Mycobacterium avium ssp. paratuberculosis (Map), agent étiologique de la paratuberculose, a évolué en deuxtypes dénommés, S pour« Sheep » et C pour « Cattle ». L’intestin grêle est le site primaire de l’infection à Map mais les mécanismes moléculaires impliqués dans l’implantation du bacille restent largement méconnus. L’objectif de mon projet de thèse visait à identifier et caractériser les adhésines exprimées par Map par des approches génétiques et biochimiques. J’ai ainsi purifié la HBHA et la LBP par chromatographie d’affinité puis les ai identifiés en spectrométrie de masse. L’originalité de ce travail repose sur le polymorphisme de ces adhésines observé entre les souches de type C et S. Cette variabilité a été mise en évidence sur le domaine d’interaction avec les sucres sulfatés de la cellule hôte influençant l’affinité des adhésines pour l’héparine. Ce travail de thèse a permis de caractériser pour la première fois ces deux adhésines produites par Map. Le polymorphisme de la HBHA et de la LBP, discriminant les types C et S, ouvre de nombreuses perspectives sur l’évolution de l’espèce M. avium et le rôle de ces adhésines sur le tropisme intestinal, la préférence d’hôte de Map ou encore leur potentiel diagnostic. / Mycobacterium avium subsp. paratuberculosis (Map), the etiological agent of paratuberculosis, has evolved into two types called, S for "Sheep" and C for "Cattle." The small intestine is the primary site of Map infection but the molecular mechanisms involved in the establishment of bacilli are still unknown. The aim of my thesis was to identify and characterize the adhesins expressed by Map by genetic and biochemical approaches. I purified HBHA and LBP by affinity chromatography then identified them by mass spectrometry. The originality of this work is based on the polymorphism of these adhesins observed between strains of type C and S. This variability has been demonstrated in the binding domain involved in interaction with sulfated sugars of host cell influences adhesins affinity for heparin. This thesis has characterized for the first time these two adhesins produced by Map. Specific polymorphism highlighted related to the evolution of the species avium, opens large number questions on their role on the pathogenesis of Map including the cellular tropism, host preference or interest of these antigens to improve diagnostic.

No description available.

The goal of this project was to incorporate bacteriophage with Bioluminescent Assay in Real-Time Loop-mediated isothermal amplification (BART-LAMP) for the rapid detection of Mycobacterium avium subspecies paratuberculosis (MAP). As the causative agent of Johne’s Disease, there are no rapid detection methods that are suitable in specificity and sensitivity. A screening assay for phage isolation was developed, and over 400 samples were screened for the isolation of a bacteriophage against MAP. One novel Mycobacterium phage was isolated and characterized using transmission electron miscroscopy, host range studies, restriction enzyme digestion, and pH and temperature stability. It was sequenced, annotated, and underwent an in silico protein analysis. No pathogenic or lysogenic genes were detected, and it was found to be related to Gordonia phage GTE2. BART-LAMP was applied to the detection of the isolated phage using purely extracted DNA and crude phage lysate, showing that phages could be detected successfully. / Beef Cattle Research Council; Agriculture and AgriFood Canada through Growing Forward initiative

No description available.

Die Paratuberkulose (Johne′sche Erkrankung) ist eine chronische bakterielle Darmerkrankung mit weltweiter Bedeutung, hervorgerufen durch Mycobacterium avium subsp. paratuberculosis (MAP). Neben selten ausgeprägten klinischen Symptomen wie Durchfall und Abmagerung führen vor allem Milchleistungsabfall, erhöhte Krankheitsanfälligkeit und verminderte Schlachterlöse zu ökonomischen Verlusten. Ein Zusammenhang zwischen einer Infektion mit MAP und Morbus Crohn, einer chronischen Darmerkrankung des Menschen, wird seit langem diskutiert. Die Anzucht auf Festkultur HEYM (Herrolds Egg Yolk Agar) ist aufgrund von mäßiger Sensitivität und langer Anzuchtdauer bis zu 16 Wochen keine optimale Referenzmethode, bietet jedoch eine hohe Testspezifität. Die Anzucht in Flüssigkultur wird als Alternative mit kürzerer Anzuchtdauer und höherer Sensitivität beschrieben. Für den Erregernachweis wird neben der Anzucht der DNA-Nachweis aus Kot mittels PCR-Verfahren (Direkt-PCR) als schnelle, automatisierbare Methode in der Paratuberkulose-Diagnostik verwendet. Die fäkal-orale Übertragung von MAP von der Kuh auf das Kalb gilt als wichtige Infektionsquelle innerhalb einer Herde. Die Kenntnis über den MAP-Status des Muttertieres vor der Geburt ist besonders hilfreich, um frühzeitig Maßnahmen zum Schutz des Kalbes treffen zu können. Vorrangiges Ziel dieser Arbeit war die optimierte Verwendung und Validierung von Methoden für einen raschen, sensitiven und automatisierbaren Nachweis von MAP innerhalb von maximal acht Wochen und unter Berücksichtigung der Anwendbarkeit für Paratuberkulose-Kontrollprogramme. Zu Beginn wurden 20 Kotproben mit Direkt-PCR-Verfahren (DNA-Extraktion und DNA-Nachweis direkt aus Kot) untersucht: Konkret wurden zwei kommerzielle Kombinatonskits (DNA-Extraktionskit für Kotproben mit real-time PCR in einem Kit) mit sechs weiteren DNA-Extraktionskits mit publizierter real-time PCR verglichen. Die zwei Kombinationskits dienten im Nachgang zur Untersuchung von 107 positiven und 50 negativen Kotproben. Parallel wurden diese Proben auf Festkultur HEYM und in Flüssigmedium M7H9C für 12 Wochen kultiviert. Für die anschließende Feldstudie wurden 245 Einzelkotproben und 12 Sockentupfer aus drei Milchviehbetrieben mit Laufstallhaltung entnommen und mit einer Direkt-PCR (Kombinationskit) und dem Flüssigmedium M7H9C nach 6-wöchiger Anzucht untersucht. Der statistische Vergleich der positiven und negativen Ergebnisse der Methoden sowie der Vergleich der Anzahl positiver Kulturröhrchen und der PCR-Signalstärke nach Kultivierung wurden mit dem Fishers Exakt-Test durchgeführt. Ein Typ 1-Fehler α von < 0,05 wurde als signifikant gewertet. Die Kombinationskits zeigten signifikant mehr positive Ergebnisse als die anderen sechs getesteten Extraktionskits (p < 0,04). Beide Kombinationskits erreichten eine Sensitivität von 86 % bzw. 89 % bei 100 % Spezifität im Vergleich zur Anzucht in dem Flüssigmedium M7H9C, welches nach 6-wöchiger Anzucht 97 % und nach 12 Wochen 100 % der positiven Proben erkannte, ebenfalls bei 100 % Spezifität. Auf Festkultur HEYM (12-wöchige Anzucht) gelang bei 74 % der Proben ein Erregernachweis. In der Feldstudie wurde MAP-DNA in Sockentupfer-Proben von allen Betrieben mit Direkt-PCR und Flüssigkultur nachgewiesen. Bei der Untersuchung der Einzelkotproben stimmten 74 % der positiven Ergebnisse beider Methoden überein. Die Stärke des PCR-Signals nach Kultivierung unterschied sich signifikant zwischen Proben mit positivem Signal in nur einem Röhrchen und Proben mit positivem Signal in zwei oder drei Röhrchen (p < 0,01). Die Direkt-PCR ist bei Verwendung von Kombinationskits gut geeignet als schnelle automatisierbare Methode für die Detektion von MAP-Ausscheidern. In Herden mit niedriger Ausscheidungsrate wird die Anzucht in dem Flüssigmedium M7H9C als Methode mit höherer Nachweisrate und Ergebnissen nach acht Wochen empfohlen. Dieses Zeitfenster ermöglicht zusätzlich die Untersuchung während der Trockenstehphase und bietet Informationen zum MAP-Status der Mutterkuh bereits vor der Abkalbung.:INHALTSVERZEICHNIS 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 2 2.1 PARATUBERKULOSE 2 2.1.1 Ätiologie 2 2.1.2 Krankheitsbild 3 2.1.3 Das zoonotische Potenzial 3 2.2 DIAGNOSTIK 4 2.2.1 Diagnostische Herausforderungen des Erregernachweises 4 2.2.2 Nachweis des Erregers mittels PCR-Verfahren und Kultivierung 5 2.2.3 Nachweis von Antikörpern mittels ELISA 5 2.2.4 Probenart und Entnahme 6 2.3 BEKÄMPFUNG DER PARATUBERKULOSE 7 2.3.1 Risikofaktoren einer Infektion 7 2.3.2 Bestehende Bekämpfungsprogramme 8 2.3.3 Hygiene und Herdenmanagement 10 3 ZIELSETZUNG 11 4 PUBLIKATIONEN 12 4.1 PUBLIKATION 1 12 Stellungnahme zum Eigenanteil der Arbeit an der Publikation 1: 12 4.2 PUBLIKATION 2 22 Stellungnahme zum Eigenanteil der Arbeit an der Publikation 2: 22 5 DISKUSSION 31 6 ZUSAMMENFASSUNG 41 7 SUMMARY 43 8 LITERATURVERZEICHNIS 45 DANKSAGUNG 54

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.

No description available.

Mycobacterium avium subsp. paratuberculosis (MAP) is a very slow growing bacterium that is the causative agent of Johne's disease (JD) in ruminants and has long been suggested to be associated with complications of Crohn's disease (CD) in humans. Although there is no direct evidence that MAP is the primary etiological agent for CD, most CD patients are found to have MAP in their intestinal tissues. The current control measures for JD in cattle, sheep, and goats have only been minimally effective, and there are only medications to treat the symptoms of mycobacterial infections associated with CD in humans. Along with not being able to cure MAP infections, there is no established laboratory animal model for testing therapeutics. When mice are infected with MAP they develop systemic infection and do not mimic disease observed in ruminants. J774A.1 murine macrophages typically have a very short lifespan of about 4-6 days, however MAP infected cell cultures can survive up to about 10 days. Using a modified protocol of Estrella et al. (2011), we have been able to establish a 45-60 day long-term MAP infected J774A.1 murine macrophage cell culture model. With the addition of retinoic acid (RA), vitamin D (VD), and phorbol myristate acetate (PMA) in combination in cell culture, we were able to screen novel therapeutics before embarking on in vivo testing in animals. This is a significant step forward in Crohn's and Johne's disease treatment research. We are not only able to test various drugs against specific strains of MAP to determine susceptibility, but we are also able to test a wide variety of drugs at the same time, with relatively minimal cost. We have evaluated the efficacy of clarithromycin, azithromycin, isoniazid, amikacin, ethambutol, ciprofloxacin, levofloxacin, rifampicin, clofazimine, as well as a combination of clarithromycin, rifampicin, and clofazimine using our MAP infected macrophage cell culture model. We were able to determine the drugs' differential ability to kill intracellular MAP in the early stages of infection, versus chronic stages of infection, and against two different strains of MAP, 43015 and 19698 that affect humans and cattle respectively. The minimal inhibitory concentration (MIC) of each drug was determined as per NCCLS protocol in vitro, and the drugs were tested at the MIC value, along with one concentration above and below the MIC in our cell culture model. The antimicrobials were found to be effective at different stages of cell culture infection and in different strains of MAP. Some drugs were more effective at early stages of MAP infection, whereas others were more effective in chronic or latent stages of infections. It is important to note that although a drug may be effective at a certain stage of infection, it may not necessarily be effective against all strains of MAP. The most promising results were seen with a combination of clarithromycin, clofazimine, and rifampicin, which was effective at all stages of infection with both strains of MAP tested. This long term cell culture model will provide researchers with important screening tools for evaluating new therapeutics before embarking on costly in vivo testing, and allow the assessment of therapeutics at different stages of MAP infection but also against an array of intracellular pathogens. / Ph. D.

Submitted by Edisangela Bastos (edisangela@ufpa.br) on 2014-02-03T21:22:32Z No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DiagnosticoParatuberculoseBovinos.pdf: 264056 bytes, checksum: 5ce068ca20fbd8e870ce142990c3a769 (MD5) / Approved for entry into archive by Ana Rosa Silva(arosa@ufpa.br) on 2014-02-04T14:47:40Z (GMT) No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DiagnosticoParatuberculoseBovinos.pdf: 264056 bytes, checksum: 5ce068ca20fbd8e870ce142990c3a769 (MD5) / Made available in DSpace on 2014-02-04T14:47:40Z (GMT). No. of bitstreams: 2 license_rdf: 23898 bytes, checksum: e363e809996cf46ada20da1accfcd9c7 (MD5) Dissertacao_DiagnosticoParatuberculoseBovinos.pdf: 264056 bytes, checksum: 5ce068ca20fbd8e870ce142990c3a769 (MD5) Previous issue date: 2005 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / 514 amostras de sangue bovino foram analisadas para detecção de anticorpos anti-Mycobacterium avium (subsp.) paratuberculosis, utilizando um kit comercial do teste ELISA indireto. Os animais eram todos mestiços, machos e fêmeas, classificados em dois grupos, de acordo com a idade, menores ou iguais a 36 meses e maiores de 36 meses, provenientes de 23 municípios do Estado do Pará. Além deste teste, também foram colhidas 100 amostras de fragmentos de intestino delgado e de linfonodo mesentérico para análise bacteriológicaatravés da coloração de Ziehl-Neelsen, a fim de identificar o Mycobacterium avium (subsp.) paratuberculosis. As amostras foram colhidas aleatoriamente em matadouro da região metropolitana de Belém. No teste ELISA indireto, das 514 amostras de sangue bovino 182 (35,4%) foram reagentes para anticorpos anti-Mycobacterium avium (subsp.) paratuberculosis e 332 (64,6%) foram não-reagentes. Em todas as mesorregiões encontraramse animais soropositivos. As fêmeas com idade superior a 36 meses mostraram maior respostas ao teste , 92,06% de animais soropositivos. Entre os machos, a maior prevalência (76,79%), foi obtida nos mais novos, com idade abaixo de 36 meses. Das 100 lâminas coradas através do método de Ziehl-Neelsen, nenhum Mycobacterium sp. foi identificado. Conclui-se assim, que o elevado número de animais com anticorpos anti-Mycobacterium avium (subsp.) paratuberculosis indica a presença da doença em todas as mesorregiões estudadas, devendo-se atentar aos riscos desta enfermidade, através de pesquisas mais abrangentes sobre a Paratuberculose, com aplicação de métodos diagnósticos diretos e indiretos. / 514 blood samples from beef cattle were examined of to find anti- M. avium (subsp.) paratuberculosis antibodies, using commercial ELISA test kit. The animals from samples were all compous from cross between Indicus x Taurus race, between male and female, with two age class, over 36 months old and below 36 months old, from of the 23 municipality of Para State. Also were collected 100 samples of small intestine and mesenteric lymph nodes of bacteriological test through Ziehl-Neelsen method to detect the M. avium (subsp.) paratuberculosis. Were at random collected in slaughterhouse of Belem. In ELISA test, through these 514 samples, 182 (35,4%) were seropositive and 332 (64,6%) samples were seronegative. All the middle region were found soropositives animals. The female with over 36 months old presented larger response test, of the 92,06% seropositives animals. Among male the prevalency larger (76,79%,) were obtained between the most new, with below 36 months old. Of 100 samples stained through Ziehl-Neelsen method, not were found either Mycobacterium spp.. The conclusion, is the biggest number of seropositive animals of anti- M. avium (subsp.) paratuberculosis sign of disease is presented in all the regions studied, watching the risks of this disease with the elaborate more covering stud ies about the paratuberculosis with the appling of directs and indirects diagnosis methods.

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2017-03-28T16:35:14Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Leticia_Fiss_Resumo.pdf: 132018 bytes, checksum: bf6cc9a5c580e6d6228123ddca70c8d1 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-03-28T21:01:19Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Leticia_Fiss_Resumo.pdf: 132018 bytes, checksum: bf6cc9a5c580e6d6228123ddca70c8d1 (MD5) / Made available in DSpace on 2017-03-28T21:01:19Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Tese_Leticia_Fiss_Resumo.pdf: 132018 bytes, checksum: bf6cc9a5c580e6d6228123ddca70c8d1 (MD5) Previous issue date: 2015-09-30 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Esta tese relata um surto de paratuberculose diagnosticado em bovinos de corte em uma propriedade na região Sul do Rio Grande do Sul. Foram descritos os aspectos epidemiológicos e clínico-patológicos da doença. Os animais eram criados extensivamente e apresentaram quadro de emagrecimento progressivo e diarreia crônica. O diagnóstico foi realizado pelas lesões macroscópicas e pela histopatologia. Fragmentos dos órgãos foram fixados em formalina 10%, incluídos em parafina, cortados com 5 μm de espessura e corados pela técnica de hematoxilina e eosina (HE) e Ziehl-Neelsen (ZN). Fezes foram encaminhadas ao Departamento de Medicina Veterinária, Área de Medicina Veterinária Preventiva da Universidade Federal Rural de Pernambuco para o cultivo de Mycobacterium avium subsp. paratuberculosis em meio Löwenstein Jensen com micobactina e para realização da PCR. Histologicamente havia enterite granulomatosa principalmente no dudeno, jejuno, íleo, ceco, colon e reto. Havia, ainda, linfangite e linfaadenite granulomatosa. Pela coloração de ZN foram observados numerosos bacilos álcool-ácido resistentes (BAAR) no interior de macrófagos, células gigantes de Langhans e nos linfonodos mesentéricos no duodeno, jejuno, íleo ceco e reto. Não houve crescimento bacteriano nas amostras de fezes e cinco amostras amplificaram a sequência genética IS900 específica do Mycobacterium avium subesp. paratuberculosis. Concluiu-se que a paratuberculose ocorre, também, em bovinos de corte criados extensivamente no sul do Rio Grande do Sul e pode ter uma prevalência maior do que se supõe na região, sendo necessárias medidas efetivas de controle desta doença que, por vezes, ainda é considerada exótica no Brasil. Por ser uma doença ainda pouco diagnosticada e pouco conhecida na região e que apresenta sinais clínicos inespecíficos, um segundo trabalho foi realizado visando determinar as principais doenças que ocorrem na região Sul do Rio Grande do Sul, que cursam com diarreia e afetam bovinos de corte maiores de dois anos de idade. Para isso realizou-se um levantamento nos arquivos do Laboratório Regional de Diagnóstico identificando-se os protocolos de necropsia nos quais era mencionada a diarreia como um sinal clínico relevante. Observou-se que as causas de diarreia em bovinos com dois anos ou mais de idade são poucas e bem conhecidas na região, sendo de fácil diagnóstico. Destaca-se a intoxicação por Senecio spp., em bovinos maiores de três anos, que causa prejuízos econômicos relevantes devido a sua ampla distribuição na região do estudo e ao seu difícil controle. Com relação aos bovinos entre dois e três anos as parasitoses de modo geral foram as mais importantes causas de diarreia representando 70% dos casos. / This thesis reports an outbreak of paratuberculosis diagnosed in beef cattle at a farm in the southern region of Rio Grande do Sul.