Zugl.: Giessen, Univ., Diss., 2009

Johne�s disease in ruminants is caused by the pathogenic bacterium Mycobacterium avium subspecies paratuberculosis. An experimental infection model in sheep was developed as a prelude to the testing of new vaccines and the development of improved diagnostic assays for Johne�s disease. The final challenge model developed used four doses of 10⁹ viable organisms given at two to three day intervals. Gross and microscopic lesions were found in a high proportion of sheep (80%) at ten months post challenge. There was considerable variation in immune responses from animals challenged with different strains of M. paratuberculosis. Sheep challenged with a low passage laboratory culture of strain (W) M. paratuberculosis, produced strong lymphocyte transformation responses and Interferon gamma (IFN-γ) production at two months post challenge. Subsequent necropsy and culture from intestinal tissues showed only a low level of infection (25%). In comparison a primary tissue isolate of M. paratuberculosis (JD3) resulted in higher (60-90%) infection rates in orally challenged animals. The immune profile from these animals showed very little reactivity for the first three months post challenge, after which IFN-γ production could be detected. Antibody production and lymphocyte transformation response could not be measured until at least seven months post challenge. Sheep challenged with the primary tissue isolate instilled directly into the tonsil resulted in equivalent levels of Johne�s disease to those obtained with oral challenge. However, intratonsillar challenge resulted in higher levels of immune reactivity than oral challenge. The proprietary Johne�s vaccines; NeoparsecTM and GudairTM and an Aqueous vaccine were tested in sheep. The immunological reactions of the sheep to these vaccines showed some variations between the two separate studies, with the NeoparasecTM and GudairTM vaccines evoking high levels of CMI and humoral reactivity within two months of vaccination. Detailed immunological examination of gut associated lymphoid tissues were carried out on subgroups of animals that were either vaccinated or non-vaccinated and went on to develop disease or were immune to experimental challenge. The results showed that the diseased animals examined had multibacillary lesions and strong CMI and humoral responses. There were decreased proportions of CD4⁺, CD8⁺ and CD25⁺ T cells in peripheral blood and gut associated lymphatics of diseased animals compared with the immune or unchallenged subgroups. Profiles from the immune subgroups showed a stronger lymphocyte transformation response than case matched diseased animals. Tissues from immune animals showed increased proportions of B cells above those seen in diseased or unchallenged animals. This study has resulted in the development of a robust experimental sheep model in which Johne�s disease occurs in a high proportion of challenged animals. Critical time points for the establishment of infection or disease have been determined. It can be used in the future to evaluate protective efficacy of vaccines or to critically chart immunological profiles that are associated with infection, disease or protective immunity. Considerable research is needed to develop improved diagnostic tests to identify patterns of immunity during the early stages of infection or while the animal has subclinical disease.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Despite the fact that E. coli supports limited posttranslational modification, this bacterium has been universally used as the expression system of choice. Expression of modified proteins in E. coli may lead to expression of recombinant proteins that lack essential immunomodulatory or catalytic components essentials for infectious processes. Previously in our laboratory, pMptb#28 plasmid containing a 4.8 kb insert from M. paratuberculosis has been identified which expressed 16 kDa recombinant protein in E. coli and 19 kDa recombinant protein in Mycobacterium smegmatis. The objective of this study is to identify the ORF sequence, investigate possible posttranslational modification and characterize the protein forms in the two hosts. Earlier in the study, the genome sequence for M. paratuberculosis was not available and therefore sequencing both the 5' and 3' ends of the 4.8 kb insert did not help in the identification of the ORF. However, unidirectional Exonuclease deletion resulted in identification of subclones containing possible ORF sequence. Later on, the publication of the M. paratuberculosis genome sequence along with BLAST analysis of sequences from the subclones resulted in the identification of 486 bp ORF with significant identity to that from M. tuberculosis and M. leprae. Cloning of the 486 ORF coding sequence in E. coli resulted in the expression of 16 kDa protein similar to the calculated predicted size of translated peptide. Cloning of the 486 bp ORF coding sequence in M. smegmatis resulted in the expression of 19 kDa protein similar to that from M. paratuberculosis. The 16/19 kDa forms of the same protein were verified using rabbit anti-M. paratuberculosis antibodies adsorbed in E. coli and M. smegmatis lysates. The size of the 19 kDa proteins was not reduced following treatment with deglycosylation enzymes in absence of any enzyme inhibitors. The 19 kDa product was confirmed not be a glycoprotein when failed to react with ConA stain. The 16/19 kDa forms of the protein were evaluated against T-lymphocytes from Crohn's disease patients and normal controls. T- proliferation assay included controls such as PHA and PPD from M. paratuberculosis. There was not a significant difference between the two forms of the protein (16/19 kDa) against T-cell response from both populations. Overall, the study identified the ORF of the 19 kDa non-glycoprotein from M. paratuberculosis. Moreover, this is the first study which reports that the zoonotic M. paratuberculosis supports posttranslational modification similar to M. tuberculosis and M. leprae pathogens. Although the posttranslational modification component in this 19 kDa nonglycoprotein did not affect T- cell response, the finding is significant toward glycoproteins from M. paratuberculosis and their role in the pathogenesis of this bacterial infection in animals and humans.

Rheumatoid Arthritis (RA) is an autoimmune disorder that results in inflamed joints and osteoporosis. Previous studies have shown that Mycobacterium avium subspecies Paratuberculosis (MAP) has a role in the downregulation of active osteocalcin in Crohn’s disease (CD). Since RA and CD have a similar pathology, we hypothesize that active inflammation following MAP infection will result in downregulation of active osteocalcin in RA patients. In this study, a total of 82 individuals (48 RA and 34 healthy) were examined for MAP status and for active osteocalcin. Out of the 48 RA patients, 18 were MAP positive compared to only 1 out of 34 healthy control (P-values < 0.05). Overall, active osteocalcin levels between RA (2.70 ± 0.87 ng/mL) were significantly lower than healthy controls (5.84 ± 1.42 ng/mL, P-values < 0.05). MAP positive RA patients active osteocalcin levels were lower (0.60 ± 0.31 ng/mL) than MAP negative RA patients (3.85 ± 1.31 ng/mL). The active osteocalcin level in healthy MAP positive controls were lower (1.65 ng/mL) than healthy MAP negative controls (5.96 ± 1.45 ng/mL). The level of active osteocalcin in all MAP positive patients was significantly lower (0.66 ± 0.29 ng/mL) than all MAP negative patients (4.95 ± 0.98 ng/mL, P-values < 0.05). The correlation between presence of MAP in RA individuals and downregulation of active osteocalcin supports a role of MAP as a pathogenic element in RA. In addition, we tested three Single Nucleotide Polymorphisms (SNPs) in TNFα, TNFRSF1A and TNFRSF1B by TaqMan genotyping from peripheral blood samples of the 82 subjects. These SNPs were used to determine whether they influence the levels of osteocalcin in RA and healthy patients. Patients with the SNP TNFRSF1B:rs3397 who had TT genotype had a significantly lower osteocalcin level compared with patients without SNP who had CC genotype (0.98 ± 0.35 ng/mL vs. 1.86 ± 0.61 ng/mL). Overall, there was a correlation of the presence of these SNPs and the downregulation of osteocalcin in RA.

Background: Type 1 Diabetes mellitus (TIDM) is a chronic disorder in which the insulin producing beta cells are selectively self-destroyed. Although the etiology of the disease has not been determined, genetic dispositions such as SLC11A1 polymorphism in suffering patients have been reported. The role of pathogenic microorganisms such as Mycobacterium avium subspecies paratuberculosis (MAP) in TIDM has also been recently debated. MAP is already known to cause paratuberculosis in cattle and now it is a strong suspect of causing autoimmune diseases in humans such as Crohn’s disease, multiple sclerosis, autoimmune Thyroiditis, rheumatoid arthritis and autoimmune diabetes. We hypothesis that molecular mimicry between MAP Heat shock protein 65K (Hsp65) and human Glutamic Acid Decarboxylase 65K (GAD65) can be the trigger which leads to the autoimmune destruction of beta cell in patients exposed to MAP . Method: To test the hypothesis, peptide sequences of MAP Hsp65 and human GAD65 were investigated using BLAST and PyMOL bioinformatics tools. Moreover, 18 blood samples from humans with TIDM and controls, and 100 sera samples from cattle with paratuberculosis and controls were evaluated for the presence of MAP, MAP DNA and its antibodies. Glucose, insulin and GAD65 antibodies were also determined in some of the clinical samples. Results: Peptide BLAST analysis revealed 44% identity between the two proteins with 75% positive identities in a 16 amino acid region. PyMOL structural analysis identified possible shared epitope regions of the proteins in its 3D conformation. Immunoblot analysis revealed a strong cross reactivity between lysate of E.coli recombinant of MAP Hsp65 and plasma from human subject with TIDM. A weak cross reactivity was also observed between healthy rat pancreatic homogenate and rabbit anti MAP IgG. Nested PCR using IS900-specific iv oligonucleotide primers did not detect MAP DNA in peripheral blood from 18 subjects with Type I Diabetes, Type II Diabetes and non-diabetic controls. Long term culture of leukocytes from blood samples from same subjects resulted in the presence of MAP in 3/10 (30%) TIDM and 4/8 (50%) control subjects. However anti MAP IgG were detected in 5/10 (50 %) TIDM samples compared to 3/8 (37.5 %) controls. Insulin level was measured in sera from paratuberculosis cattle and controls. In MAP infected cattle, insulin level ranged from below 0.1ng/ml to 2.456 ng/ml with an average of 0.36 +/- 0.57ng/ml compared to below 0.1ng/ml to 13.47ng/ml with an average of 2.86 +/- 3.00ng/ml in healthy cattle. Conclusion: Bioinformatics analysis between MAP Hsp65 and human GAD65 through BLAST and PyMOL analysis revealed a homology of 16 amino acid motif and possible shared epitope regions; immunohistochemistry analysis revealed a cross reactivity between rabbit antiMAP IgG and pancreatic cell homogenate from a healthy rat. Moreover, plasma from patient with TIDM (TD8), who was confirmed to be positive for MAP DNA and MAP IgG, reacted strongly with MAP Hsp65 in MAP protein lysate and MAP Hsp65 recombinant clone pmptb20. Culture of MAP from human leukocytes is significant despite the lack of correlation between MAP in samples from TIDM and controls. It is worth noting that some of the control subjects have not been evaluated for other autoimmune diseases possible MAP role. Additionally, antiMAP IgG levels in TIDM subjects compared to controls have raised a suspicion on the involvement of MAP in TIDM. The poor correlation of MAP in blood versus either the antiMAP IgG or the insulin level may be related with the fastidious nature of MAP and in vitro cultivation. Since MAP is the sole causative agent of Johne’s disease, it is significant that the insulin level is 8 folds less in MAP infected cattle compared to MAP free cattle. Overall, the data v is mixed and suggest that further study is needed to investigate the intriguing question to whether MAP is involved in TIDM or not.