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Early macrophage response to Mycobacterium avium subspecies paratuberculosisMathie, Heather January 2018 (has links)
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis that has a damaging economic and welfare impact on the livestock industry. Johne's disease in cattle is known to reduce milk yield and carcass value, making it of economic concern to both dairy and beef farmers. In addition, there is cause for concern regarding zoonotic transmission, as there is an unconfirmed but potential relationship between MAP infection and human Crohn's disease, which presents similar clinical symptoms. MAP is most often contracted by neonates through the faecal-oral route, but can also be spread through contact with contaminated milk and colostrum, as well as in utero. Once the host receives an oral dose, the bacteria traverse the gut epithelium and are phagocytosed by gut macrophages residing in the lamina propria and Peyer's patches. MAP are able to evade the macrophage response by resisting intracellular degradation within phagosomes. Infected macrophages respond to the infection by secreting several pro-inflammatory cytokines that drive the downstream immune response and granuloma formation. This work aimed to elucidate key early responses of bovine monocyte derived macrophages (MDM) to MAP infection, and determine the reliability of using the reference strain, K10 (which is likely to have undergone lab adaptation) to model the infection in vitro, by comparing the MDM response to K10 with the response to a recent clinical isolate, C49. At a multiplicity of infection of 5 (MOI 5), there was a significant decrease in K10 intracellular survival (~90%), compared to C49 intracellular survival, over a 24 hour infection time-course. This suggests that K10 may have lost some virulence mechanism through lab adaptation. Understanding the mechanisms of how MDM respond to these two strains could be informative for the design of targeted vaccines When further investigating the MDM response to both strains, it was found that, at MOI 5, MDM infected with K10 secreted higher levels of IL-1β and IL-10, compared to MDM infected with C49. Both cytokines are associated with mycobacterial infection and could perhaps indicate that MDM are more responsive to the K10 strain at early time-points. In addition, MDM infected with K10 produced significantly higher levels of reactive nitrogen species (RNS). RNS are antimicrobial products that can destroy invading pathogens, and have been shown to have bactericidal effects on MAP. The production of RNS could, therefore be a potential mechanism by which MDM are able to kill K10 more efficiently than C49. An additional aim of this project was to understand the importance of the route of phagocytosis in determining the outcome of MAP infection. MDM express several phagocytic receptors, including Fc receptors (FcRs), complement receptors (CR), Ctype lectin receptors and scavenger receptors. This project mainly focused on the role of the mannose receptor (MR) on bacterial uptake and downstream immune responses, as past studies have suggested that other species of mycobacteria such as M. tuberculosis, target the mannose receptor in order to regulate macrophage immune responses. Blocking the MR reduced intracellular survival for both strains of MAP; however, the mechanism by which the MR influences intracellular survival remains poorly understood The effect of opsonisation on MAP prior to uptake by phagocytic cells was also investigated, as presence of opsonins, such a complement proteins and antibody, can change the mechanism by which pathogens are phagocytosed. MAP were incubated in serum from either MAP- negative or MAP- positive cattle, prior to infection and the percentage uptake and survival assessed by performing colony counts. Opsonisation in serum from Johne's negative cattle resulted in marked increase in MAP uptake but not intracellular survival, whereas opsonisation in serum from Johne's positive cattle did not increase uptake but decreased the intracellular survival rate by 24 HPI. This finding highlights a potential protective role of antibody early in the infection process, and could significantly impact how the infection is modelled in future, as anti-MAP antibody may be present in contaminated milk at the point of infection. Taken together, the data presented in this thesis show that bacterial strain has a significant impact on MDM response to MAP infection, which may have important implications for the interpretation of previous studies and the design of future studies investigating host-pathogen interactions in the context of paratuberculosis. Additionally, this work has shown that RNS production and the mechanism of uptake can affect intracellular survival rates, and although this needs further investigation, the findings could have implications for the design of future vaccines.
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Comparaison du Ziehl-Neelsen, de l'immunohistochimie et du PCR dans le diagnostic de la paratuberculose chez le moutonGalarneau, Jean-René January 2004 (has links)
Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.
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Aspectos cl?nico-patol?gicos da paratuberculose em rebanho bovino leiteiro no munic?pio de Rio Claro, RJ. / Clinic-pathological aspects of paratuberculosis in dairy cattle in Rio Claro county, Rio de Janeiro State.Yamasaki, Elise Miyuki 21 January 2010 (has links)
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Previous issue date: 2010-01-21 / The epidemic and clinic-pathological aspects of paratuberculois in a dairy cattle herd in the
Rio Claro county, southern Rio de Janeiro region, are described. In the years 2006-2009, eight
adult cows presented chronic-intermittent diarrhea, chronic weight loss and normal appetite.
At necropsy, the subserosal lymphatics were proeminent and dilated, mesenteric nodes were
enlarged, and intestinal mucosa was corrugated, thickened and of microgranular aspect.
Especially, in duodenum, was observed polipoids lesions in mucosa surface. Histopathology
revealed, from the duodenum to the rectum, severe and diffuse granulomatous inflammation
of the lamina propria and submucosa, broadened and distorced villi, marked dilatation of the
lymphatic vessels in their apex, lymphangioectasia and granulomatous lymphangitis in the
submucosa. Ziehl-Neelsen stain revealed variable amounts of acid-fast bacilli in
macrophages, Langhan s giant cells and freely in the musosa and submucosa of the small
intestine and colon, and in lymphnodes. Lesions in the lamina propria, particularly in the jejun
and ileum, in some animals, were severe hypertrophy; polipoids lesions observed in
duodenum mucosa was markedly muscularis mucosa hipertrophy , intestinals and duodenal
glands hiperplasia. Mycobacterium avium subsp. paratuberulosis was isolated by bacterial
cultivation of samples from feces, intestinal mucosa and milk, and identified through IS900
PCR. From 298 cows, older than three years, the percentage of reactive animals was 40%, in
indirect ELISA test. The diagnosis of paratuberculosis was based on clinic-epidemiological
data, sorology, bacterial isolation and IS900 PCR. After the adoption of control measures, as
slaughter of sick cows and selective slaughter of soropositive animals, was observed reduction
of clinical cases in the herd, from six cases to one case per year, in three years of study. / Descreve-se os aspectos epidemiol?gicos e cl?nico-patol?gicos da paratuberculose em um
rebanho bovino leiteiro no munic?pio de Rio Claro, regi?o Sul-Fluminense, Rio de Janeiro.
No per?odo de 2006 a 2009, oito vacas adultas da ra?a Girolanda apresentaram diarreia
cr?nico-intermitente, perda de peso progressivo e apetite normal. ? necropsia observou-se
vasos linf?ticos subserosos proeminentes, linfonodos mesent?ricos aumentados de volume,
?midos ao corte, serosa do intestino com aspecto anelado e cerebr?ide, mucosa espessada,
pregueada e com aspecto microgranular. Em especial, no duodeno havia les?es polip?ides na
supef?cie da mucosa. ? microscopia, desde o duodeno at? o intestino grosso, havia acentuada
inflama??o granulomatosa difusa, marcada dilata??o dos vasos linf?ticos no ?pice das
vilosidades, linfangiectasia e linfangite granulomatosa na submucosa, muscular e serosa,
altera??es tamb?m vistas nos linfonodos mesent?ricos. A colora??o de Ziehl-Neelsen revelou,
vari?vel quantidade de bacilos ?lcool-?cido resistentes no interior de macr?fagos, c?lulas
gigantes de Langhans e livres na mucosa e submucosa do intestino delgado e grosso e em
linfonodos mesent?ricos. A l?mina pr?pria da mucosa, principalmente do jejuno e ?leo de
alguns animais, apresentava acentuada hipertrofia; as les?es polip?ides correspondiam ?
marcada hipertrofia da muscular da mucosa, hiperplasia de gl?ndulas duodenais e intestinais.
Mycobacterium avium subsp. paratuberculosis foi isolado em cultivo bacteriano a partir de
amostras de fezes, raspado de mucosa intestinal e leite e identificado pela t?cnica de PCR
IS900. Atrav?s da avalia??o sorol?gica semestral, foram analisadas 298 vacas, a partir de tr?s
anos de idade, e observou-se cerca de 40% de animais reagentes ao teste ELISA indireto no
per?odo estudado. O diagn?stico da paratuberculose foi baseado nos dados cl?nicopatol?gicos,
sorologia, isolamento e identifica??o do agente em amostras de fezes, raspado de
mucosa e leite, atrav?s do cultivo bacteriano e PCR IS900. Ap?s implementa??o de medidas
de controle, tais como elimina??o de animais doentes, abate seletivo dos soropositivos,
separa??o dos bezerros ao nascer com utiliza??o de banco de colostro, observou-se
diminui??o da ocorr?ncia de casos cl?nicos no rebanho, de seis casos por ano para cerca de um
caso por ano, em tr?s anos de estudo.
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The evolution of the pathogen Mycobacterium avium subsp paratuberculosisTurenne, Christine Yvette, January 1900 (has links)
Thesis (Ph.D.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2009/06/11). Includes bibliographical references.
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Studies on the interaction between Mycobacterium avium subsp. paratuberculosis and bovine mastitis associated Escherichia coli in a mammary epithelial cell model and identification of passive shedding in small ruminants / Estudos da interação entre Mycobacterium avium subsp. paratuberculosis e Escherichia coli associada à mastite bovina em um modelo de células epiteliais mamárias e identificação de transmissores passivos em pequenos rumantesSchwarz, David Germano Gonçalves 09 December 2016 (has links)
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Previous issue date: 2016-12-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mastite causada por Escherichia coli tem a capacidade de estimular intensamente o sistema imunológico e desencadear rapida inflamação na glândula mamária. Em contraste, Mycobacterium avium subsp. paratuberculosis (MAP), agente etiológico da paratuberculose, caracterizada por enterite granulomatosa crônica, pode infectar a glândula mamária sem estimular intensamente a resposta inflamatória. A interação dessas duas bactérias na glândula mamária ainda é desconhecida. Em alguns casos, tanto a eliminação de MAP pelo leite como pelas fezes podem ocorrer de forma passiva, após a infecção ascendente da glândula mamária ou após ingestão de MAP, respectivamente. Estes animais, chamados passive-shedders, são importantes como uma fonte de infecção a animais suscetíveis no rebanho. O objetivo deste estudo foi investigar a relação entre MAP e E. coli em células de glândula mamária sob condições experimentais e verificar a presença de animais passive-shedders. A relação entre uma cepa K-10 de MAP e E. coli isolada de leite mastítico em linhagem de células epiteliais mamárias (MAC-T) foi avaliada. As células previamente infectadas com MAP diminuíram a invasão de E. coli durante 120 min de experimentação. Contudo, a eficiência da translocação de E. coli e a viabilidade das células MAC-T não foram comprometidas. Ao contrário, células previamente infectadas por E. coli aumentaram a capacidade de translocação baso-apical de MAP até os 30 min e diminuiu aos 120min pós-infecção. A quantificação de citocinas relevou que a expressão de IL-1β aos 120min foi significativa (P<0.05) para células infectadas por MAP + E. coli e E. coli apenas. As expressões de MAPKp 38 e IL-10 não foram significativas, independente do tempo pós-infecção. Para detectar a ocorrência de animais passive-shedders, 10 propriedades foram previamente investigadas para a presença de MAP. Treze cabras foram positivas por cultura de fezes e/ou PCR de leite. Dentre os animais positivos, quatro (4/13) foram adquiridas e avaliados por IS900-PCR, cultura de fezes, de leite e de tecido, e sorologia (ELISA). Todos os resultados foram negativos no período de um ano, demonstrando que os animais realizaram o fenômeno pass-through e a contaminação ascendente da glândula mamária, sem tornarem-se infectados. No geral, esses resultados indicam que a presença de MAP nas células mamárias pode dificultar a capacidade de invasão de E. coli, mas quando no interior da célula mamária, translocam-se mais eficientemente. No entanto, quando as células são previamente infectadas por E. coli, MAP é rapidamente atraído da região subepitelial para a superfície celular. A produção de IL-1β intensifica a atração de macrófagos para o sítio de infecção, onde MAP se beneficia, infectando-os. / Mastitis caused by Escherichia coli can intensely stimulate the immune system and rapidly trigger inflammation in the mammary gland. In contrast, Mycobacterium avium subsp. paratuberculosis (MAP), the etiological agent of paratuberculosis, characterized by chronic granulomatous enteritis, can infect the mammary gland without intensely stimulating the inflammatory response. The interaction of these two species in the mammary gland is still unknown. In some cases, both the elimination of MAP by milk and faeces may occur passively, either through ascending infection of the mammary gland or through ingestion of MAP, respectively. These animals are called passive- shedders and are important as a source of infection to susceptible animals in the herd. The aim of this study was to investigate the relationship between MAP and E. coli in mammary gland cells under experimental conditions and verify the presence of passive- shedder animals. The relationship between a K-10 strain of MAP and E. coli isolated from mastitic milk in mammary epithelial cell lines was evaluated. Cells previously infected by MAP decreased E. coli invasiveness during 120min experimentation. However, the efficiency of E. coli translocation was not compromised, nor was the viability of the MAC-T cells. In contrast, cells previously infected by E. coli showed increased basal-apical translocation capacity of MAP up to 30 min and decreased at 120 min postinfection. Quantification of cytokines showed that IL-1β expression at 120 min was significantly increased in cells infected by MAP + E. coli and E. coli only. Expression of MAPKp 38 and IL-10 were not significant, regardless of time postinfection. To determine the occurrence of passive-shedders, 10 properties were previously investigated for MAP detection. Thirteen goats were positive by faeces culture and/or milk PCR. Among the positive animals, four (4/13) were evaluated by IS900-PCR, feces culture, milk and tissue culture and serology (ELISA). All the results were negative over a one-year period, demonstrating that the animals performed pass- through phenomenon and upward contamination of the mammary gland without becoming infected. Together, these results indicate that the presence of MAP in mammary cells may hamper capacity of E. coli invasion, but when within the mammary cell, the bacteria evade more efficiently. However, when the cells are pre-infected by E. coli, MAP is rapidly attracted from the subepithelial region to the cell surface. IL-1β production enhances the attraction of macrophages to the site of infection, where MAP benefits by infecting them.
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Epidemiologic investigations of mycobacterium avium subspecies paratuberculosis infections in Ohio dairy herdsNaugle, Alecia Larew 06 August 2003 (has links)
No description available.
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La paratubercolosi bovina causata dal Mycobacterium avium subsp paratuberculosis: un modello in vitro per studiare la risposta precoce all'infezione / Johne's disease in cattle caused by Mycobacterium avium subsp paratuberculosis: an in vitro model to study early response to infectionMARINO, ROSANNA 18 July 2013 (has links)
La malattia di Johne o paratubercolosi è un’enterite cronica granulomatosa provocata dal Mycobacterium avium subsp paratubercolosis (MAP), che colpisce i ruminanti ed in particolare i bovini da latte ed ha un grande impatto economico a livello mondiale. Il MAP sembra anche avere un ruolo nella malattia umana di Crohn.
Tale patogeno è capace di sopravvivere molto bene all’interno dei macrofagi dell’ospite dove previene la loro attivazione, blocca l’acidificazione e la maturazione del fagosoma, e interferisce con la presentazione degli antigeni al sistema immunitario.
Al fine di analizzare la complessa interazione tra l’ospite e il patogeno, è stata valutata la risposta dopo 2h, 6h, e 24h di macrofagi derivati da monociti bovini (MDM), coltivati in vitro e infettati con il ceppo L1 di MAP utilizzando un approccio di RNA-Seq.
L’analisi statistica dei dati di sequenza ha mostrato un aumento del numero di geni differenzialmente espressi durante l’esperimento in risposta all’infezione. Inoltre i geni sottoespressi negli MDM infettati sono stati individuati solo a 24h post-infezione.
L’analisi dei pathway ha evidenziato tre network che sono associati alla risposta immunitaria e al processo infiammatorio. Inoltre lo studio dei geni sottoespressi a 24h ha mostrato il ruolo centrale del complemento e del complesso maggiore di istocompatibilità nella patogenesi della malattia. / Johne’s disease (paratuberculosis) is a chronic granulomatous enteritis caused by Mycobacterium avium subsp paratubercolosis (MAP), affecting ruminants worldwide with a significant economic impact. MAP has also been speculated as a cause of human Crohn’s disease.
MAP is a pathogen highly adapted for survival within host macrophages due to the organism's capacity to prevent macrophage activation, block phagosome acidification and maturation, and attenuate presentation of antigens to the immune system. The consequence is a very long silent infection and subclinical phases.
To decipher the complex interaction between host and MAP, the response of in vitro bovine monocyte-derived macrophages (MDM) after 2h, 6h and 24h of infection with L1 strain of MAP was explored using RNA-Seq approach.
Statistical analysis of sequence data revealed an increasing number of differentially expressed genes in MDM following infection through the three time points analysed. Furthermore down-regulated genes were only found at 24 h post-infection.
Ingenuity Pathways Analysis of differentially expressed genes showed that “cell-mediated immune response” was the most significant network related to 2hpi dataset, “immune cell trafficking” for 6hpi, and “inflammatory response” for 24hpi. Finally the analysis of down-regulated genes at 24hpi confirmed the role of complement and major histocompatibility complex (MHC) in the pathogenesis of MAP in cattle.
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Paratuberkulose-Diagnostik beim Rind: Optimierung des Erregernachweises aus Kot mittels Kultivierung und PCRSchwalm, Anna Katharina 31 March 2020 (has links)
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 (Herrolds 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 Fishers 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
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Establishment of a Long Term Cell Culture Model for Testing Anti-Infectives against Mycobacterium avium subsp. paratuberculosisKimsawatde, Gade Carolyn 05 May 2015 (has links)
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.
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Paratuberculose em bovinos de corte na região Sul do Rio Grande do Sul: ocorrência e diagnóstico diferencial / Paratuberculosis in beef cattle in the southern region of Rio Grande do Sul: occurrence and differential diagnosisFiss, Letícia 30 September 2015 (has links)
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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.
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