Made available in DSpace on 2015-03-26T13:47:19Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1625922 bytes, checksum: 8e4e59f3e5a2481a04957bd2da9aa918 (MD5) Previous issue date: 2012-09-04 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of paratuberculosis, a disease which causes chronic granulomatous enteritis preferably in domestic and wild ruminants. It is considered a disease of economic and social impact, because of losses in livestock and importance in human health, since there is evidence of possible correlation of MAP with Crohn's disease. However, much is still unknown about the developing the pathophysiology and immunopathogenesis this disease, in the sites of infection. Thus, this study has proposed verify the response of cytokines proinflammatory and anti-inflammatory by quantitative PCR and associate the expression of these cytokines with the lesions and the presence of the agent in organs of C57BL/6 mice infected intraperitoneal with strain MAP66115-98. It was applied 250μl of inoculum at 3 x 108 UFC/ml in 20 mice, and it was applied saline phosphate buffers (PBS) in eight control species. The animals were sacrificed at 30, 60, 90 and 120 days post-inoculation. In each period were collected spleen, liver, intestine (ileum and colon) and Peyer's patches of five challenged mice and two controls. It was found in the majority an upregulation in the expression profiles of proinflammatory cytokines IL-2, IFN-, and TNF-α ү, and anti-inflammatory IL-4 and IL-10 analyzed in the organs compared to controls during period of the experiment. It was observed also negative regulations in the fourth week in spleen and colon, twelfth in the spleen, ileum and Peyer's patch and the sixteenth week in the liver, colon and ileum. Statistical differences were only observed at the eighth week after the challenge for IFN-ү in liver, in spleen for expression of IL-2 and IFN-ү and in the ileum for IL-4 and TNF-α. In the fourth week, it was possible to identify increased levels of mRNA transcripts of the cytokines studied. In the liver, ileum and colon IFN-ү showed the highest level of mRNA transcripts compared to the negative control, however at higher levels spleen were observed for IL- 10. Only eight weeks it was possible to observe a statistically significant difference for the cytokine IFN-ү in the liver and spleen samples for TNF-α and IL-4 in the ileum and IL-2 in spleen. The highest levels of mRNA transcript were observed in the liver for IFN- ү, spleen for IL-2, and in the ileum Peyer's patch for IL-4 and colon for IL-10. In the twelfth week, the major mRNA transcript levels were observed in liver for IL-10 in spleen and ileum for IL-2 in colon for TNF-α and Peyer's patch for IL-4. In the sixteenth week, the major mRNA transcript levels were observed in liver and colon for IL-10 in spleen and ileum for IFN-ү and Peyer's patch for TNF-α. The animals showed increased expression of mRNA transcripts of cytokines studied organs after challenge with MAP, although not verified gross and microscopic changes (hematoxylin and eosin staining). However it was possible to observe the presence of alcohol-acid resistant rods in two samples of Peyer's patches (Ziehl-Neelsen) and nested-PCR technique was possible to observe the presence of MAP in every organ analyzed. Thus, these results contribute to a better understanding of the expression of proinflammatory and anti-inflammatory in local infection at different post-inoculation in both organs taken as site specific as MAP intestine (colon and ileum) and Peyer's patch and organs extraintestinal, and thus find a better understanding of the immunopathogenesis of infection in the local. / Mycobacterium avium subespécie paratuberculosis (MAP) é o agente etiológico da paratuberculose, uma enfermidade que causa enterite granulomatosa crônica preferencialmente em ruminantes domésticos e silvestres. É considerada uma doença de impacto econômico e social, devido às perdas no rebanho e na saúde humana, uma vez que se têm indícios da possível relação de MAP com a doença de Crohn. Contudo, muito ainda se desconhece sobre o desenvolvimento da patofisiologia e imunopatogênese desta doença, nos locais da infecção. Desse modo, este estudo propôs verificar a resposta de citocinas pró e anti-inflamatórias por meio da técnica de PCR quantitativo e associar a expressão destas citocinas com as lesões e a presença do agente em órgãos de camundongos C57BL/6 infectados por via intraperitoneal com a cepa MAP66115-98. Em 20 camundongos foram aplicados 250μl de inóculo a 3 x 10 8 UFC/ml e, em oito controles foram aplicados PBS. Os animais foram sacrificados aos 30, 60, 90 e 120 dias pós-inoculação. Em cada período, foram coletado baço, fígado, intestino (cólon e íleo) e placas de Peyer de cinco camundongos desafiados e de dois controles. Verificou-se na maioria, uma regulação positiva nos perfis de expressões das citocinas pró-inflamatórias IL-2, IFN-ү e TNF-α, e anti-inflamatórias IL-4 e IL-10 nos órgãos analisados em relação aos controles durante o período do experimento. Observaram-se também regulações negativas na quarta semana no baço e colón, na décima segunda no baço, íleo e placa de Peyer e na décima sexta semana no fígado, colón e íleo. Diferenças estatísticas foram observadas somente na oitava semana após o desafio no fígado para IFN-ү, no baço para expressão da IL-2 e IFN-ү e no íleo para IL-4 e TNF-α. Na quarta semana, foi possível identificar aumento dos níveis de transcritos de RNAm das citocinas estudadas. No fígado, íleo e colón o IFN-ү apresentou maior nível de transcritos de RNAm em relação ao controle negativo, no entanto no baço os maiores níveis foram observados para a IL-10. Somente na oitava semana foi possível observar diferença estatística significativa para a citocina IFN-ү, em amostras do fígado e baço, para o TNF-α e IL-4 no íleo e para a IL-2 no baço. Os maiores níveis de transcrito de RNAm foram observados no fígado, para o IFN-ү , no baço para a IL-2 , no íleo e placa de Peyer para a IL-4 e no colón para a IL-10. Na décima segunda semana, os maiores níveis de transcrito de RNAm foram observados no fígado para a IL-10, no baço e íleo para a IL-2, no colón para o TNF-α e na placa de Peyer para a IL-4. Na décima sexta semana, os maiores níveis de transcrito de RNAm foram observados no fígado e colón para a IL-10, no baço e íleo para o IFN-ү e na placa de Peyer para o TNF-α. Os animais apresentaram uma maior expressão dos transcritos de RNAm das citocinas estudadas nos órgãos após o desafio com MAP, embora não tenham sido verificadas alterações macro e microscópica (coloração de Hematoxilina e Eosina). No entanto foi possível observar presença de bastonetes álcool ácido resistentes em duas amostras de placas de Peyer, (coloração de Ziehl-Neelsen) e pela técnica de nested-PCR foi possível observar a presença de MAP em todos os órgão analisados. Assim, estes resultados contribuem para a melhor compreensão da expressão de citocinas pró e anti-inflamatória nos locais da infecção em diferentes tempos pós-inoculação tanto em órgãos tidos como sitio específicos de MAP como intestino (colón e íleo) e placa de Peyer e órgãos extra-intestinais, e com isso buscar o melhor entendimento da imunopatogênese nos locais da infecção.

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

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.

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.

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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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.

Ziel der vorliegenden Arbeit war es, Screening–Methoden zu prüfen, um auf einfache, kostensparende Weise und mit ausreichender statistischer Sicherheit festzustellen, ob der Erreger der Paratuberkulose (Mycobacterium avium ssp. paratuberculosis) in einer Herde vorhanden ist oder nicht. Dazu wurden zwei auf dem kulturellen Erregernachweis beruhende Verfahren, die Untersuchung individueller Kotproben auf der Basis von Stichproben und die Untersuchung von Umgebungskotproben, einem serologischen Untersuchungsansatz, dem Nachweis von MAP–Antikörpern nach Aufkonzentrierung in gepoolten Einzelmilchproben, gegenübergestellt. Als Referenzmethode diente die kulturelle Einzeltieruntersuchung aller über 24 Monate alten Tiere des jeweiligen Bestandes. In 20 Thüringer Milchviehherden mit bekannter Einzeltierprävalenz wurden 5063 Einzeltierkotproben, 200 Umgebungskotproben und 262 aufkonzentrierte Milchpools aus 4337 laktierenden Rindern untersucht. Zusätzlich wurde eine systematische retrospektive Stichprobenkalkulation (nStichprobe = 1458 Einzeltierkotproben) vorgenommen. Die Kultivierung der Einzeltierkotproben und der Umgebungskotproben erfolgte über 12 Wochen auf HEYM–Nährmedium mit anschließender Speziesidentifizierung durch PCR und Ziehl–Neelsen–Färbung. Die Umgebungskotproben wurden zu zwei Untersuchungszeitpunkten (Frühjahr und Sommer) an jeweils fünf Lokalisationen eines Betriebes entnommen: Abkalbebereich, Melkbereich einschließlich Vorwartehof, Laufbereich, Haupttriebweg, Übergang zum Kälberbereich. Die Untersuchung der Milchpools erfolgte nach vorheriger Aufkonzentrierung mittels zweier verschiedener ELISAs. Im Frühjahr entnommene Umgebungskotproben aus 16 MAP–positiven Betrieben detektierten das Vorhandensein des Erregers in neun Betrieben (56,3 %). Betriebe mit einer Einzeltierprävalenz von über 4,5 % wurden in neun von zehn Fällen (90 %) sicher erkannt. Im Sommer entnommene Umgebungskotproben fielen durch eine sehr starke Kontamination auf. Von den 16 MAP–positiven Beständen wurden 15 Herden (93,7 %) mittels Stichprobenuntersuchung als Bestand mit Paratuberkulosevorkommen identifiziert, wobei lediglich ein Bestand mit einer Einzeltierprävalenz von 0,49 % nicht detektiert wurde. Die serologische Untersuchung der Milchpools lieferte keine verwendbaren Ergebnisse. Mit Hilfe der Untersuchung von Umgebungskotproben lassen sich Herden mit einer durch kulturelle Untersuchung ermittelten Einzeltierprävalenz von 4,5 % und darüber mit hinreichender Sicherheit auffinden. Bei der Bewertung dieses Schwellenwertes ist zu beachten, dass bei Verwendung von nur einem Kulturröhrchen je Kotprobe von einer Sensitivität der Methode von 60 % im Vergleich zur Verwendung von drei Kulturröhrchen auszugehen ist. Für eine Überwachung unverdächtiger Herden ist die Sensitivität dieses Untersuchungsansatzes jedoch zu gering. Die individuelle kulturelle Untersuchung einer Stichprobe zeigte eine ausreichend hohe Sensitivität, um bei der Überwachung größerer unverdächtiger Herden eingesetzt werden zu können. Ein Einsatz serologischer Milchuntersuchung ist zur Bewertung von Beständen zur Ermittlung des MAP–Infektionsstatus gegenwärtig nicht zu empfehlen. Für die Überwachung größerer, als Paratuberkulose–unverdächtig anerkannter Bestände ist somit ein wechselnder Einsatz von Einzeltierkotproben aller Rinder über 24 Monaten und einer aussagekräftigen systematischer Stichprobenuntersuchung möglich und trägt damit zur Erleichterung der derzeit noch zeit– und kostenintensiven Paratuberkulose–Diagnostik bei.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic, debilitating enteritis of cattle, other domestic livestock and some wildlife species. JD was first identified in the late 1800s and today it is a worldwide problem in dairy cattle. Heavily infected cows have reduced milk production, a higher risk of removal from the herd and low slaughter value. Several countries have implemented national level control strategies. In New Zealand, JD was first reported in 1912 and today the prevalence of infected dairy herds is thought to be high. To improve our understanding of the epidemiology of JD and to evaluate the feasibility of a national control strategy, four studies were conducted. The first study was a questionnaire based case-control study to identify associations between management practices and the occurrence of clinical JD on farms from four regions of New Zealand. The second study was on the effect of sub-clinical JD on milk production and the risk of removal from the herd in four dairy herds over four milking seasons. The effect of misclassification of disease status on productivity estimates was also studied. In the third study diagnostic test result data from the productivity study was combined with a novel Bayesian regression model to estimate performance of the ELISA and faecal culture tests as a function of covariates and utilising repeated tests on individual cows. Finally, results from these three studies were used to adapt an existing JD simulation model, 'JohneSSim', to represent the epidemiological behaviour of JD in New Zealand dairy herds. Control strategies for the disease were simulated and evaluated based on their cost effectiveness. Of the 427 farmers responding to the questionnaire, 47% had suspected clinical cases of JD in their herd in the preceding 5 years. Only 13% of suspected infected herds had an average incidence of greater than 0.5 cases per 100 cow years at risk. The disease was not considered a serious problem by 20% of herd managers who reported the presence of disease in the preceding 5 years. The presence of Jersey cows in the herd and the purchase of bulls had strong positive associations with the presence of clinical JD. Grazing calves in the hospital paddock, larger herds, the purchase of heifers, and the use of induction were also positively associated with JD. In the productivity study the herd-level prevalence of JD by ELISA and/or faecal culture ranged from 4.5% (95% CI 2.6-6.9) to 14.2% (95% CI 9.2-20.6). Daily milksolids production by JD positive cows was 0.8% (95% CI -6.1%-4.5%) less than that of JD negative cows. However in herd D, JD positive cows produced 15.5%, (95% CI 6.75%-24.2%) milksolids less than JD negative herd mates daily. This equates to a loss of 53kg of milksolids/305 day lactation, or NZD 265/lactation, given a price of NZD 5/kg of milksolids. In herd D only, the annual hazard ratio of removal for JD positive cows was significantly increased. It was 4.7 times and 1.4 times higher in cows older than 5 years and younger than 5 years. The results were insensitive to misclassification. Analysis of the diagnostic test data demonstrated the strengths of our Bayesian regression model. While overall estimates of sensitivity and specificity by this method were comparable to estimates by existing methods, it showed a broad trend of increasing sensitivity in higher parity groups and higher sensitivity in early, relative to late, lactation. It also showed that estimates of prevalence may in fact decline with repeated, relative to single, testing. Our novel approach demonstrated trends that could not be shown by existing methods, but could be improved by application to a larger data set. Simulation showed that control strategies for JD based on either test-and-cull, vaccination, breeding for genetic resistance, or removal of offspring from clinically affected cows, were not cost effective for the average infected herd. Improvement of the hygiene associated with calf management provided the greatest reduction in the within-herd prevalence of JD. While JD is present in a high proportion of New Zealand dairy herds, the incidence of clinical cases is usually low, and most farmers consider it to be of little importance. However, JD causes significant losses in productivity in some herds. The disease would probably be best controlled on a herd-by-herd basis, given the limited success of national-scale control programs for JD in other countries. The education of dairy farmers regarding risky management practices, and the offer of a risk assessment to farmers wishing to control the disease, would provide a combination of wide reaching and targeted approaches, of low cost, for JD control. It seems likely that JD will persist in some capacity in the years ahead, but will remain of minor concern next to major animal health issues, such as infertility and mastitis. Clarification of the effect of genetic strain on the virulence of MAP may help explain differences in the effect of the disease between herds. This knowledge could then be used to further improve the efficiency of JD control.

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