Browsing by Subject "Diagnostics"

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    Epidemiology of Mycoplasma hyorhinis in U.S swine production systems
    (2014-08) Clavijo Michelangeli, Maria Jose
    Mycoplasma hyorhinis has recently emerged as one of the main concerns of swine veterinarians dealing with post-weaning morbidity and mortality. Since the introduction of PCR in 2009, M. hyorhinis is detected in about 50% of polyserositis and 12% of arthritis cases submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) every year. In the field, swine practitioners are applying different antibiotic and autogenous vaccine protocols with varying degrees of success. Part of the minimal success is due to the lack of essential epidemiological information, which is scarce and obsolete. The main goal of this dissertation was to advance the knowledge on the infection dynamics and the epidemiology of M. hyorhinis in swine populations in the US. Such information will contribute to the better implementation of control and prevention strategies for this pathogen. The lack of accurate and thoroughly evaluated diagnostic tools represents a major limitation to study the dynamics of M. hyorhinis infection in swine populations. A high-throughput, sensitive and specific quantitative PCR was developed for the detection of M. hyorhinis DNA in diverse clinical samples. The qPCR showed suitable precision within and between runs. The qPCR will be a valuable tool to characterize infection dynamics and to quantify bacterial load in order to evaluate selected intcccerventions targeting M. hyorhinis. Essential information, such as the prevalence of pigs in commercial populations is absent. The prevalence of M. hyorhinis infection was estimated in different age groups across three commercial swine populations by means of a cross-sectional study. The results consistently showed an extremely low prevalence in the sows and suckling piglets. The prevalence of nasal infection after weaning increased in two distinct patterns. In two herds a high proportion of PCR-positive pigs were detected shortly after weaning while in the third herd the prevalence of nasal infection remained extremely low until the last week in the nursery when all pigs tested PCR-positive. In addition, this study provided the opportunity to confirm the role of M. hyorhinis as a cause of systemic disease. While M. hyorhinis was detected in the upper respiratory tract of both healthy and diseased pigs, it was only detected in systemic sites of diseased pigs and frequently associated with the typical lesions of fibrinous serositis. Information related to M. hyorhinis transmission and spread within infected populations is largely unknown. The prevalence and incidence of M. hyorhinis in naturally infected pigs and the relationship between sow infection and serological status with piglet infection was evaluated through a longitudinal study. While the prevalence of M. hyorhinis infection observed in suckling piglets and sows was low (<5%), over 90% of the pigs became colonized during the nursery phase. Moreover, the incidence rate estimations demonstrated a relatively high number of pigs acquiring the microorganism within a short period of time, indicating that a rapid transmission occurred after placement in the nursery.The development of an MLST-s typing scheme for epidemiological and genetic characterization of M. hyorhinis field isolates was described. This study demonstrated genetic variation within M. hyorhinis strains circulating in US swine production systems. While no clear relationships were observed between ST and state of origin, sample type and pig age, the majority of isolates from each system clustered together. The application of the MLST-s protocol described will allow studying M. hyorhinis epidemiology and dynamics of infection in U.S swine herds. Molecular typing will also aid in better understanding transmission routes, in assessing sources of infection and in evaluating interventions such as vaccination and use of antibiotics.The information generated in this thesis provided essential M. hyorhinis epidemiological information applicable to modern US swine production systems. Forthcoming M. hyorhinis research could focus on identifying risk factors for pathogen transmission and disease manifestation. The tools developed, evaluated and presented here can aid in such studies. Moreover, new information on the prevalence and incidence of infection could be used to better target control measures such as antibiotic and vaccine administration.
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    Laser Induced Gold Nanoparticle Heating: Thermal Contrast in Lateral Flow Immunoassays
    (2014-06) Qin, Zhenpeng
    Nanomaterial research has grown exponentially for biomedical applications in imaging, diagnostics and therapeutics. Within these areas laser nanoparticle heating uniquely enables important applications including molecular delivery or destruction, endosomal release of genes or siRNA, and selective cell or tumor destruction, with nano to macro-scale spatiotemporal control and precision. While our studies were initially motivated to support in vitro and in vivo biomedical applications, further study of laser nanoparticle heat transfer at a fundamental level, suggested a further opportunity for use in point-of-care diagnostics, in particular for gold nanoparticle (GNP) based lateral flow assays. The lateral flow immunoassay (LFA) is a point-of-care diagnostic test that has found broad applications in medicine, agriculture, and over-the-counter personal use such as for pregnancy testing. Within the LFA, antibody-coated GNPs are used as reporters due to accumulation (i.e. antigen-antibody recognition) on a test line that leads to a visually detectable signal due to a deep red color indicative of GNP accumulation. However, one universally recognized limitation of LFA is the low sensitivity of this visual readout. In this work, we developed a low cost solution to this sensitivity using laser GNP heating. Specifically, metallic nanoparticles generate heat upon optical (i.e. laser) stimulation. This in turn can be used to enhance detection of GNPs, creating a thermal contrast amplification (TCA). We have shown that TCA improves the analytical sensitivity on several existing, commercial LFAs. For instance, our results show a 32-fold improvement in analytical sensitivity using an FDA-approved cryptococcal antigen LFA. My dissertation then describes the development of TCA devices and components that will ultimately allow clinical, laboratory and eventually lay people alike to use and benefit from the technology. In particular, a benchtop TCA prototype device is described and engineering efforts continue to place TCA in a number of clinical laboratories and eventually create a product for infectious disease detection. The absorption and heat generation from nanoparticles eventually determine the magnitude of TCA. Thus, the use of higher heat generating nanoparticles can further improve the LFA sensitivity. Literature suggests that nanoparticle morphology plays an important role in the optical absorption, and nanorods absorb more light energy than nanospheres with the same amount of gold per particle based on previous calculations. Our study suggests that the optical absorption and extinction of gold nanorods are significantly reduced (more than 70%) by polydispersity (i.e. distribution of size and shape) while relatively unaffected for gold nanospheres (less than 10% change). This indicates that the expected enhancement due to absorption of gold nanorods over nanospheres may be greatly diminished by the presence of polydispersity in real nanoparticle samples. Further work incorporating higher heating gold nanoparticles (i.e. larger gold spheres and well characterized gold nanorods) to improve existing lateral flow assays such as to one day rival the sensitivity of more costly, time and labor intesive laboratory testing is underway. In summary, this dissertation describes the foundation of a new technology entitled: Thermal Contrast Amplication (TCA). TCA has been patented and licensed by a start up that is pursuing commercialization and broader societal impact of the technology. Future work including the development of a handheld TCA device for point-of-care diagnostics, and a next generation LFA optimized for TCA are underway. TCA and LFA together represent a potential disruptive platform technology that can improve early diagnosis of infectious diseases and general biomolecular screening in areas of medicine, agriculture, and biodefense where a quick and sensitive detection is needed.