass condemnation, and medication costs. Avian mycoplasma strain differentiation is a cornerstone for outbreak investigation, understanding infection dynamics, and epidemiology as well as planning future prevention and control strategies. Currently, avian mycoplasma strain differentiation is based on two main approaches; DNA fingerprinting and single or multiple locus based sequence typing. Despite that sequence typing approaches have many advantages over DNA fingerprinting approaches, they have many limitations. Recently due to the advanced next generation sequencing technologies, microbial whole genome sequencing (WGS) has become more feasible and resulted in a paradigm shift in the efficiency of outbreak investigation, understanding infection dynamics and epidemiology as well as planning future prevention and control strategies for many human and animal pathogens. The efficiency of WGS based strain differentiation approaches have superseded that of single or multiple loci based sequence typing approaches. The aim of this work was to improve and upgrade the currently used molecular typing tools for avian mycoplasma strain differentiation and in turn, epidemiological outbreak investigation via the development and application of state-of-the-art molecular typing approaches. This should be reflected in better prevention and control strategies and ultimately increasing the chances of successful eradication of avian mycoplasma spp. from commercial poultry. This dissertation is organized into an introduction and five chapters; the first chapter of this dissertation pres (open full item for complete abstract)

Committee: Mohamed El-Gazzar (Advisor) Subjects: Animal Diseases; Epidemiology; Microbiology; Veterinary Services

Master of Science, The Ohio State University, 2016, Chemical Engineering

Clostridium formicoaceticum is a Gram-negative homoacetogenic bacterium for acetic acid production and is also considered as a promising heterologous host for biofuel production. Here, its genome has been completely sequenced and consists of a 4.5-Mbp chromosome without containing any plasmid. The results of sequence analysis indicate that C. formicoaceticum is able to grow on several sugars and other organic substrates. In addition, it also exhibits a highly conserved Wood-Ljungdahl pathway gene cluster, which shows an identical arrangement as that in other autotrophic acetogens, such as Clostridium aceticum, Clostridium carboxidivorans, and Clostridium ljungdahlii. Differently from these homoacetogens, C. formicoaceticum contains a Na+-translocating ATPase and Rnf system for energy conversion. Such ATP generation system has also been sufficiently elucidated in Acetobacterium woodii. However, no growth defects were observed when C. formicoaceticum was grown on the heterotrophic and autotrophic medium under low sodium ion concentration, respectively. Otherwise, unlike Moorella thermoacetica and C. aceticum, neither completely cytochromes nor quinones synthesis genes are identified in the genome. Therefore, like C. ljungdahlii, C. formicoaceticum generates proton gradient via Rnf system and belongs to a H+-type homoacetogen. Moreover, ethanol formation may proceed by acetaldehyde dehydrogenases, alcohol dehydrogenases, and reversible aldehyde oxidoreductases under excessive reducing equivalent condition. Such information will be valuable in developing strategies for fermentation and metabolic engineering to produce bio-based chemicals and fuels in C. formicoaceticum.

Committee: Shang-Tian Yang (Advisor); Jeffrey Chalmers (Committee Member) Subjects: Chemical Engineering