Investigation of Rapid Diagnostic Tests for Characterization of Mycobacterium Avium Complex (MAC) from various isolates and Identification of Virulence Factors of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Vitro
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Mycobacterium avium complex (MAC) has become one of the major problems in public health and livestock. Members of MAC, such as M. avium subsp paratuberculosis (MAP) and M. avium subsp hominissuis (MAH), are responsible for many opportunistic infections and the loss of livestock. MAP is economically significant to the beef and dairy industries because it is the etiologic agent of Johnes’s disease, a chronic and fatal enteritis in ruminants. Tracing the infection sources of MAC could be difficult since it infects many types of hosts in the natural environment. Furthermore, there is less information known about MAP pathogenicity in specific hosts, but some strains often predominate in certain hosts. Being able to differentiate subspecies of MAC, as well as to trace MAP host species of origin, would be important tools for researchers to assess the potential pathogenicity, transmissibility, and environmental persistence of the strains to limit outbreaks. Thus, this study aimed to characterize M. avium subspecies and to identify MAP host species of origin using MALDI TOF MS (Matrix Assisted Laser Desorption/Ionization- Time of Flight Mass Spectrometry) library and FT-IR (Fourier Transform-Infrared Spectroscopy) biomarker based proteomic approaches. MALDI-TOF MS provided more sensitive characterization of MAC subspecies through matching mass fingerprint of isolates with a custom main consensus library (MSP). The manufacturer’s database (BDAL) discriminated 14 out of 47 MAC isolates (29 %). Our new data database (MSP) could identify 80% of the total isolates (40/47). Our Kappa Analysis was 0.73, signifying good agreement between the PCR and MALDI-TOF results. The FT-IR offered visualization of unique IR spectra that could be analyzed based on the cell wall components present in the cell, such as mycolic acid and peptidoglycan. Both instruments could successfully classify each subspecies of MAC, but it failed to track the host origin of species of MAC.
Porcine reproductive and respiratory syndrome (PRRS) is a major viral disease responsible for huge economic losses and morbidity in the swine industry. Clinical signs of PRRS include chronic respiratory disease among young pigs and reproductive failure of pregnant sows. The etiologic agent of PRRS is a member of Arterivirus, and known as PRRS virus (PRRSV). There are two types of PRRSV circulating worldwide, classified as PRRSV-1 (European) and PRRSV-2 (North American). Genetic variations among PRRSVs are common as their RNA genome is prone to mutation. Multiple types of vaccines have been created, including a synthetic vaccine candidate (PRRSV-CON) which can provide broader protection against the two types of PRRSV (Vu et al., 2015). Nevertheless, the mechanism of how PRRSV provides immune protection remains unclear regarding which viral proteins trigger optimum immune responses. Hence, in this project, our goal was to determine the relative contribution of PRRSV-2 structural proteins located in Open Reading Frames (ORFs) 2-7 to immune responses. We used PRRSV-1 as a backbone for our construct and inserted PRRSV-2 structural proteins. Our construct was not successfully recovered in the cell culture, possibly because the replacement of ORF2-7 from a divergent genotype may interfere with viral replication.
Faculty Advisors: Dr. Hiep Vu, Dr. John Dustin Loy, Dr. Sarah Sillman,