Browsing by Author "Maughan, Michele Nancy"
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Item Molecular detection and identification of avian influenza viruses by cDNA microarray(University of Delaware, 2006) Maughan, Michele NancyAvian influenza (AI) is a respiratory viral pathogen of major concern to poultry producers and public health officials across the world. Rapid detection and subtyping of influenza viruses is necessary in order to control outbreaks and maintain routine surveillance. Microarray technology is a relatively new means of detecting pathogens and characterizing their genomic content. An avian influenza virus-specific cDNA microarray has been created and shown to correctly detect and identify the H5, H7, and H9 hemagglutinin subtypes, the N1, N2, and N3 neuraminidase subtypes, and the matrix gene of AI. ☐ Our prototype AI cDNA microarray contains 16 elements representing the matrix, hemagglutinin, and neuraminidase genes of avian influenza isolates and a negative control from the F gene of Newcastle disease virus. These elements are spotted in duplicate in four subarrays yielding 8 spots per element and 128 spots total. To validate our microarray, an unknown panel comprised of 10 avian influenza isolates was tested. Of the ten isolates, 100% (10/10) were correctly identified as type A influenza viruses and 70% (7/10) were fully subtyped by their hemagglutinin and neuraminidase genes. Further characterization of 100% (4/4) of the H5 isolates was accomplished by quantifying hybridization signal strength between the extensive phylogenetic representation of the H5 hemagglutinin gene sequences on our microarray and the H5 isolates in the unknown panel. ☐ Our results demonstrate the ability of a cDNA microarray to detect, identify, subtype, and phylogenetically/geographically group various avian influenza isolates. Our method as validated here can identify type A influenza via the conserved matrix gene, differentiate between the H5, H7, and H9 hemagglutinin subtypes, and differentiate between the N1, N2, and N3 neuraminidase subtypes of avian influenza. Furthermore, our AI cDNA microarray demonstrates the ability to determine the phylogenetic/geographic group from which an H5 hemagglutinin subtype originates based on hybridization signal strength. ☐ This method can be applied to clinical situations pending further validation experiments to determine the sensitivity of the array and increasing the number of representative HA and NA subtypes on the array (i.e. H1-4, H6, H8, and H10-16 and N4-9). Also, other viral and/or bacterial pathogens could be added to the array to increase its diagnostic power and aid the medical community in differential diagnoses.Item Pathogenomic approaches to characterizing the avian host innate immune response to microbial infection(University of Delaware, 2012) Maughan, Michele NancyThe avian innate immune response is activated within hours of infection. While it is difficult to prevent infection by a pathogen, clinical signs of disease can be ameliorated once the host-pathogen interactions are elucidated. Host-pathogen interaction research has predominantly focused on the adaptive immune response and cell signaling events later in infection; recently however, the innate immune response and early signaling events have garnered increased attention as another area worthy of investigation and intervention. Cells of innate immunity serve as the first-responders to infection, their signaling and antigen presentation is critical to the development of a protective adaptive immune response, and the cellular products of their activity (cytokines, reactive oxygen species, complement, etc.) are responsible for many of the clinical signs associated with disease. ☐ Avian immunology research is expanding quickly due to the growing knowledge base of the chicken cytokines, Toll-Like Receptors (TLRs) and their immune signaling pathways. Our aim was characterize the avian innate immune response to microbial infection by utilizing a pathogenomics approach. By performing microarray experiments using our Avian Innate Immune Microarray (AIIM), we were able to measure the transcriptional host immune response to several important avian pathogens. Furthermore, by performing immunotherapeutic interventions using TLR agonists prior to challenge with highly pathogenic avian influenza virus, we were able to extend survival time of treated birds by 14% (p<0.01). This project has led to the characterization of the avian innate immune in different avian species, to different pathogens, at early time points throughout infection, and with and without the aid of a pre-treatment.