Orientador: Antonio Carlos Paes / Resumo: A tuberculose causada por Mycobacterium bovis (bTB) é uma zoonose de distribuição mundial com ampla gama de hospedeiros. Nos países onde a bTB é prevalente, 10 a 20% dos casos de tuberculose humana são causados por M. bovis. São escassos em todo o mundo estudos que investigam a resistência à isoniazida (INH) e rifampicina (RMP) em linhagens de M. bovis de origem bovina, reservatórios silvestres, e em casos humanos de tuberculose. Foi investigada a diversidade genotípica de 67 linhagens de M. bovis isoladas de bovinos de abatedouro, obtidas de 100 linfonodos com lesão caseosa, pelas técnicas de Spoligotyping e MIRU-VNTR, bem como foi determinado o perfil fenotípico de resistência à INH e RMP pela técnica de REMA, e pesquisadas possíveis bases genéticas para resistência aos antimicrobianos. Dentre os isolados, 11 (16%) foram classificados como MDR-TB, 8 (12%) resistentes à INH e 2 (3%) resistentes à RMP. A pesquisa pelo GenoType MTBDRplus ver. 2.0 não acusou a presença de mutações em nenhum dos isolados fenotipicamente resistentes. Foram identificados 16 spoligotipos entre as linhagens. A subfamília BOV_1 predominou com 52 (77,6%) isolados, com os SIT 481, 482, 594, 665, 691, 698, 1021, 1667, 1852, 2141 e dois isolados sem shared type. A BOV_2 foi identificada em 8 (11,9%) isolados, com o SIT 683. Os SIT 982, 1851 e 1853 foram agrupados na família BOV. Dois isolados não foram classificados em família ou subfamília. A análise de MIRU-VNTR com painel de 12 MIRUs, identificou 31 i... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Tuberculosis caused by Mycobacterium bovis (bTB) is a zoonosis of worldwide distribution with broad host range. In countries where bTB is prevalent, 10-20% of the human cases of tuberculosis are caused by M. bovis. All over the world there are few studies investigating the resistance to isoniazid (INH) and rifampicin (RMP) in M. bovis strains from cattle, wild reservoirs, and human cases of tuberculosis. The genotypic diversity of 67 M. bovis strains obtained out of 100 lymph nodes with caseous lesions from slaughtered animals was investigated by Spoligotyping and MIRU-VNTR techniques, as well as the assessment of their phenotypic profile of resistance to INH and RMP by REMA method and the search of possible genetic basis for antimicrobial resistance. Among the obtained isolates, 11 (16%) were classified as MDR-TB, 8 (12%) INH-resistant and 2 (3%) RMP-resistant. The use of GenoType MTBDRplus ver. 2.0 did not pointed the presence of genetic mutations in any of the phenotypically resistant isolates. Sixteen different spoligotype patterns were identified. The BOV_1 subfamily predominated with 52 (77.6%) isolates, with SITs 481, 482, 594, 665, 691, 698, 1021, 1667, 1852, 2141 and two isolates without a given SIT. BOV_2 was identified in 8 (11.9%) isolates, within SIT 683. The SITs 982, 1851 and 1853 were grouped in BOV family. Two isolates were not classified in family or subfamily. The MIRU-VNTR analysis using the 12 classical MIRUs, identified a cluster of 31 isolates belonging... (Complete abstract click electronic access below) / Doutor

Yes / Background: Tracking recent transmission is a vital part of controlling widespread pathogens such as Mycobacterium tuberculosis. Multiple methods with specific performance characteristics exist for detecting recent transmission chains, usually by clustering strains based on genotype similarities. With such a large variety of methods available, informed selection of an appropriate approach for determining transmissions within a given setting/time period is difficult. Methods: This study combines whole genome sequence (WGS) data derived from 324 isolates collected 2005–2010 in Kinshasa, Democratic Republic of Congo (DRC), a high endemic setting, with phylodynamics to unveil the timing of transmission events posited by a variety of standard genotyping methods. Clustering data based on Spoligotyping, 24-loci MIRU-VNTR typing, WGS based SNP (Single Nucleotide Polymorphism) and core genome multi locus sequence typing (cgMLST) typing were evaluated. Findings: Our results suggest that clusters based on Spoligotyping could encompass transmission events that occurred almost 200 years prior to sampling while 24-loci-MIRU-VNTR often represented three decades of transmission. Instead, WGS based genotyping applying low SNP or cgMLST allele thresholds allows for determination of recent transmission events, e.g. in timespans of up to 10 years for a 5 SNP/allele cut-off. Interpretation: With the rapid uptake of WGS methods in surveillance and outbreak tracking, the findings obtained in this study can guide the selection of appropriate clustering methods for uncovering relevant transmission chains within a given time-period. For high resolution cluster analyses, WGS-SNP and cgMLST based analyses have similar clustering/timing characteristics even for data obtained from a high incidence setting. / ERC grant [INTERRUPTB; no. 311725] to BdJ, FG and CJM; an ERC grant to TS [PhyPD; no. 335529]; an FWO PhD fellowship to PM [grant number 1141217N]; the Leibniz Science Campus EvolLUNG for MM and SN; the German Centre for Infection Research (DZIF) for TAK, MM, CU, PB and SN; a SNF SystemsX grant (TBX) to JP and TS and a Marie Heim-Vögtlin fellowship granted to DK by the Swiss National Science Foundation. The computational resources and services used in this work were provided by the VSC (Flemish Supercomputer Center), funded by the Research Foundation - Flanders (FWO) and the Flemish Government – department EWI.

Die Rindertuberkulose ist eine chronische Erkrankung, die von Mycobacterium (M.) bovis und M. caprae, Mitgliedern des Mycobacterium-tuberculosis-Komplex (MTC), ausgelöst wird. Tuberkulose-Erregern werden sowohl mittels kultureller als auch molekulare Untersuchungsmethoden nachgewiesen. Ziel der vorliegenden Studie war es, die Sensitivität des DNA-Nachweises von Tuberkulose-Erregern zu steigern. Dafür wurden drei Fragestellung im Bereich der molekularen Mykobakterien-Diagnostik bearbeitet. I) Zur Verbesserung der Lyse der mykobakteriellen Zellwand als Voraussetzung für eine Zunahme der Freisetzung von DNA wurden im Vergleich zu einer standardisierten DNA-Isolierungsmethode vier verschiedene Lyseprotokolle (thermische, enzymatische, thermo-enzymatische und mechanische Lyse) entwickelt und mit M. bovis BCG durchgeführt. Die Verbesserung wurde anhand der cycle threshold (Ct)-Werte einer MTC-spezifischen Real-Time (rt) Polymerase-Kettenreaktion (PCR) geprüft. Zwei Lyseprotokolle (thermische und mechanische Lyse) wurden bei zehn Gewebeproben (Lymphknoten, Leber und Lunge) von zehn Tieren (acht Rinder, ein Lama und ein Luchs) mit nachgewiesener Tuberkulose ange-wendet. II) Ausserdem, wurde eine rt-PCR mit dem 16S rRNA Gen als Zielgen (16S-rt-PCR) für den direkten Nachweis von Erregern der Gattung Mycobacterium im Gewebe entwickelt. III) Ein neu entwickelter Spoligotyping-Microarray wurde mit der konventionellen Spoligoty-ping-Methode verglichen, um die neue Methode in Bezug die Sensitivität des Nachweises und des diskriminatorischen Potenzials direkt bei infizierten Gewebeproben zu analysieren. Bei der konventionellen Methode erfolgt die Hybridisierung des PCR-Produktes auf einer Nylon Membran, auf der spezifische Oligonukleotide fixiert sind. Bei der Microarray-Methode sind diese auf einem Microarray-Chip fixiert. Die Ergebnisse der Untersuchungen (I) zur Lyse der Zellwand bei M. bovis BCG zeigten, dass bei der mechanischen Lyse eine Zunahme um 14 % und bei der thermischen Lyse eine Zunahme an PCR-Produkt von 6 % im Vergleich zur Standardlyse erbrachte. Bei beiden Lyseprotokollen wurde eine statistische Signifikanz von α = 1 % (Mann-Whitney-Test) im Vergleich zur Standardlyse errechnet. Bei den tuberkulösen Gewebeproben wurde bei der mechanischen Lyse eine durchschnitliche Zunahme an PCR-Produkt um circa 9 % im Vergleich zur Standardlyse erzielt. Dieser Unterschied war jedoch auf Grund der geringen Probeanzahl nicht statistisch signifikant. II) Bei der Untersuchung von 43 Mykobakterien-Spezies, sechs Mitgliedern des MTC (unter anderen M. bovis BCG) und 37 Non Tuberculous Mycobacteria (NTM) Spezies, konnten alle mit der entwickelten Real-Time PCR (16S-rt-PCR) nachgewiesen werden. DNA-Extrakte von acht nicht zur Gattung Mycobacterium gehörenden Spezies wurden mit der 16S-rt-PCR nicht erfasst. Ein Erreger der Gattung Gordonia und ei-ner der Gattung Rhodococcus wurden auf Grund ihres engen Verwandtschaftsgrades jedoch ebenfalls mit der 16S-rt-PCR detektiert. Die oben erwähnten mittels MTC-spezifischer rt-PCR (Zielgen IS 1081) als infiziert identifizierten zehn Gewebeproben, wurden mittels 16S-rt-PCR untersucht. Die Ergebnisse zeigten, dass beiden rt-PCR Systeme eine vergleichbare Sensitivität aufwiesen. III) Bei dem Vergleich zwischen den Spoligotyping-Methoden zeigte sich die neue Methode um einen Faktor von 100 bei der M. bovis BCG-Reinkultur und um einen Fak-tor von 10 bei DNA-Extrakten aus tuberkulösen Gewebeproben sensitiver als die konventionelle Methode. Im Rahmen dieser Arbeit hat sich der Einsatz der mechanischen Lyse für die Verbesserung der Freisetzung von mykobakterieller DNA als routinefähig erwiesen. Die entwickelte 16S-rt-PCR erwies sich als brauchbare Methode für den Nachweis von Erregern der Gattung Mycobacterium. Die Microarray-Methode stellte sich wesentlich einfacher, sensitiver und schneller dar als die konventionelle Methode. Zusammenfassend kann gesagt werden, dass alle drei Ansätze dieser Arbeit einen Beitrag zur Verbesserung der molekularen Labordiagnostik der Tuberkulose leisten. / Bovine tuberculosis is a chronic disease, that results from infection of Mycobacterium (M.) bovis and M. caprae, members of Mycobacterium tuberculosis complex (MTC), respectively. The laboratory diagnosis of bovine tuberculosis is possible with culture as well as considerate fast molecular methods. The aim of this study was to improve the sensitivity of DNA detec-tion of tuberculosis-causing pathogens. Therefore, three different issues were addressed in the complex molecular procedure targeted. I) four different lytic protocols (thermal, enzymatic, thermo-enzymatic and mechanical lysis) were developed and compared to a standardized DNA isolation protocol that was performed on pure culture of Mycobacterium (M.) bovis BCG in order to improve the mycobacterial cell wall lysis leading to an increase of DNA release. The efficiencies of the lysis protocols were assessed by the resulting cycle threshold (Ct) values of a MTC-specific real time (rt) Polymerase Chain Reaction (PCR). Two lysis protocols (thermal and mechanical) were selected to fur-ther testing of ten tuberculosis-infected tissue samples (lymph nodes, liver and lungs) from ten animals (eight cattle, one lama and one lynx). II) In addition, a real time PCR using the 16S rRNA gene as target sequence was developed, which is also suitable to detect pathogens of Genus Mycobacterium on tissue samples. III) A comparison of a newly developed microarray and the conventional spoligotyping method was realised, to analyse the applicability of the microarray in relation of the sensitivity of the method and to analyse discriminatory potential directly from infected tissue samples. During the conventional spoligotyping method, the PCR product is hybridized with specific oligonucleotides, fixed on a nylon membrane. Using the newly developed method these oligonucleotides are fixed on a microarray-chip. The results I) of the mycobacterial cell wall lysis experiment with pure culture of M. bovis BCG showed an increase of 14 % by using mechanical lysis and an increase by 6 % of the PCR product by using thermal lysis compared to the standard protocol. Using the mechanical lysis as well as the thermal lysis a statistically significant (α = 1 % (Mann-Whitney-Test)) im-provement compared to the standard lysis was achieved. Mechanical lysis was performed on tuberculous tissue samples and the results of the lysis were improved by 9 % compared to the standard lysis. However, the difference between mechanical and standard lysis was not statistically significant due the small sample number. II) Forty-three different mycobacterial species, six members of MTC (among them M. bovis BCG) and 37 Non Tuberculous Mycobacteria (NTM), were detected using the newly developed real time PCR (16S-rt-PCR). Eight non-mycobacterial species were not detected using this rt-PCR, whereas one of genus Rhodococcus and one of genus Gordonia were detected by the 16S-rt-PCR due to their close genetic similarity to the genus Mycobacterium. The ten tuberculosis-infected tissue samples (see above) testing positive using a MTC-specific real time rt-PCR (target gene IS 1081) were sub-jected to the 16S-rt-PCR. Both rt-PCR systems showed a comparable sensitivity. III) By comparing the two spoligotyping-methods, the ArrayStrip™–format method was more sensitive than the conventional method by a factor of 100 applied to pure culture and by a factor of 10 when applied to DNA extracts from infected tissue samples. In conclusion, the mechanical lysis proved to be a practical method to liberate mycobacterial DNA. The newly developed rt-PCR was suitable to detect members of the genus Mycobacterium. The spoligotyping ArrayStrip™–format method appeared to be substantially easier to perform, more sensitive, and less time-consuming than the conventional method. The three methods described were suitable to improve the molecular laboratory diagnosis of tuberculosis.

Made available in DSpace on 2016-12-23T13:55:58Z (GMT). No. of bitstreams: 1 Solange Alves Vinhas.pdf: 4494197 bytes, checksum: c05b3a06ae983246a835977f36eb32e3 (MD5) Previous issue date: 2013-08-30 / Background: Based on the hypothesis that genetic variability of Mycobacterium tuberculosis (MTB) could influence virulence and immunopathology we analyzed genetic profiles of different MTB strains in order to detect relatedness between genetic diversity and presence of cavity (disease severity). Methods: We conducted a retrospective molecular study in Vitória ES, based on TB strains (2003 to 2006, n = 214) from patients with pulmonary cavitary and non-cavitary TB using IS6110-RFLP, Spoligotyping and MIRU-VNTR methodologies. RESULTS: Initially, we compared the association of the demographic and clinical characteristics of patients with the presence of cavities. After logistic regression the variables that most contributed to explain the model of the disease were smear positive (ORajust = 5.96; IC= 2.58-13.73) and sputum production (ORajust = 4.55; IC= 1.28-16.12), there was no statistically significant association with the remaining variables. The LAM family was the most frequent within the samples of the two groups analyzed, representing 65 (62%) of the isolates in the cavitary group and 40 isolates (38%) of the non-cavitary. After comparing the proportions of LAM and other spoligotyping families there was no statistically significant difference between the groups (p=0.17). In relation to deletions RDRio (p=0.65) and RD174 (p=0.65) there were no statistically significant difference between the groups. Amongst the 205 isolates analyzed, 25 (12%) belonging to the non-cavitary group and 43 (21%) belonging to the cavitary group, were grouped in clusters. The statistical analysis of the association of the occurence of clusters with the presence of cavity showed no statistically significant difference between the quantity of clusters and the groups that were analyzed, (p= 0.4). Conclusion: The genotipic profile for the isolates from patients with cavitary and non-cavitary disease was determined. Our data showed that LAM9 was the most frequent among the strains between cavitary and noncavitary groups, corroborating findings that this family is the most frequent in Brasil. There were no statistical differences that could show association among the variables analyzed related to presence of cavity or disease severity / Introdução: Baseado na hipótese de que a variabilidade genética de Mycobacterium tuberculosis (MTB) pode influenciar a virulência e a gravidade da doença os perfis genéticos de isolados clínicos de MTB foram avaliados para detectar associação entre diversidade genética e gravidade da doença. Objetivos: Analisar características genéticas de isolados de MTB e verificar sua possível associação com a gravidade da TB pulmonar. Métodos: Estudo retrospectivo, caso controle, conduzido em Vitória-ES, utilizando isolados de MTB (2003 a 2006, n=214) de pacientes com TB pulmonar, cavitária (127) e não cavitária (87). Realizou-se genotipagem por meio de RFLP-IS6110, Spoligotyping, MIRU-VNTR 24 loci, e a análise de deleções e inserções, como RDRio, RD174 utilizando PCR multiplex, bem como a detecção do Ag85C103. Realizou-se análise estatística, para verificação dos padrões de distribuição das variáveis, seguida de análises bivariadas para verificação de associações entre elas, empregando-se os teste exato de Fisher ou Chi-quadrado, ambos com 95% de intervalo de confiança e nível de significância (&#61554;) < 0,05. Resultados: Após a regressão logística, as variáveis que contribuíram no modelo explicativo da doença foram baciloscopia (ORajust = 5,96; IC= 2,58-13,73) e produção de escarro (ORajust = 4,55; IC= 1,28- 16,12). Não houve associação estatisticamente significativa com o restante das variáveis.A família LAM foi a mais frequente entre os dois grupos analisados, representando 65 (62%) dos isolados no grupo cavitário e 40 isolados (38%) do grupo não cavitário. Não houve diferença estatisticamente significativa entre os grupos em relação à deleção RDRio (p=0,65) e com relação à deleção RD174 (p=0,65). Dentre os 205 isolados analisados, 25 (12%) isolados do grupo não cavitário e 43 (21%) do grupo cavitário, estavam em cluster. não houve diferença estatisticamente significativa entre a quantidade de clusters e os grupos analisados (p= 0,4). Conclusões: Foi determinado o perfil genotípico dos isolados de pacientes com doença pulmonar, cavitária e não cavitária. Não houve associação entre a presença de cavidade e os genótipos encontrados. Não houve associação do genótipo com nenhum dos marcadores moleculares avaliados

Die Rindertuberkulose ist eine chronische Erkrankung, die von Mycobacterium (M.) bovis und M. caprae, Mitgliedern des Mycobacterium-tuberculosis-Komplex (MTC), ausgelöst wird. Tuberkulose-Erregern werden sowohl mittels kultureller als auch molekulare Untersuchungsmethoden nachgewiesen. Ziel der vorliegenden Studie war es, die Sensitivität des DNA-Nachweises von Tuberkulose-Erregern zu steigern. Dafür wurden drei Fragestellung im Bereich der molekularen Mykobakterien-Diagnostik bearbeitet. I) Zur Verbesserung der Lyse der mykobakteriellen Zellwand als Voraussetzung für eine Zunahme der Freisetzung von DNA wurden im Vergleich zu einer standardisierten DNA-Isolierungsmethode vier verschiedene Lyseprotokolle (thermische, enzymatische, thermo-enzymatische und mechanische Lyse) entwickelt und mit M. bovis BCG durchgeführt. Die Verbesserung wurde anhand der cycle threshold (Ct)-Werte einer MTC-spezifischen Real-Time (rt) Polymerase-Kettenreaktion (PCR) geprüft. Zwei Lyseprotokolle (thermische und mechanische Lyse) wurden bei zehn Gewebeproben (Lymphknoten, Leber und Lunge) von zehn Tieren (acht Rinder, ein Lama und ein Luchs) mit nachgewiesener Tuberkulose ange-wendet. II) Ausserdem, wurde eine rt-PCR mit dem 16S rRNA Gen als Zielgen (16S-rt-PCR) für den direkten Nachweis von Erregern der Gattung Mycobacterium im Gewebe entwickelt. III) Ein neu entwickelter Spoligotyping-Microarray wurde mit der konventionellen Spoligoty-ping-Methode verglichen, um die neue Methode in Bezug die Sensitivität des Nachweises und des diskriminatorischen Potenzials direkt bei infizierten Gewebeproben zu analysieren. Bei der konventionellen Methode erfolgt die Hybridisierung des PCR-Produktes auf einer Nylon Membran, auf der spezifische Oligonukleotide fixiert sind. Bei der Microarray-Methode sind diese auf einem Microarray-Chip fixiert. Die Ergebnisse der Untersuchungen (I) zur Lyse der Zellwand bei M. bovis BCG zeigten, dass bei der mechanischen Lyse eine Zunahme um 14 % und bei der thermischen Lyse eine Zunahme an PCR-Produkt von 6 % im Vergleich zur Standardlyse erbrachte. Bei beiden Lyseprotokollen wurde eine statistische Signifikanz von α = 1 % (Mann-Whitney-Test) im Vergleich zur Standardlyse errechnet. Bei den tuberkulösen Gewebeproben wurde bei der mechanischen Lyse eine durchschnitliche Zunahme an PCR-Produkt um circa 9 % im Vergleich zur Standardlyse erzielt. Dieser Unterschied war jedoch auf Grund der geringen Probeanzahl nicht statistisch signifikant. II) Bei der Untersuchung von 43 Mykobakterien-Spezies, sechs Mitgliedern des MTC (unter anderen M. bovis BCG) und 37 Non Tuberculous Mycobacteria (NTM) Spezies, konnten alle mit der entwickelten Real-Time PCR (16S-rt-PCR) nachgewiesen werden. DNA-Extrakte von acht nicht zur Gattung Mycobacterium gehörenden Spezies wurden mit der 16S-rt-PCR nicht erfasst. Ein Erreger der Gattung Gordonia und ei-ner der Gattung Rhodococcus wurden auf Grund ihres engen Verwandtschaftsgrades jedoch ebenfalls mit der 16S-rt-PCR detektiert. Die oben erwähnten mittels MTC-spezifischer rt-PCR (Zielgen IS 1081) als infiziert identifizierten zehn Gewebeproben, wurden mittels 16S-rt-PCR untersucht. Die Ergebnisse zeigten, dass beiden rt-PCR Systeme eine vergleichbare Sensitivität aufwiesen. III) Bei dem Vergleich zwischen den Spoligotyping-Methoden zeigte sich die neue Methode um einen Faktor von 100 bei der M. bovis BCG-Reinkultur und um einen Fak-tor von 10 bei DNA-Extrakten aus tuberkulösen Gewebeproben sensitiver als die konventionelle Methode. Im Rahmen dieser Arbeit hat sich der Einsatz der mechanischen Lyse für die Verbesserung der Freisetzung von mykobakterieller DNA als routinefähig erwiesen. Die entwickelte 16S-rt-PCR erwies sich als brauchbare Methode für den Nachweis von Erregern der Gattung Mycobacterium. Die Microarray-Methode stellte sich wesentlich einfacher, sensitiver und schneller dar als die konventionelle Methode. Zusammenfassend kann gesagt werden, dass alle drei Ansätze dieser Arbeit einen Beitrag zur Verbesserung der molekularen Labordiagnostik der Tuberkulose leisten. / Bovine tuberculosis is a chronic disease, that results from infection of Mycobacterium (M.) bovis and M. caprae, members of Mycobacterium tuberculosis complex (MTC), respectively. The laboratory diagnosis of bovine tuberculosis is possible with culture as well as considerate fast molecular methods. The aim of this study was to improve the sensitivity of DNA detec-tion of tuberculosis-causing pathogens. Therefore, three different issues were addressed in the complex molecular procedure targeted. I) four different lytic protocols (thermal, enzymatic, thermo-enzymatic and mechanical lysis) were developed and compared to a standardized DNA isolation protocol that was performed on pure culture of Mycobacterium (M.) bovis BCG in order to improve the mycobacterial cell wall lysis leading to an increase of DNA release. The efficiencies of the lysis protocols were assessed by the resulting cycle threshold (Ct) values of a MTC-specific real time (rt) Polymerase Chain Reaction (PCR). Two lysis protocols (thermal and mechanical) were selected to fur-ther testing of ten tuberculosis-infected tissue samples (lymph nodes, liver and lungs) from ten animals (eight cattle, one lama and one lynx). II) In addition, a real time PCR using the 16S rRNA gene as target sequence was developed, which is also suitable to detect pathogens of Genus Mycobacterium on tissue samples. III) A comparison of a newly developed microarray and the conventional spoligotyping method was realised, to analyse the applicability of the microarray in relation of the sensitivity of the method and to analyse discriminatory potential directly from infected tissue samples. During the conventional spoligotyping method, the PCR product is hybridized with specific oligonucleotides, fixed on a nylon membrane. Using the newly developed method these oligonucleotides are fixed on a microarray-chip. The results I) of the mycobacterial cell wall lysis experiment with pure culture of M. bovis BCG showed an increase of 14 % by using mechanical lysis and an increase by 6 % of the PCR product by using thermal lysis compared to the standard protocol. Using the mechanical lysis as well as the thermal lysis a statistically significant (α = 1 % (Mann-Whitney-Test)) im-provement compared to the standard lysis was achieved. Mechanical lysis was performed on tuberculous tissue samples and the results of the lysis were improved by 9 % compared to the standard lysis. However, the difference between mechanical and standard lysis was not statistically significant due the small sample number. II) Forty-three different mycobacterial species, six members of MTC (among them M. bovis BCG) and 37 Non Tuberculous Mycobacteria (NTM), were detected using the newly developed real time PCR (16S-rt-PCR). Eight non-mycobacterial species were not detected using this rt-PCR, whereas one of genus Rhodococcus and one of genus Gordonia were detected by the 16S-rt-PCR due to their close genetic similarity to the genus Mycobacterium. The ten tuberculosis-infected tissue samples (see above) testing positive using a MTC-specific real time rt-PCR (target gene IS 1081) were sub-jected to the 16S-rt-PCR. Both rt-PCR systems showed a comparable sensitivity. III) By comparing the two spoligotyping-methods, the ArrayStrip™–format method was more sensitive than the conventional method by a factor of 100 applied to pure culture and by a factor of 10 when applied to DNA extracts from infected tissue samples. In conclusion, the mechanical lysis proved to be a practical method to liberate mycobacterial DNA. The newly developed rt-PCR was suitable to detect members of the genus Mycobacterium. The spoligotyping ArrayStrip™–format method appeared to be substantially easier to perform, more sensitive, and less time-consuming than the conventional method. The three methods described were suitable to improve the molecular laboratory diagnosis of tuberculosis.

A legislação permite o uso de leite cru para fabricar queijos com maturação superior a 60 dias em temperatura acima de 5°C, mas falta comprovação científica sólida sobre a eficácia desse processo quanto à inativação de importantes patógenos que podem estar presentes no leite, como o Mycobacterium bovis; além disso, não há metodologia oficial para pesquisa deste agente em alimentos. Desta forma, este trabalho se propôs a estabelecer um protocolo para estudar a curva de inativação do Mycobacterium bovis durante a cura do queijo. Foram fabricadas três partidas de queijo do tipo parmesão com leite pasteurizado e contaminado com uma cepa de Mycobacterium bovis isolada de bovinos abatidos no estado de São Paulo. O queijo foi curado a 18°C e analisado semanalmente até o 63ºdia. As amostras foram submetidas à diluição decimal seriada, semeadas em duplicata em meio Stonebrink-Leslie, acrescido de antibióticos, e incubadas a 37°C por 45 dias. Os resultados de um queijo foram perdidos por contaminação por fungos. O valor D18°C médio, ponderado pelas incertezas, foi de 37,5 dias ± 5,3 dias. Esse resultado indica a necessidade de outros estudos para ampliar o número de queijos estudados e obter, portanto, um resultado mais representativo do efeito da cura sobre o decaimento da população de M. bovis. Há também necessidade de melhorar o poder inibitório do meio de cultura para evitar perdas devido ao crescimento de fungos. / The legislation that regulates the specific conditions for the consumption of food allows the use of raw milk to produce cheese matured over 60 days at temperatures above 5°C; however, the effectiveness of this process in the inactivation of important pathogens that may be present in milk, such as Mycobacterium bovis, lacks solid scientific evidence. In addition, there is no official methodology for the research of this pathogen in foods. Considering that context, this study proposes to establish a protocol to study the inactivation curve of Mycobacterium bovis during cheese curing. Three matches were made with parmesan cheese and pasteurized milk contaminated with a strain of Mycobacterium bovis isolated from cattle slaughtered in the state of São Paulo. The cheese was cured at 18°C and analyzed weekly until the 63th day. The samples were submitted to decimal serial dilution, plated in duplicate in the Middle Stonebrink-Leslie, plus antibiotics, and incubated at 37° C for 45 days. The results of a single cheese were lost to fungal contamination. The value D 18°C average, weighted by the uncertainties, was 37.5 days ±5.3 days. This result indicates the need for further studies to expand the number of cheeses studied and to therefore obtain a more representative result of the effect of healing on the decay of the population of M. bovis. There is also the need to improve the inhibiting power of the culture medium to avoid losses due to fungal growth.

A legislação permite o uso de leite cru para fabricar queijos com maturação superior a 60 dias em temperatura acima de 5°C, mas falta comprovação científica sólida sobre a eficácia desse processo quanto à inativação de importantes patógenos que podem estar presentes no leite, como o Mycobacterium bovis; além disso, não há metodologia oficial para pesquisa deste agente em alimentos. Desta forma, este trabalho se propôs a estabelecer um protocolo para estudar a curva de inativação do Mycobacterium bovis durante a cura do queijo. Foram fabricadas três partidas de queijo do tipo parmesão com leite pasteurizado e contaminado com uma cepa de Mycobacterium bovis isolada de bovinos abatidos no estado de São Paulo. O queijo foi curado a 18°C e analisado semanalmente até o 63ºdia. As amostras foram submetidas à diluição decimal seriada, semeadas em duplicata em meio Stonebrink-Leslie, acrescido de antibióticos, e incubadas a 37°C por 45 dias. Os resultados de um queijo foram perdidos por contaminação por fungos. O valor D18°C médio, ponderado pelas incertezas, foi de 37,5 dias ± 5,3 dias. Esse resultado indica a necessidade de outros estudos para ampliar o número de queijos estudados e obter, portanto, um resultado mais representativo do efeito da cura sobre o decaimento da população de M. bovis. Há também necessidade de melhorar o poder inibitório do meio de cultura para evitar perdas devido ao crescimento de fungos. / The legislation that regulates the specific conditions for the consumption of food allows the use of raw milk to produce cheese matured over 60 days at temperatures above 5°C; however, the effectiveness of this process in the inactivation of important pathogens that may be present in milk, such as Mycobacterium bovis, lacks solid scientific evidence. In addition, there is no official methodology for the research of this pathogen in foods. Considering that context, this study proposes to establish a protocol to study the inactivation curve of Mycobacterium bovis during cheese curing. Three matches were made with parmesan cheese and pasteurized milk contaminated with a strain of Mycobacterium bovis isolated from cattle slaughtered in the state of São Paulo. The cheese was cured at 18°C and analyzed weekly until the 63th day. The samples were submitted to decimal serial dilution, plated in duplicate in the Middle Stonebrink-Leslie, plus antibiotics, and incubated at 37° C for 45 days. The results of a single cheese were lost to fungal contamination. The value D 18°C average, weighted by the uncertainties, was 37.5 days ±5.3 days. This result indicates the need for further studies to expand the number of cheeses studied and to therefore obtain a more representative result of the effect of healing on the decay of the population of M. bovis. There is also the need to improve the inhibiting power of the culture medium to avoid losses due to fungal growth.

Bovine tuberculosis (TB) is a contagious neglected zoonosis of cattle that is prevalent but under-investigated in Cameroon, hence this study was designed to assess the epidemiology of bovine TB in cattle, risks for M. bovis infection in cattle and humans; and public health implications of zoonotic bovine TB in the highlands of Cameroon. A retrospective study of meat inspection records (1994 – 2010) was done to estimate the prevalence of TB lesions in slaughtered cattle in the North West region. The prevalence of bovine TB and anti-bovine TB antibodies in live cattle based on tuberculin skin tests (2 surveys) and immune-chromatographic assays respectively were carried out in the Western and Adamawa highlands of Cameroon. The performance of the tuberculin tests for bovine TB diagnosis in cattle using various tuberculin skin test cut-off points against the detection of anti-bovine TB antibodies (hypothesised risks of exposure) was compared. Suspected TB lesions from slaughtered cattle and infected human sputa were cultured on Lowentein – Jesen and Middlebrook 7H9 media to isolate mycobacteria agents for molecular genotyping using genomic deletion analysis and spoligotyping. Risk factors for exposure and transmission of zoonotic bovine TB infection of cattle and cattle professionals, and its public health significance were determined using structured questionnaires. Seventeen years of meat inspection record revealed that suspect TB lesions were identified in 599 of 129,165 slaughtered cattle at the Bamenda abattoir. The lungs and associated lymph nodes (over 60%) were the most affected tissues. Other results showed that the prevalence of anti-bovine TB antibodies in cattle in the study regions was 37.17%. Chi square statistics revealed that irrespective of the tuberculin test cut-off value (P<0.05; χ2>48), strong associations existed between the detection of anti-bovine TB antibodies and disease status. A 95% confidence interval analysis of the comparative cervical tuberculin tests revealed that the prevalence rates were 4.67% – 7.15%, 12.02% – 15.67% and 20.56% – 24.98% at the ≥ 4mm, ≥ 3mm and ≥ 2mm cut-off points, respectively. Overall, the best test performance was realised at ≥ 3-mm, though the ≥ 2-mm cut-off point predicted more positive reactors. Age, sex, breed and husbandry practices served as significant (P<0.05) risks to the prevalence and exposure of bovine TB in cattle. The feedbacks from cattle professionals suggested that there was high possibility of cattle to cattle and cattle to human transmission of bovine TB such as intimate and repeated animal / animal and animal / human interactions, consuming unpasteurised milk and eating raw meat. Genomic deletion analysis of cultured isolates showed evidence of M. tuberculosis from cattle and M. bovis from human while spoligotyping identified five cattle M. bovis strains; and four spoligotype patterns that had not been previously described anywhere. The study has important epidemiological and public health implications requiring prompt and decisive actions from the Cameroonian authority towards controlling zoonotic bovine TB in both humans and animals. A multidisciplinary approach is needed for further collaborative research and effective control strategies such as enhancing the awareness of people to this deadly disease through continuous education, proper food handling and personal hygiene, healthy husbandry practices and maintenance of the environment.