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CHARACTERIZATION OF OUTER MEMBRANE PROTEINS AND OUTER MEMBRANE VESICLES AND COMPARATIVE GENOMICS TO IDENTIFY VACCINE CANDIDATES IN FUSOBACTERIUM NECROPHORUMPrabha K Bista (14206271) 02 December 2022 (has links)
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<p><em>Fusobacterium necrophorum</em> is a Gram-negative, anaerobic, opportunistic pathogen that causes necrotic infections in cattle leading to liver abscess, foot rot, and calf diphtheria. Particularly, liver abscess in cattle is reported at 20.7% annually, and leads to liver condemnation and an annual economic burden of about 62 million dollars to the feedlot industry. Antibiotic administration is the mainstay of treating these infections, but antibiotic resistance is unavoidable and demand for antibiotic-free, natural, and organic beef has demanded alternative therapies and preventatives. Vaccination is one of the best alternatives to prophylactic antibiotic administration. In this study, we have explored outer membrane proteins (OMPs) and outer membrane vesicles (OMVs) for potential vaccine candidates. OMPs and OMVs are vaccine targets because of their antigenic properties and host specificity. Additionally, we performed comparative genomic analysis of <em>F. necrophorum</em> species to identify additional virulence genes with vaccine potential, unique to the <em>F. necrophorum</em> and its virulent subspecies <em>necrophorum</em>. </p>
<p>Protein- protein interaction investigation through binding assay and pulldown assay identified novel OMPs, namely 17kDa, 22kDa, and 66.3 kDa proteins, which were further characterized as OmpH, OmpA and Cell Surface Protein (CSP), respectively. In this study, these novel OMPs including previously characterized 43kDa OMPs were cloned, and recombinant proteins were expressed and purified. These recombinant proteins were used to generate polyclonal antibodies in rabbits, and their efficacy was studied using <em>in vitro</em> adhesion inhibition assays. The combination of two or more antibodies raised against the recombinant OMPs was significantly effective in reducing/neutralizing bacterial binding to bovine endothelial cells compared to individual antibody treatment. This suggests that a multiple subunit vaccine could be effective and provide sufficient evidence to perform <em>in vivo</em> studies. </p>
<p>Similarly, we purified OMVs of <em>F. necrophorum</em> subspecies <em>necrophorum</em> 8L1 and analyzed its content using proteomics and lipidomics. Out of 342 proteins identified by tandem liquid chromatography mass spectrometry (LC-MS), OMPs and toxins were the most abundant. These included OMPs and toxins namely, 43 kDa OMP, OmpH, OmpA, CSP, FadA, leukotoxin family filamentous adhesin, N-terminal domain of hemagglutinin and other OMP transport and assembly factor protein. The presence of a subset of these proteins was further confirmed by western blot analysis. Lipidomics analysis showed that OMVs contained phospholipid, sphingolipid, and acetyl carnitine as the main lipid contents. Cytotoxicity assay on BL-3 cell line showed that these OMVs have a toxic effect on host immune cells and could impart immunomodulatory effect. All these findings suggest the vaccine potential of OMVs and demand dose-based <em>in vivo</em> study.</p>
<p>In addition, we identified and characterized 5 clinical isolates of <em>F. necrophorum</em> using comparative genomics, UBCG (Up-to-date Bacterial Core Gene) based analysis enabled phylogenetic characterization of 46 <em>F. necrophorum</em> genomes into subspecies specific clades. The pangenome and recombination analysis showed the extensive disparity in accessory genes resulting in species divergence. Strikingly, we detected antimicrobial resistance gene for macrolides and tetracycline in one strain of <em>F. necrophorum</em>, a harbinger of the start of resistance and necessitating search for an alternative prophylactic method. We also noted common virulence genes, including toxins, outer membrane adhesion proteins, cell envelope, type IV secretion system, ABC (ATP-binding cassette) transporters and transporter proteins in <em>F. necrophorum</em> strains. A focused study on these genes could help identify the main genes of virulence and inform effective vaccination strategies against fusobacterial infections. </p>
<p>Overall, the studies suggest adhesins and toxin and/or OMV-based subunit vaccine could be potential targets for vaccine development against fusobacterial infections. </p>
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APPLIED BACTERIAL ECOLOGY IN LIVESTOCK SYSTEMCarmen L Wickware (14003562) 26 October 2022 (has links)
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<p>Microbiome studies are varied and involve the examination of microorganisms at different levels: individual cells to determine individual functions, populations of specific microorganisms to determine interactions between organisms, and/or communities of microorganisms for a broader investigation of interactions between organism and environment. These studies are typically done within the context of a particular niche or environment. There are two parts to this dissertation, separated by the types of research involved. First, the analysis of bacterial communities using 16S rRNA sequencing and analysis. In this first part the bacterial communities of the reproductive tract of bulls and the gastrointestinal tract of weanling pigs were studied. The reproductive organs of the male, domestic species had not been studied from an ecological perspective prior to the study. As such, the research was mainly focused on characterizing the bacterial communities found within the prepuce of bulls that were considered to be healthy, or that the breeding soundness exam was satisfactory and the bulls had no clinical disease in the urogenital tract. Through this study two distinct types of bacterial communities were found based on the diversity of the observed taxa; the groups were split into a low diversity group identified by the presence of <em>Bradyrhizobium</em> and a high diversity group distinguished by the abundance of mucosal-associated bacteria found in oral, respiratory, and vaginal communities of cattle. Second, the effects of supplementary, soluble fiber on the intestinal bacterial communities of piglets pre- and/or post-weaning were studied. The rationale behind this study was to determine if pre-weaning fiber could alter the microbiome prior to weaning and the change of diet from liquid to solid. Pre-weaning, supplementary, soluble fiber was found to increase short-chain fatty acid concentrations and bacterial taxa potentially involved in their production. Additionally, bacterial taxa implicated in an increased inflammatory response were reduced in groups fed supplementary fiber. Taken together, the two bacterial community studies highlight the gaps in knowledge for reproductive communities in male animals as well as the potential for reducing weaning stress in pigs. Part two of this dissertation focuses on whole genome sequence analysis as a way to study bacterial populations associated with bovine respiratory disease (BRD), a common and potentially fatal disease in cattle. Identification of BRD has low accuracy and the presence of antibiotic resistant bacteria increases the chance of treatment failure. Using machine learning, the prediction of antibiotic resistance in bacterial isolates from animals with BRD was performed to find potential sequences for use in future molecular assays. While using known resistance genes was helpful for some antibiotics, several of the antibiotics used in treating BRD were better predicted using the machine learning models. Model output sequences should be further tested using molecular methods to determine function and importance before using as an assay target. Put together, the contents of this dissertation should serve as an introduction to bacterial ecology as well as how the concepts can be applied to food animal production systems.</p>
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Bridging the TB data gap: in silico extraction of rifampicin-resistant tuberculosis diagnostic test results from whole genome sequence dataNg, K.C.S., Ngabonziza, J.C.S., Lempens, P., de Jong, B.C., van Leth, F., Meehan, Conor J. 05 November 2019 (has links)
Yes / Background: Mycobacterium tuberculosis rapid diagnostic tests (RDTs) are widely
employed in routine laboratories and national surveys for detection of rifampicinresistant (RR)-TB. However, as next-generation sequencing technologies have
become more commonplace in research and surveillance programs, RDTs are being
increasingly complemented by whole genome sequencing (WGS). While comparison
between RDTs is difficult, all RDT results can be derived from WGS data. This
can facilitate continuous analysis of RR-TB burden regardless of the data generation
technology employed. By converting WGS to RDT results, we enable comparison of
data with different formats and sources particularly for low- and middle-income
high TB-burden countries that employ different diagnostic algorithms for drug
resistance surveys. This allows national TB control programs (NTPs) and
epidemiologists to utilize all available data in the setting for improved RR-TB
surveillance.
Methods: We developed the Python-based MycTB Genome to Test (MTBGT) tool
that transforms WGS-derived data into laboratory-validated results of the primary
RDTs—Xpert MTB/RIF, XpertMTB/RIF Ultra, GenoType MDRTBplus v2.0, and
GenoscholarNTM+MDRTB II. The tool was validated through RDT results of
RR-TB strains with diverse resistance patterns and geographic origins and applied on
routine-derived WGS data.
Results: The MTBGT tool correctly transformed the single nucleotide polymorphism
(SNP) data into the RDT results and generated tabulated frequencies of the RDT
probes as well as rifampicin-susceptible cases. The tool supplemented the RDT
probe reactions output with the RR-conferring mutation based on identified SNPs.
The MTBGT tool facilitated continuous analysis of RR-TB and Xpert probe reactions
from different platforms and collection periods in Rwanda.
Conclusion: Overall, the MTBGT tool allows low- and middle-income countries to
make sense of the increasingly generated WGS in light of the readily available RDT. / Erasmus Mundus Joint Doctorate Fellowship grant 2016- 1346.
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