Crohn’s disease (CD) is a chronic inflammatory bowel disease, primarily affecting the young, which causes marked morbidity and reduced quality of life. Currently there is no cure for CD, and the causes of this disease are poorly understood. In ruminants, Johne’s disease (JD) is characterised by chronic intestinal inflammation similar to CD and is caused by the pathogen Mycobacterium avium subspecies paratuberculosis (MAP), which invades and replicates within the phagocytes of infected animals, leading to chronic disease. There is increasing molecular and microbiological evidence of Map bacteria in CD patients. However, little is known regarding the role of Map in the aetiology of CD. This thesis demonstrated that a human isolate of Map traffics through THP-1 human monocytes via a similar path to that taken by pathogenic mycobacteria. Flow cytometry demonstrated that Map are phagocytosed via a cholesterol-dependant mechanism, potentially mediated by a cell wall constituent. Once internalised, live Map reside in cholesterol-rich areas of the cell. These compartments exhibit reduced acidity compared to the compartments containing killed-Map, and have atypical retention of markers including the late endosomal marker Rab 7 and cellular TACO protein. Both of these markers were also present on phagosomes of pathogenic mycobacteria, where they interrupt fusion of the compartment with lysosomes. This was confirmed by visualisation of these proteins on phagosomes containing M. bovis,a known mycobacterial pathogen. Cholesterol depletion using simvastatin affected Map persistence in THP-1 cells at 1 and 2 weeks post infection, a finding similar to other studies with M. tuberculosis. Spheroplast-like forms were evident after long term culture of Map with THP-1 monocytes, visualised by light and electron microscopy. These were similar to forms observed in peripheral blood leukocytes from a CD patient. Collectively, these results support the hypothesis that Map may be involved in the aetiology of at least a subset of CD cases.

Graduation date: 2005

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / "August 1996" Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (PH.D.)--Michigan State University. Large Animal Clinical Sciences, 2008. / Title from PDF t.p. (viewed on Aug. 28, 2009) Includes bibliographical references (p. 260-281). Also issued in print.

To advance crop monitoring techniques in horticultural tree crops, earlier research has examined the relationship between crop vigor (height, canopy den- sity, health) as assessed by remote sensing technologies and aspects such as fruit quality and yield requirements. In recent years, structure-from-motion image pro- cessing techniques have been widely used to generate orthomosaics and 3D point clouds from RGB and multispectral (MS) imagery acquired by unmanned aerial vehicles. However, this process requires a lot of computing power and can be expensive, especially for large commercial orchards. However, studies have been scarce comparing the accuracy of different remote sensing technologies in deter- mining tree height and plant area index. Light detection and ranging (LiDAR) is an alternative method to generate 3D point clouds that requires less compu- tational power. This study assessed the accuracy, processing parameters, and limitations of UAV-based RGB, MS, and LiDAR data for measuring cherry trees’ height and plant area index in a high-density orchard in Malauc`ene, southeastern France. Furthermore, the plant area index changes of 5 different cherry culti- vars were assessed during the growth cycle. Overall, the LIDAR data provided the highest accuracy for tree height measurements around harvest (R² = 0.923, RMSE = 0.215 m) and the beginning of leaf senescence (R² = 0.863, RMSE = 0.218 m). LiDAR-derived plant area index also produced the best accuracy at May (R² = 0.48 and RMSE = 0.42) and October (R² = 0.45 and RMSE = 0.59). Our findings demonstrate that UAV-based LiDAR data provide an effective and rapid means for measuring cherry tree height and plant area index over time. Such information can serve as a general indicator of tree health and aid growers in making informed agricultural crop monitoring and management decisions.

Mycobacterium avium isolates resistant to clarithromycin and azithromycin have been recovered from patients undergoing antibiotic therapy. Comparison of DNA fingerprints of sensitive and resistant isolates showed that resistance resulted from mutation of the original, sensitive isolate in five of seven patients. In the other two patients, the clarithromycin-resistant isolates were unrelated to the sensitive isolate, suggesting that the resistant isolate resulted from either superinfection or selection of a resistant strain from a polyclonal population. Investigation of the mechanisms of clarithromycin and azithromycin resistance in M. avium showed that high-level resistance resulted from a point mutation at position A-2058 in the 23S rRNA. Based on this finding, a rapid screen for clarithromycin-resistance in M. avium was developed based on PCR. Twenty-three clinical isolates were analyzed, seven of which were clarithromycin-resistant. The target product was amplified only in clarithromycin-resistant strains, all of which had mutations at position 2058. A polyuridylic acid (poly U)-dependent in vitro translation system from M. avium was developed to investigate the effect of antibiotics on protein synthesis. Clarithromycin was an effective inhibitor of protein synthesis in cell-free extracts of a susceptible M. avium strain, whereas a high-level resistant strain was less susceptible to clarithromycin in vitro. Mixtures of extracts from sensitive and resistant strains showed a pattern of clarithromycin inhibition similar to the resistant strain, suggesting that resistance may be dominant in partial diploids. Three M. avium strains exhibiting step-wise, intermediate resistance to azithromycin were characterized in comparison to the sensitive parent. All strains were similar in hydrophobicity, growth medium requirements, and growth response to temperature. The azithromycin-resistant strains were resistant to several unrelated agents, including ciprofloxacin, rifabutin, and ethidium bromide. Addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) did not lower minimal inhibitory concentrations (MICs) for ciprofloxacin or ethidium bromide. Cell-free extracts of the strains were as sensitive to azithromycin in vitro as the parent strain. The results rule out inactivation, efflux, and mutations in the target as resistance mechanisms, and suggest intermediate resistance may be due to altered permeability of the cell wall or membrane. / Ph. D.

Clofazimine is a member of the phenazine pigment family which has been successfully used in chemotherapy against a variety of mycobacteria including M. avium. The presence of clofazimine in growth medium resulted in higher carotenoid pigmentation in M. avium cells. Carotenoid pigments have been shown to quench superoxide radicals supporting the hypothesis that pigmentation possibly protected cells against superoxide. Clofazimine caused the generation of superoxide radicals in M. intracellulare strain LR163 represented by cyanide-resistant oxygen consumption. The amount of oxygen consumed was dependant upon the clofazimine concentration. This supports the hypothesis that clofazimine is antibiotic via its ability to generate toxic oxygen metabolites. Higher catalase activity was found in extracts of cells grown in the presence of a low concentration of clofazimine. At a higher concentration, the amount of catalase and superoxide dismutase activities were lower than the basal level. This finding did not agree with the hypothesis. At this point the reason for the drop in the activities (i.e. lower than basal level) is not known. Clofazimine was mildly synergistic with rifampicin. This result supports hypothesis that the defense mechanism of M. intracellulare to clofazimine was enzymatic. Clofazimine-resistant derivatives of M. intracellulare strain LR163 have been isolated. Their characterization will provide a direct approach towards determining the mode of action of clofazimine in cells. / Master of Science

Background: Crohn's Disease (CD), a chronic inflammatory bowel disease (IBD), is thought to be multifactorial, involving an interaction between genetic susceptibility, undefined environmental triggers, and immune-mediated tissue injury. Biochemical and other molecular approaches identified isolates from intestinal tissues of patients with CD as Mycobacterium avium subsp paratuberculosis (MAP), the causative agent of Johne's disease, a granulomatous bowel disease in ruminants similar to CD. MAP has been identified directly in resected tissues of increasing numbers of CD patients at a frequency significantly higher than those of controls. Treatment of CD patients, which depends on the location and severity of disease, complication, and response to previous treatment is most often to control the disease. There is no cure. Diagnosis of this disease requires a series of tests including upper gastrointestinal endoscopy and colonoscopy. These tests are expensive, inconvenient and require hospitalization. Objective: A blood serologic test is sought for diagnosis of CD patients infected with MAP. Methods: The recombinant E. coli clone pBl 1 containing a 1,302 bp MAP DNA insert and expressing a 20 kD protein has been grown, induced by arabinose and then harvested by centrifugation. Protein extracts were prepared, quantitated and then subjected to Isoelectricfocussing (IEF) in ampholyte buffer pH 3-10. Twenty fractions were collected, quantitated and then analyzed on SDS-P AGE by silver staining and Imrnuboblotting. The immunoblots were screened with anti-express IgG monoclonal antibodies. Fractions containing the semipurified 20 KD protein were analyzed by immnoblot against 85 sera specimens with 1:30 dilution (43 CD patients and 42 controls). Both IgG and IgA response in each patient was determined. Results: Of 20 fractions collected, fractions 5 and 6 with a PI ranging from 4.18 to 5.01 reacted with the anti-express IgG antibodies. p20 with a 20 kD molecular weight was confirmed. These fractions contained fewer proteins bands with p20 being dominant. Of 43 CD sera specimens, 74% contained IgG response and only 50% contained IgA response to p20. On the other hand, of 42 controls, only 17% contained IgG and. 50 % contained IgA response.against p20 antigen. Conclusion: p20 reacted with CD IgG sera with frequency much higher than control sera (74% versus 17%) indicating a great potential for using p20 as a reagent in a quantitative ELISA assay for specific diagnosis of CD patients. Additionally, the data add strong support to MAP role in CD pathogenesis.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) has been suggested as a causative agent of Crohn's disease (CD). Despite a long-term debate to prove this possibility, the role of this bacterium in the pathogenesis of CD is still a subject of controversy. The objective of the study was to develop a serodiagnostic assay for the diagnosis of CD in humans. METHODS: In the present study, five different ELISA assays were accessed: 1) IDEXX, a commercially available kit for the diagnosis of Johne's disease in ruminants; 2) an in-house developed assay based on total MAP cytoplasmic proteins, and three other assays based on recombinant MAP recombinant antigens a) a 23 kDa antigen, pB11/B7, b) a 35 kDa antigen, P35 and c) a 36 kDa antigen, P36. The last three proteins were identified from an expression genomic library of MAP that was constructed in our laboratory. A total of 43 sera samples were analyzed in this study, which included 14 CD patients, 14 Ulcerative Colitis (UC) patients, and 13 non-inflammatory bowl disease (IBD) patients. lmmunoblot and silver stain analyses were performed to confirm protein identity and purity. ELISA was developed and used to analyze the level of anti-MAP lgG antibodies in sera from patients. RES UL TS: The rate of positive ELISA results is based on previously published interpretation criteria. ELISA results using the IDEXX kit showed 12/14 (85.7%) positive for CD as compared to 7/13 (53.8%) for non-lBD and 6/14 (42.9%) for uc.· 8/14 (57.1%) of the CD sera were positive with the ELISA results based on MAP cytoplasmic proteins compared with 6/13 (46.2%) of non-lBD and 10/14 (71.4%) of UC. Further analyzing the recombinant proteins, when two out of three assays were used 12/14 (85.7%) CD (P<0.05), 0/13 (0.0%) non-lBD, and 1/14 (7.7 %) UC were positive. Moreover, when all three recombinant proteins are utilized for analysis, the specificity of the test greatly increased, giving 13/14 (92.9%) positive for CD, 3/14 (21.4%) for UC and 2/14 (14.3%) for non-lBD. CONCLUSION: MAP recombinant proteins, pB11/B7, p35, and p36 showed a strong reactivity with diagnosed CD patients while excluding healthy individuals and other IBD patients. In addition, they served as a great tool to distinguish between CD and UC patients. A larger sample size needs to be tested, none the less this data strengthens the role of MAP in CD etiology and suggests a great potential for using the recombinant-based assays for diagnosis and treatment monitoring of patients with inflammatory bowel disease.