No description available.

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.

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.

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.

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.

Lic.-avh. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 2 uppsatser.

Paratuberculosis, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a chronic wasting disease mainly of domestic and wild ruminants. It occurs worldwide, causing significant economic losses through decreased productivity, low fertility, increased cull rates and mortality. It is listed by the OIE (World Organization for Animal Health) as a disease of concern to trade in animals. Prevalence of this disease can be studied by detecting anti-MAP antibodies by Enzyme linked immunosorbent Assay (ELISA). The aim of this study was to investigate the current prevalence of MAP infection in cattle in Khartoum State. The overall apparent prevalence of MAP infection was found to be 6.3% and 18.9% at animal and herd levels, respectively. All seropositive animals were cross-bred females of good body condition; most of them (>90%) were >3 years old and >50% were from medium-sized herds in Omdurman. No significant association (p > 0.05) was found between seropositivity and animal herd size. The prevalence of MAP infection in Khartoum State is still low to medium compared to other parts of the world, but it is comparable to those reported from other African countries. Further studies with the view of designing nationwide surveys in domestic ruminants and camels in other states of the country are needed for establishing control programmes.

No description available.

Crohn's disease (CD) is a chronic granulomatous inflammatory bowel disease involving Mycobacterium avium subspecies paratuberculosis (MAP). Other microorganisms such as adherent-invasive Escherichia coli (AIEC) have also been proposed in CD association. To date, only one study investigated both MAP and AIEC simultaneously using peripheral blood but not in affected intestinal tissues. A standardized and effective antibiotic therapy against MAP and/or AIEC is needed for better treatment. Three antibiotic drugs – Clarithromycin (CLA), Rifabutin (RIF), and Clofazimine (CLO) have been used to treat CD patients suspected with MAP infection. However, the outcome has been controversial. The treatment dosage is high, the duration is long, and the reported drug side effects resulted in patient non-compliance; therefore, a lower and effective drug dosage is needed. In this study, we developed two aims 1) to evaluate RHB 104, a drug formula comprised of low dosages of CLA, RIF, and CLO, against clinical MAP strains in-vitro using fluorescence quenching method, and 2) to develop a fluorescence in-situ hybridization method to detect both MAP and AIEC simultaneously in intestinal tissues of CD patients. A total of 16 clinical MAP strains and 19 non-MAP strains were tested against varied concentrations of RHB 104, CLA, RIF, and CLO. Although the MIC for all drugs ranged between 0.5-20 ?g/ml, the MIC for RHB 104 was significantly lower against most MAP strains. The effect of RHB 104 against MAP was bactericidal. Unlike RHB-104 formula, CLA, CLO, and RIF dosage similar to those in RHB-104 did not inhibit MAP growth when trialed individually and in dual-drug combinations. The data illustrated the presence of synergistic anti-MAP activity of low dosage of the three antibiotics in RHB-104. We also developed a rapid and sensitive multicolor in-situ hybridization technique that can detect MAP and AIEC using tagged-oligonucleotide probes. Non-pathogenic Escherichia coli (npEC) was used as a control for the study. Specifically, cultured MAP and npEC were fixed and hybridized with MAP488 and EC647 probes, respectively. Confocal laser scanning microscope (CLSM) revealed specific signals at 488nm for MAP and 647nm for npEC, indicating probe binding to each bacteria. This was confirmed with hybridization of MAP with EC647 and npEC with MAP488 resulting in absence of signals. Intestinal tissue samples from 9 CD patients were then analyzed using our technique. Preliminary data indicated positive results in 6/6 samples for MAP, 6/6 for npEC, 3/3 for AIEC, and 2/2 for both MAP and AIEC with MAP being more dominant. This protocol shortened the FISH procedure from multiple days to short-hours. The protocol allows the investigation of more than one pathogen simultaneously in the same clinical sample. A quantitative measurement of the signals is needed.