Currently, about one third of the world's population is latently infected with Mycobacterium tuberculosis and about 4 million people die from the disease annually worldwide. Although treatment with antimicrobials can be curative, many people fail to complete the prescribed therapeutic regimen which can increase the risk of disease re-emergence, spread of infection to others and development of drug resistance. An improved approach is urgently needed for patient compliance. Development of safe and effective colloidal drug delivery systems may reduce the amount and frequency of antimicrobial therapy needed. The major goal of this research effort is to explore the safety and efficacy of antimicrobial loaded nanoparticles against M. avium. Various in vitro efficacy studies were done with a) amikacin-loaded nanoparticles, b) clarithromycin-loaded nanoparticles, and c) with aerogel nanoparticles loaded with rifampicin, clarithromycin and ethambutol. Clarithromycin (CLA) and amikacin (AMK) loaded nanoparticles showed a significant reduction in viable M. avium compared to free antibiotics and untreated controls. Cytotoxicity assays revealed that all types of drug-laden nanoparticles were non-toxic to J774A.1 mouse macrophage cells at therapeutic doses. In vivo efficacy studies showed that only amikacin-loaded polymeric nanoparticles improved clearance compared to free amikacin in M. avium infected BALB/c mice. In general, none of the nanoparticle formulations elicited any significant microscopic lesions in the organs of infected mice at tested doses. Each nanoparticle formulation was analyzed physicochemically for size, zeta potential, amount of drug load, minimum inhibitory concentration (MIC) and stability. Both the AMK and CLA polymeric nanoparticles were below 200 nm in size and had a slightly negative overall surface charge, aerogel nanoparticles were somewhat larger in size. The amount of drug load varied between all three nanoparticles and is largely dependent on the chemical structure and interactions between the nanoparticle and drug. The AMK and CLA nanoparticles were relatively stable under varying environmental conditions and time points and had MIC ranges equivalent to the respective free drugs. / Ph. D.

Mycobacter avium complex serovars 4 and 20 were cultured in the presence of [3H] fucose, [3H]-methionine, and [3H]-mannose to specifically radiolabel the oligosaccharide of the glycopeptidolipid (GPL) antigens. Distribution of radioactivity in lipid was determined by thin-layer chromatographic methods. Examination of acid hydrolysates from radiolabeled antigens revealed that [3H]-methionine incorporated into methylated sugars in polar and apolar GPL components, whereas [3H]-mannose incorporated exclusively into the oligosaccharide of polar GPL antigens. Least incorporation of radiolabel into antigens was observed with [3H]-fucose. Use of radiolabeled serovar 4 antigens in macrophage uptake studies revealed maximum uptake to be slightly above 250 gg/ 3.2 x 105 cells. Timed experiments demonstrated that GPL antigens were relatively inert to degradation by resident peritoneal macrophages.

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Thesis (Ph. D.)--Ohio State University, 2005. / Document formatted into pages; contains xxi, 216 p. Includes bibliographical references. Abstract available online via OhioLINK's ETD Center; full text release delayed at author's request until 2006 March 9.

Thesis (M.S.)--Michigan State University. Dept. of Large Animal Clinical Sciences, 2008. / Title from PDF t.p. (viewed on July 29, 2009) Includes bibliographical references. Also issued in print.

Memoria para optar al Título Profesional de Ingeniero Agrónomo Mención: Fruticultura / En la Zona Sur de Chile los cerezos han ido desplazando cultivos tradicionales con variedades de maduración tardía, de las cuales ‘Kordia’ y ‘Regina’ son las variedades autoincompatibles más importantes y de mayor interés comercial, tanto por su tolerancia a la partidura, buena firmeza y calidad organoléptica, como por su cosecha tardía, alcanzando los mejores precios en los mercados internacionales. En el Sur de Chile y en particular en la localidad de Angol estas variedades han presentado bajos rendimientos, esto debido, en parte, a la falta de polinizantes que coincidan en las floraciones y que sean compatibles. Debido a la falta de información de estas variedades en el país se hace necesario determinar polinizantes apropiados para incluirlos en los huertos comerciales y en los diseños de nuevas plantaciones con el fin de aumentar los rendimientos. El objetivo de este estudio fue determinar la intensidad del cuajado de frutos de ‘Kordia’ y ‘Regina’, polinizadas con distintas variedades compatibles en la localidad de Angol (IX Región). Se realizaron tres ensayos, uno para ‘Kordia’ y dos para ‘Regina’ donde se utilizaron como polinizantes las variedades Sam, Staccato, Summit, Sunburst, Schneider y además Kordia y Regina entre sí, donde se evaluó la intensidad del cuaje, la concordancia de los estados fenológicos y la viabilidad del polen utilizado. ‘Sunburst’ se comportó como un buen polinizante de ‘Kordia’ con un 37% de cuaje y con una coincidencia completa en la floración, presentando diferencias significativas con ‘Sam’ (4,9% de cuaje). Otras variedades tuvieron coincidencias completa y parciales en la floración, pero con bajo porcentaje de cuaje. En ‘Regina’, las variedades utilizadas presentaron una coincidencia parcial con la floración y un cuaje bajo a muy bajo, no presentando diferencias significativas entre los tratamientos.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis that has a damaging economic and welfare impact on the livestock industry. Johne's disease in cattle is known to reduce milk yield and carcass value, making it of economic concern to both dairy and beef farmers. In addition, there is cause for concern regarding zoonotic transmission, as there is an unconfirmed but potential relationship between MAP infection and human Crohn's disease, which presents similar clinical symptoms. MAP is most often contracted by neonates through the faecal-oral route, but can also be spread through contact with contaminated milk and colostrum, as well as in utero. Once the host receives an oral dose, the bacteria traverse the gut epithelium and are phagocytosed by gut macrophages residing in the lamina propria and Peyer's patches. MAP are able to evade the macrophage response by resisting intracellular degradation within phagosomes. Infected macrophages respond to the infection by secreting several pro-inflammatory cytokines that drive the downstream immune response and granuloma formation. This work aimed to elucidate key early responses of bovine monocyte derived macrophages (MDM) to MAP infection, and determine the reliability of using the reference strain, K10 (which is likely to have undergone lab adaptation) to model the infection in vitro, by comparing the MDM response to K10 with the response to a recent clinical isolate, C49. At a multiplicity of infection of 5 (MOI 5), there was a significant decrease in K10 intracellular survival (~90%), compared to C49 intracellular survival, over a 24 hour infection time-course. This suggests that K10 may have lost some virulence mechanism through lab adaptation. Understanding the mechanisms of how MDM respond to these two strains could be informative for the design of targeted vaccines When further investigating the MDM response to both strains, it was found that, at MOI 5, MDM infected with K10 secreted higher levels of IL-1β and IL-10, compared to MDM infected with C49. Both cytokines are associated with mycobacterial infection and could perhaps indicate that MDM are more responsive to the K10 strain at early time-points. In addition, MDM infected with K10 produced significantly higher levels of reactive nitrogen species (RNS). RNS are antimicrobial products that can destroy invading pathogens, and have been shown to have bactericidal effects on MAP. The production of RNS could, therefore be a potential mechanism by which MDM are able to kill K10 more efficiently than C49. An additional aim of this project was to understand the importance of the route of phagocytosis in determining the outcome of MAP infection. MDM express several phagocytic receptors, including Fc receptors (FcRs), complement receptors (CR), Ctype lectin receptors and scavenger receptors. This project mainly focused on the role of the mannose receptor (MR) on bacterial uptake and downstream immune responses, as past studies have suggested that other species of mycobacteria such as M. tuberculosis, target the mannose receptor in order to regulate macrophage immune responses. Blocking the MR reduced intracellular survival for both strains of MAP; however, the mechanism by which the MR influences intracellular survival remains poorly understood The effect of opsonisation on MAP prior to uptake by phagocytic cells was also investigated, as presence of opsonins, such a complement proteins and antibody, can change the mechanism by which pathogens are phagocytosed. MAP were incubated in serum from either MAP- negative or MAP- positive cattle, prior to infection and the percentage uptake and survival assessed by performing colony counts. Opsonisation in serum from Johne's negative cattle resulted in marked increase in MAP uptake but not intracellular survival, whereas opsonisation in serum from Johne's positive cattle did not increase uptake but decreased the intracellular survival rate by 24 HPI. This finding highlights a potential protective role of antibody early in the infection process, and could significantly impact how the infection is modelled in future, as anti-MAP antibody may be present in contaminated milk at the point of infection. Taken together, the data presented in this thesis show that bacterial strain has a significant impact on MDM response to MAP infection, which may have important implications for the interpretation of previous studies and the design of future studies investigating host-pathogen interactions in the context of paratuberculosis. Additionally, this work has shown that RNS production and the mechanism of uptake can affect intracellular survival rates, and although this needs further investigation, the findings could have implications for the design of future vaccines.

Memoria para optar al Título Profesional de Ingeniero Agrónomo Mención Fruticultura / En algunas localidades de la Zona Central de Chile, cuando la disponibilidad de frío invernal ha sido insuficiente para el cerezo, se han observado problemas como desuniformidad, extensión y retraso de la brotación, lo cual afecta negativamente el desarrollo de la planta, el manejo de los huertos y también el rendimiento. Para evitar o minimizar estas consecuencias se realizan aplicaciones de cianamida hidrogenada (CH), para promover artificialmente la salida del receso. El algunos casos, aún cuando el frío es suficiente, la CH también puede ser usada para adelantar y/o concentrar la brotación. Sin embargo, el éxito de esta aplicación depende del frío acumulado previamente, de la concentración utilizada y de las condiciones térmicas durante y después de la aplicación. Con el objetivo de determinar el efecto de la acumulación de frío previo a la aplicación de CH y de la exposición a bajas temperaturas con posterioridad a la aplicación, se estableció un ensayo de campo en cerezos ‘Bing’ con aplicaciones de CH (Nexus®) al 1,5% i.a. en 3 oportunidades, definidas según la acumulación de unidades frío Richardson (UF): 1100; 1200 y 1300 UF aproximadamente, más un tratamiento de 1% CH a los 1300 UF y un control sin aplicación. Posteriormente, se sometieron ramas con madera de uno y dos años a 5 °C hasta acumular 100 y 200 UF; y luego fueron llevadas a una cámara de crecimiento en condiciones de temperatura y luz controladas, para estimular la brotación. Se evaluó la dinámica de la brotación en campo y en cámara. El frío acumulado antes de la aplicación de CH (1,5%) no concentró la brotación, mostrando un patrón similar en todos los tratamientos. Sin embargo, la CH aplicada a las 1200 UF anticipó la brotación aproximadamente 7 días con respecto al tratamiento control, reduciendo también los requisitos de calor desde la aplicación hasta la brotación. Por otro lado, la aplicación tardía (1300 UF) de CH al 1,5% fue inefectiva en el adelanto de la brotación, sin embargo, cuando fue aplicada a una concentración del 1%, se produjo una disminución de los requisitos de calor, siendo similar al tratamiento de CH (1,5%) aplicado a las 1200 UF. Se pudo constatar que el frío posterior a la aplicación de CH al 1,5% tiene un efecto positivo en reducir los requisitos de calor para la brotación, pero resulta ser negativo cuando la acumulación de frío previo a la aplicación de CH es de 1300 UF.

Creation of a reliable and easy to use serologic test would greatly improve ante mortem diagnosis of Mycobacterium avium subsp. avium and aid in the control of avian mycobacteriosis, particularly in captive birds. In order to determine whether serodiagnostics could be of value in testing ring-neck doves (Streptopelia risoria) for M. a. avium infection, Western blot analysis was used to assess the humoral response of ring-neck doves exposed to M. a. avium, and to evaluate whether an association could be made between humoral response and necropsy findings, histopathology, culture, and PCR testing. Western blot results were examined for reactivity patterns associating the humoral response with infection status, severity and type of lesions (diffuse vs multifocal granulomatous inflammation) and phenotype (white vs non-white). A sensitivity of 88.24% and a specificity of 100% were achieved utilizing Western blot analysis to detect M. a. avium infection in ring-neck doves, offering a negative predictive value of 93% and a positive predictive value of 100%. While Western blot analysis results did not reflect lesion severity, lesion type did partially correspond with the humoral response. The findings of the present study indicate that serologic testing can be used as a valuable ante mortem screening tool for identifying ring-neck doves infected with M. a. avium.