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.

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.

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

Dada a existência de quatro famílias molecularmente distintas de estirpes de Mycobacterium avium isoladas de suínos da região Sul do Brasil e a verificação de diferentes virulências em hamsters de um representante por família, o presente trabalho objetiva relacionar a virulência de isolados de M. avium aos seus perfis genéticos obtidos em experimentos de RFLP. Foram selecionados três representantes por família que foram inoculados pela via intraperitoneal em hamsters distribuídos em doze grupos (cada grupo recebeu uma estirpe diferente). Foi mantido um grupo controle que recebeu solução salina estéril pela mesma via. Após 16 dias da inoculação, os animais foram eutanasiados; os baços foram colhidos, pesados, macerados, suspendidos em solução salina estéril e diluídos. Cada uma das diluições foi semeada em duplicata em placas com meio de Petragnani, que foram incubadas a 37°C. Após 30 dias de incubação, foram realizadas as contagens de UFC e os resultados foram expressos em UFC de M. avium por grama de baço. As famílias genéticas apresentaram capacidade de virulência semelhante (p=0,49). As estirpes dentro das famílias PIG B e PIG D não apresentaram diferença na virulência (p=0,15 e p=0,87, respectivamente). Dentro da família PIG A, o isolado A52 foi mais virulento do que A1 e A162; a estirpe C122 se apresentou menos virulenta do que as estirpes C44 e C68 dentro da família PIG C. Independente das famílias, todos os isolados apresentaram diferenças na virulência. A estirpe A52 mostrou maior capacidade de virulência em relação às estirpes A1, A162, B72, C122, D242 e 243. Diferentes contagens de UFC significam diferentes capacidades de produzir infecção, ou seja, diferentes virulências. Concluiu-se que isolados de M. avium com diferentes perfis de RFLP podem apresentar diferentes virulências, independentemente de pertencerem a uma mesma família genética definida com base na similaridade dos padrões de RFLP; não houve diferença de virulência entre as quatro famílias genéticas de M. avium estruturadas com base na similaridade dos padrões de RFLP. / Knowing the existence of four molecularly distinct families of Mycobacterium avium strains isolated from swine populations of Brazil Southern and the corroboration of diverse virulence in experimentally infected hamsters by one representative of each family, this work intend to establish relation between virulence of M. avium isolates and their genetic patterns obtained in prior RFLP analyses. We selected three representatives of each family, totalizing twelve strains, which were introduced by intraperitoneal route into hamsters distributed in twelve groups (each group received a different strain). A control group was maintained and received buffer solution by the same route. Sixteen days after the inoculation, animals were euthanized and their spleens were collected, weighted, triturated, suspended in buffer solution and then diluted. Two plates containing Petragnani medium were seeded with each dilution and were incubated at 37ºC. At the 30th day, the CFU counting was performed and the results were expressed in M. avium CFU/g of spleen. All genetic families presented similar capacity of virulence (p=0,49). Strains of PIG B and PIG D families presented similar virulence within their families (respectively, p=0,15 and p=0,87). Inside the PIG A family, strain A52 was more virulent than strains A1 and A162; the strain C122 presented the lowest virulence compared with the strains C44 and C68 within the PIG C family. All strains, independent of their family, presented diverse virulence. Strain A52 was more virulent than strains A1, A162, B72, C122, D242 and D243. Distinct counting of CFU means different capacity of producing infection, i.e. diverse virulence. We concluded that, independent fo the genetic family established by similarity of RFLP patterns, M. avium isolates with diverse RFLP profiles may present different virulence; there was not difference in virulence; among all of four M. avium genetic families structured according to the similarity of RFLP patterns.

Haja vista a existência de quatro famílias de M. avium molecularmente distintas circulando na população de suínos do Sul do Brasil, a diferença de virulência constatada entre essas quatro famílias, a influência da virulência nos mecanismos de transmissão, as dúvidas existentes a respeito da existência e da importância da transmissão horizontal de M. avium em suínos e do significado desse conhecimento para o estabelecimento de métodos de controle eficientes, o presente projeto tem por objetivos: 1) Padronizar método de isolamento de micobactérias a partir de fezes suínas; 2) Caracterizar a eliminação de M.avium pelas fezes em suínos experimentalmente infectados pela via oral; 3) Verificar se existe transmissão horizontal entre suínos durante a fase de eliminação ativa, através de experimentos envolvendo infecção oral e exposição de animais contactantes; 4) Estudar, através de modelagem matemática, a dinâmica da infecção por M.avium em uma população suína. Como resultados, para cada um dos itens obteve-se: 1) Houve diferença significativa entre os protocolos de recuperação de micobactérias a partir de fezes de suínos (p< 0,05) e o método ácido com ressuspensão em solução de anfotericina B e semeadura em meio de Lowenstein-Jensen com antibióticos apresentou o maior percentual de recuperação (87%); 2) Foram constados dois períodos de eliminação fecal de MAC em suínos: um inicial, relativo à eliminação residual do inóculo, do 1º ao 4º, e um segundo, com início no 18º e término no 62º dias pós-inoculação, este último é resultado de suposta lesão aberta para a luz do intestino; 3) Cinco, dos sete animais contactantes, infectaram-se com o M.avium, estirpe PIG B e 4) A simulação matemática da doença, considerando a transmissão horizontal como mecanismo principal da ocorrência de condenações em matadouro por linfadenite granulomatosa é inconsistente com o que se observa na população. Portanto, o componente ambiental tem papel preponderante na dinâmica das infecções micobacterianas dos suínos produzidos no Brasil. / Considering four genetic distinct M. avium families within the swine population of the Southern Brazil, the virulence difference detected among them, the virulence influence on the transmission mechanisms, the current doubts concerning M. avium horizontal transmission existence and importance in swine and about its knowledge meaning in order to stablish efficient control programs, the objectives of the present study were: 1) to set in a standard mycobacterial isolation method from swine feces; 2) to characterize M. avium excretion through feces in swine infected orally; 3) to verify the existence of horizontal transmission among swine during the active elimination phase, by experiments involving oral infection and exposure of contacting animals; 4) to study, through mathematical modelling, M. avium infection dynamics in a swine population. These are the attained results for each of the items: 1) there was a significant difference (p<0.05) between the mycobacterial recovery protocols from swine feces and the acid method with suspension in amphotericin solution and inoculation in Lowenstein-Jensen media with antibiotics presented the greatest recovery percentage (87%); 2) two fecal elimination periods of M. avium in swine feces were observed: an initial one, relative to the residual elimination of the inoculum, within days 1 and 4, and a second one, starting at day 18 and ending at day 62 post inoculation - the last one results from a supposed lesion opened to the intestinal lumen. 3) Five out of seven contacting animals were infected with M. avium; 4) The mathematical simulation of the disease, considering the horizontal transmission as the major mechanism of occurrence of condemnation at slaughterhouses due to granulomatous lymphadenitis is not consistent with what is observed in the population. Therefore, the environmental component plays a preponderant role in the mycobacterial infections dynamics of swine raised in Brazil.

Tendo sido comprovada a existência da famílias molecularmente distintas de M. avium circulando em suínos de região sul do Brasil, e havendo dúvidas a respeito da importância da transmissão horizontal como mecanismo de manutenção da doença, o presente teve por objetivo estudar a virulência dessas estirpes, informação importente para o aperfeiçoamento dos métodos de controle. As estirpes emergiram do estudo caso-controle, onde as tipagens moleculares por RFLP mostraram a existência de quatro famílias de M. avium (PIG-A, B, C e D). Um estirpe representante de cada família foi inoculada pela via intra-peritoneal em 48 hamsters com uma dose de 30.000 U.F.C. por animal. Após 2, 13, 26 e 40 dias da inoculação, 12 hamsters inoculados de cada família foram anestesiados, sacrificados e os agentes foram quantificados no fígado, baço e pulmão. A presença das estirpes foi verificada no sangue e também foram realizados exames histológicos. As estipers PIG-A, B, C e D desenvolveram lesões granulomatosas no fígado e baço nos quatro tempos experimentais; disseminaram-se pela via linfo-hemática, multiplicando-se em fígado, baço e pulmão. Nos quatro tempos experimentais houve diferença entre as contagens de U.F.C./g entre os órgãos (T1: p<0,001; T2: p<0,001; T3: p<0,001 e T4: p<0,001) e as obtidas do baço foram sempre superiores às do fígado e pulmão. Nos quatro tempos experimentais houve diferença entre as contagens de U.F.C./g entre as estirpes (T1: p<0,001; T2: <0,001; T3: p<0,001 e T4: p<0,001) e foi possível construir a seguinte escala decrescente de virulência: PIG-B > PIG-A > PIG-D > PIG-C. / Given that the existence of molecularly different families of M. avium circulating in swine of the south area of Brazil has been proved, and that some doubts remain regarding the importance of the horizontal transmission as mechanism of maintenance of the disease, this work aimed to study the virulence of those strains, an important information for the improvement of the control methods. The strains emerged from a case-control study, when the RFLP molecular typification showed the existence of four families of M. avium (PIG-A, B, C and D). A strain representative of each family was inoculated by intra-peritoneal route in 48 hamsters with a dose of 30.000 C.F.U./animal. After 2, 13, 26 and 40 days post-infection, 12 inoculated hamsters of each family were anesthetized, euthanized and the bacteria were quantified in the liver, spleen and lung. The presence of the strains was verified in the blood and also histological exams were accomplished. The strains PIG-A, B, C and D developed granulomatous lesions in the liver and spleen in the four experimental times; they were disseminated by the linfo-haematic route, multiplying in liver, spleen and lung. In the four experimental times there was difference among the countings of C.F.U./g among the organs (T1: p<0,001; T2: p<0,001; T3: p<0,001 and T4: p<0,001) and that obtained of the spleen were always superiors to the one of the liver and lung. In the four experimental times there was difference among the countings of C.F.U./g among the ancestries (T1: p<0,001; T2: p<0,001; T3: p<0,001 and T4: p<0,001) and was possible to build the following order of decreasing virulence: PIG-B > PIG-A > PIG-D > PIG-C.

Mycobacterium avium subsp. hominissuis (MAH) is a pervasive environmental bacterium that can cause opportunistic infections in humans. Among the most robust and hardy members of the Mycobacterium genus, M. avium can persist and thrive in a range of challenging environments, including many which place it in direct contact with humans. Surface-exposed proteins are central to the bacterial processes involved in both environmental persistence and pathogenesis. These proteins also play a critical role in how the immune system of the host recognizes and responds to pathogens. Mycobacteria have evolved a specialized mechanism for protein export, a Type VII Secretion System (T7SS), in order to transport their proteins through their thick and impermeable cell envelope. This system is responsible for the export of several classes of proteins, many of which play an integral role in virulence. A central focus of this dissertation is the characterization of a conserved element of the T7SSs in pathogenic mycobacteria, a PPE family protein, whose deletion attenuates virulence in M. avium. Specifically, we examined the localization of this PPE protein (MAV_2928) within the bacterium, screened potential protein-protein interactions with other conserved elements in the adjacent T7SS loci and analyzed the transcriptional regulation of the gene in response to environmental changes. Seeking to more thoroughly characterize the surface-exposed proteome of M. avium, particularly in the context of early infection, we then developed a method, based on selective biotinylation and affinity purification, to profile the of surface-exposed proteome of the bacterium. We employed this method to analyze the surface-exposed proteomes of M. avium 109 that had been exposed to macrophages to those of M. avium 109 that had been cultured in media. This comparison detected several proteins whose presence at the bacterial surface appeared to be dependent on particular growth conditions. Lastly, in order to establish a more efficient method to isolate biotinylated surface proteins from complex mixtures, we developed a testing paradigm to identify modifications to the original method that might improve our coverage of identified proteins. Through this process, we developed a more robust methodology that yielded improved coverage and depth. We then utilized this technology to profile the surface-exposed proteome of another clinical isolate of M. avium subsp. hominissuis, M. avium 104. Beyond improving our understanding of the basic biology of M. avium, this new data provides independent evidence that PPE family proteins are indeed exported to the surface of M. avium, where they remain associated with the bacterial cell envelope. In total, this analysis represents the most comprehensive profile of the surface-exposed proteins of M. avium generated to date. / Graduation date: 2012