A tuberculose continua sendo uma importante doença infecciosa, tanto nos humanos quanto nos animais, com índices de morbidade e mortalidade significativos e perdas econômicas em todo o mundo. A tuberculose bovina é uma doença infecciosa causada pela bactéria Mycobacterium bovis, que gera perdas na produção nos rebanhos infectados, sendo também considerada uma importante zoonose. Os métodos de diagnóstico direto têm fundamental importância para um sistema de vigilância para a tuberculose bovina e a agregação de métodos moleculares, notadamente aqueles que têm aplicação em epidemiologia, traz maior precisão diagnóstica para esses sistemas. Dentre as técnicas moleculares, destacam-se o TB Multiplex PCR, o RD Multiplex PCR e o Multispacer Sequence Typing, para a identificação dos isolados e o Variable Number Tandem Repeat (VNTR) e o Spoligotyping, como técnicas de fingerpint de M. bovis. Assim, o presente estudo teve como objetivo a identificação molecular de amostras oriundas de várias regiões do Brasil utilizando estes padrões de técnicas moleculares. Os espoligotipos identificados em maior abundância foram o SB0121, o qual apresentou-se amplamente distribuído entre as amostras, seguido pelo SB0295, SB1380, SB0140 e SB1050. Além disso, foram detectados quatro perfis nunca antes descritos na literatura, sendo que um deles foi o terceiro mais frequente entra as amostras pesquisadas. Os resultados observados neste trabalho demonstraram ainda que a tipagem pelo MIRU-VNTR revelou-se superior ao Spoligotyping para discriminar os isolados. Nesta perspectiva, acredita-se que as pesquisas moleculares voltadas a identificação de micobactérias, aliadas as técnicas epidemiológicas tradicionais, possam melhorar sensivelmente a performance dos sistemas de vigilância para tuberculose bovina no Brasil. / Tuberculosis remains a major infectious disease in both humans and animals, with morbidity and mortality and significant economic losses. Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis, with yield losses in infected herds and is also considered an important zoonosis. The methods of direct diagnosis are important for a surveillance system for bovine tuberculosis and aggregation of molecular methods brings greater diagnostic accuracy for these systems, especially those that have application in epidemiology. Among them, TB Multiplex PCR, RD Multiplex PCR, and Multispacer Sequence Typing for the identification of strains, and Variable Number Tandem Repeat (VNTR) and Spoligotyping, for fingerprint of M. bovis. Thus, the present study aimed to identify molecular samples from different regions of Brazil using these molecular techniques. The most abundant were the spoligotype SB0121, which has become widely distributed among the samples, followed by SB0295, SB1380, SB0140, and SB1050. In addition, four profiles never before described in the literature were detected, one of which was the third most frequent. The results of this study also showed that the MIRU-VNTR typing has proved superior to Spoligotyping to discriminate the isolates. In this perspective, it is believed that the research focused on molecular identification of mycobacteria, combined traditional epidemiological techniques, can significantly improve the performance of surveillance systems for bovine tuberculosis in Brazil.

A tuberculose continua sendo uma importante doença infecciosa, tanto nos humanos quanto nos animais, com índices de morbidade e mortalidade significativos e perdas econômicas em todo o mundo. A tuberculose bovina é uma doença infecciosa causada pela bactéria Mycobacterium bovis, que gera perdas na produção nos rebanhos infectados, sendo também considerada uma importante zoonose. Os métodos de diagnóstico direto têm fundamental importância para um sistema de vigilância para a tuberculose bovina e a agregação de métodos moleculares, notadamente aqueles que têm aplicação em epidemiologia, traz maior precisão diagnóstica para esses sistemas. Dentre as técnicas moleculares, destacam-se o TB Multiplex PCR, o RD Multiplex PCR e o Multispacer Sequence Typing, para a identificação dos isolados e o Variable Number Tandem Repeat (VNTR) e o Spoligotyping, como técnicas de fingerpint de M. bovis. Assim, o presente estudo teve como objetivo a identificação molecular de amostras oriundas de várias regiões do Brasil utilizando estes padrões de técnicas moleculares. Os espoligotipos identificados em maior abundância foram o SB0121, o qual apresentou-se amplamente distribuído entre as amostras, seguido pelo SB0295, SB1380, SB0140 e SB1050. Além disso, foram detectados quatro perfis nunca antes descritos na literatura, sendo que um deles foi o terceiro mais frequente entra as amostras pesquisadas. Os resultados observados neste trabalho demonstraram ainda que a tipagem pelo MIRU-VNTR revelou-se superior ao Spoligotyping para discriminar os isolados. Nesta perspectiva, acredita-se que as pesquisas moleculares voltadas a identificação de micobactérias, aliadas as técnicas epidemiológicas tradicionais, possam melhorar sensivelmente a performance dos sistemas de vigilância para tuberculose bovina no Brasil. / Tuberculosis remains a major infectious disease in both humans and animals, with morbidity and mortality and significant economic losses. Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis, with yield losses in infected herds and is also considered an important zoonosis. The methods of direct diagnosis are important for a surveillance system for bovine tuberculosis and aggregation of molecular methods brings greater diagnostic accuracy for these systems, especially those that have application in epidemiology. Among them, TB Multiplex PCR, RD Multiplex PCR, and Multispacer Sequence Typing for the identification of strains, and Variable Number Tandem Repeat (VNTR) and Spoligotyping, for fingerprint of M. bovis. Thus, the present study aimed to identify molecular samples from different regions of Brazil using these molecular techniques. The most abundant were the spoligotype SB0121, which has become widely distributed among the samples, followed by SB0295, SB1380, SB0140, and SB1050. In addition, four profiles never before described in the literature were detected, one of which was the third most frequent. The results of this study also showed that the MIRU-VNTR typing has proved superior to Spoligotyping to discriminate the isolates. In this perspective, it is believed that the research focused on molecular identification of mycobacteria, combined traditional epidemiological techniques, can significantly improve the performance of surveillance systems for bovine tuberculosis in Brazil.

BACKGROUND:Bovine tuberculosis (TB) caused by Mycobacterium bovis is primarily a disease of ruminants, particularly cattle (Bos primigenius) and buffalo (Syncerus caffer), and is endemic in most developing countries. To date, studies done in Uganda have documented the prevalence of M. bovis in cattle, humans and wild life, in addition to non-tuberculous mycobacteria in pigs. Pigs are increasingly becoming an important component of the livestock sector and share the human ecosystem in rural Uganda. It is therefore of public health interest that they are not a source of human infections. As a follow up to previously published findings on mycobacteria in pigs, this study was aimed at investigating the occurrence and molecular characteristics of M. bovis detected in slaughter pigs in Mubende district, Uganda. One hundred fifty mesenteric lymph nodes with lesions suggestive of mycobacterial infections were collected from approximately one thousand slaughtered pigs in Mubende district over a period of five months. The isolation and identification of M. bovis was done using conventional mycobacteriological methods. Mycobacteria belonging to the Mycobacterium tuberculosis complex (MTC) were identified to species level using deletion analysis. Molecular typing was done using Spoligotyping and MIRU-VNTR analysis. Molecular data were analysed and interpreted using MIRU-VNTR plus, SpolDB4.0 and the Mycobacterium bovis spoligo database.RESULTS:Of the examined animals, one boar and two sows from Madudu Sub County were infected with M. bovis which presented as lesions of a deep yellow colour and a grit-like texture in the mesenteric lymph nodes. This represents 2% (3/150) of the lymph nodes where lesions suggestive of mycobacterial infections were detected. Molecular analysis revealed that the isolates from the infected pigs showed identical MIRU-VNTR profile and spoligotype (SB1469).CONCLUSIONS:This is the first study documenting the occurrence of M. bovis in slaughter pigs in Uganda, revealing that one in fifty slaughter pigs with suspected lesions in mesenteric lymph nodes were infected. Molecular analysis revealed that the isolates were identical, showing a spoligotype previously reported from humans and cattle in the north eastern part of the Uganda cattle corridor. This finding is of public health importance, therefore there is a need for close cooperation between medical and veterinary professionals in designing and implementing control and prevention measures that safeguard the public from this potential source of zoonotic TB in Uganda.

The evolutionary relationship between Salmonella enterica serovar Typhi, other typhoid-like enteric fever causing serovars and 10 non-Typhoid serovars from S. enterica subspecies I, could not be determined by comparative nucleotide sequences of six genes. Phylogenetic analyses of the dataset showed that the genes of interest underwent frequent recombination, suggesting a low level of clonality within subspecies I of S. enterica. To establish the evolutionary relationships within serovar Typhi, genome-wide Single Nucleotide Polymorphism (SNP) was explored as a marker for both typing purposes and phylogenetic analysis. Thirty eight SNPs were typed in 73 global Typhi isolates, including 18 isolates expressing the special flagellar antigen z66, using restriction enzyme digestion method. The isolates were differentiated into 23 SNP profiles and grouped into four distinct clusters. The z66 isolates were divided into four SNP profiles and were all grouped into one cluster, suggesting a single origin. An alternative SNP typing method using the hairpin real time PCR assay was investigated to type four additional SNPs, termed as biallelic polymorphisms (BiP). These BiPs were found to classify 481 global Typhi isolates into five major clusters (Roumagnac et al., 2006). Typing four BiPs resulted in the identification of four additional SNP profiles. We proposed nine SNPs were required to type Typhi isolates into 13 subclusters for global epidemiology. An enzymatic-based method using CelI nuclease was evaluated to discover more SNPs from other Typhi genomes. The efficiency of the CelI was shown to be unsatisfactory and we were unable to demonstrate the effectiveness of the proposed method. Nine Variable Number of Tandem Repeats (VNTRs) were typed in the 73 Typhi isolates using fluorescent-labelled universal primers, and analysed on an automated DNA sequencer. Five isolates were unable to give PCR products in one or more VNTR loci. Nine VNTRs could differentiate 68 Typhi isolates into 65 MLVA profiles, suggesting a higher discriminating power than SNP typing. SNPs were shown to be a more appropriate marker for phylogenetic tracing for Typhi while VNTRs were highly discriminating but could not be used to establish the evolutionary relationships of diverse Typhi isolates.

The evolutionary relationship between Salmonella enterica serovar Typhi, other typhoid-like enteric fever causing serovars and 10 non-Typhoid serovars from S. enterica subspecies I, could not be determined by comparative nucleotide sequences of six genes. Phylogenetic analyses of the dataset showed that the genes of interest underwent frequent recombination, suggesting a low level of clonality within subspecies I of S. enterica. To establish the evolutionary relationships within serovar Typhi, genome-wide Single Nucleotide Polymorphism (SNP) was explored as a marker for both typing purposes and phylogenetic analysis. Thirty eight SNPs were typed in 73 global Typhi isolates, including 18 isolates expressing the special flagellar antigen z66, using restriction enzyme digestion method. The isolates were differentiated into 23 SNP profiles and grouped into four distinct clusters. The z66 isolates were divided into four SNP profiles and were all grouped into one cluster, suggesting a single origin. An alternative SNP typing method using the hairpin real time PCR assay was investigated to type four additional SNPs, termed as biallelic polymorphisms (BiP). These BiPs were found to classify 481 global Typhi isolates into five major clusters (Roumagnac et al., 2006). Typing four BiPs resulted in the identification of four additional SNP profiles. We proposed nine SNPs were required to type Typhi isolates into 13 subclusters for global epidemiology. An enzymatic-based method using CelI nuclease was evaluated to discover more SNPs from other Typhi genomes. The efficiency of the CelI was shown to be unsatisfactory and we were unable to demonstrate the effectiveness of the proposed method. Nine Variable Number of Tandem Repeats (VNTRs) were typed in the 73 Typhi isolates using fluorescent-labelled universal primers, and analysed on an automated DNA sequencer. Five isolates were unable to give PCR products in one or more VNTR loci. Nine VNTRs could differentiate 68 Typhi isolates into 65 MLVA profiles, suggesting a higher discriminating power than SNP typing. SNPs were shown to be a more appropriate marker for phylogenetic tracing for Typhi while VNTRs were highly discriminating but could not be used to establish the evolutionary relationships of diverse Typhi isolates.

Introduction : Mycobacterium avium ssp. paratuberculosis (Map) est l’agent étiologique de la paratuberculose, qui affecte les ruminants et est suspectée d’être transmissible à l’homme en lien avec la maladie de Crohn, une inflammation chronique de l’intestin. Le contrôle de cette maladie à forte prévalence, nécessite de mieux identifier et connaître son agent. Aujourd’hui, le typage des souches repose sur la RFLP-IS900 qui est peu discriminante et peu sensible. Objectif : Dans cette optique, mon projet de thèse a eu pour objectif de développer une méthode de typage moléculaire novatrice utilisant les minisatellites afin d’évaluer le degré de diversité génétique de la sous-espèce paratuberculosis. Ce travail nécessite la construction d’une collection de souches et le développement d’une méthode de typage hautement discriminante. Résultats : Nos résultats obtenus avec huit marqueurs MIRU-VNTR (Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat) montrent que cette méthode est rapide, adaptée à Map et plus discriminante de la RFLP-IS900. Par ailleurs, ces mêmes marqueurs nous ont permis de typer une collection de souches de M. avium ssp. avium et M. avium ssp. hominissuis. Le typage MIRU-VNTR a également permis de différencier la souche vaccinale Map 316F, en fonction de son origine, laboratoire Weybridge (Royaume-Uni) et laboratoire Mérial (France). Dans le but d’affiner la discrimination de ces souches, nous nous sommes intéressés aux marqueurs microsatellites SSR (Short Sequence Repeat) analysés par séquençage. Onze loci SSR ont été analysés et évalués sur une collection de 127 souches de Map préalablement typées par MIRU-VNTR et RFLP- IS900. Les résultats suggèrent que la stratégie la plus adaptée pour la discrimination des souches consiste à utiliser dans un premier temps le typage MIRU-VNTR ; le typage SSR et la RFLP-IS900 peuvent permettre une discrimination complémentaire. Conclusion : Ce travail de thèse nous a permis de développer une méthode de typage rapide, fiable et discriminante pour l’étude de la sous-espèce paratuberculosis, mais également du complexe MAC. / Background: Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of paratuberculosis, affecting wide range of domestic ruminants and suspected to be involved in Crohn’s disease in human. The control of this disease, highly prevalent, requires better identification methods and better knowledge of the etiological agent. Currently, the gold standard method to type Map strains is the IS900-RFLP. But this technique is not very sensitive and not very discriminating. Objective: In this context, the project of my thesis aimed to develop an innovative method of molecular typing using the minisatellites in order to evaluate the degree of diversity of Map. This work requires the construction of a collection of strains and the development of a new molecular typing method. Results: Our results obtained with eight MIRU-VNTR markers (Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat) show that MIRU-VNTR typing is a fast typing method, adapted to Map and more discriminative than IS900-RFLP. In addition, these 8 markers could be applied to type a collection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. hominissuis. MIRU-VNTR typing differentiated the vaccine strain 316F according to the laboratory of origin Weybridge (UK) or Mérial (France). In order to improve the discrimination of the strains, we used the microsatellites markers SSR (Short Sequence Repeat) using sequencing analysis of the Map genome. Eleven SSR were analysed and evaluated on a collection of 127 Map strains previously typed by MIRU-VNTR and IS900-RFLP typing. Our results suggested that the most adapted strategy to discriminate the Map strains consisted in using MIRU-VNTR typing, and that SSR and RFLP-IS900 typing to better discriminate the clustered isolates. Conclusion: The work of my thesis enabled to develop fast, reliable and robust typing method for the study of Map and also MAC subspecies.

[EN] In the last years, various genotyping techniques were developed for isolation of Mycobacterium tuberculosis complex (MTBC) that has demonstrated a high discriminatory power. In this study, after the identification of selected strains at level of species by Genotype MTBC technique, we evaluated the profit of the simplified amplified-fragment Length Polymorphism technique (AFLPs) and the Mycobacterial Interspersed Repetitive Units technique (MIRU-15). A total of 131 mycobacterium tuberculosis isolates were analyzed, 68 isolates were collected in Ecuador, from the Clinical Laboratory of Hospital Alli Causai located in city of Ambato, and the Laboratory of Bacteriology in Carlos Andrade Marín Hospital located in the capital city Quito. The remaining 63 isolates were harvested in Spain and belong to microorganism's collection of Microbiology Services of Consorcio Hospital General Universitario and Hospital Clínico Universitario of the city of Valencia. Among these isolates, 126 were identified by conventional methods such as molecular MTBC, corresponding to 106 patients. The Mycobacterium tuberculosis control strain ATCC 25177 was also identified as such by this method. The AFLPs technique allowed as to group the strains in twelve patterns (P1 to P8, P10, P12, P13, P14), of which the most prevalent were patterns P1 with 77 (61.1%) and P2 with 27 (21, 4%) isolates, representing 82.5% of the same. These were followed by the pattern P5 with 5 (3.9%) isolates, the patterns P3, P4 and P6 grouped 3 isolates each one (2.4%), the patterns P8 and P12 with 2 isolates (1.6% ) and finally the patterns P7, P10, P13 and P14 with 1 isolated each one (0.8%). The control strain M. tuberculosis ATCC 25177, showed a restriction profile that prevented their inclusion in any of the patterns described. The discriminatory power of the Hunter-Gaston discriminatory index (HGDI) method was 0.5812 against to 0.9843 of the MIRU-15 technique, which grouped 69 strains (54.8%) in 20 clonal complex and 57 unique patterns (45.2%). In the case of Spain, the strains were related mostly to the lineage 4 or Euro-American including: Cammeroon (1.59%), Haarlem (36.51%), S (31.75%), and LAM (19.05%); the lineage 6 or West Africa I (9.53%), the lineage 1 or EIA (1.59%) In the case of Ecuador the strains were related to the lineage 4: Haarlem (42.86%), S (33.33%) and LAM (22.22%) and Beijing lineage 2 (1.59%) from Asia. The MIRU-VNTR Variable-Number Tandem Repeats (15 loci) technique proved to be a stable system, reproducible and high discriminatory power in comparison with AFLPs system, allowing the use of it to conduct prospective population studies with the aim of contributing to the public health programs to control Tuberculosis (TB). / [ES] En los últimos años se han desarrollado diversas técnicas de genotipificación para aislados de Mycobacterium tuberculosis complex (MTBC) que han demostrado tener un alto poder discriminatorio. En este estudio, tras identificación de las cepas seleccionadas al nivel de especie mediante la técnica comercial GenoType MTBC, se ha evaluado la utilidad de la técnica simplificada del Polimorfismo de Longitud de Fragmentos Amplificados (AFLPs) y la técnica de Unidades Repetitivas Intercaladas Micobacterianas (MIRU-15). Se analizaron un total de 131 aislados clínicos de los cuales 68 aislados fueron recolectados en Ecuador, provenientes tanto del Laboratorio Clínico del Hospital Alli Causai ubicado en la ciudad de Ambato, provincia de Tungurahua como del Laboratorio de Bacteriología del Hospital Carlos Andrade Marín ubicado en la ciudad capital Quito, provincia de Pichincha. Los 63 aislados restantes fueron recolectados en España y pertenecían colección de microorganismos de los Servicios de Microbiología del Consorcio Hospital General Universitario y Hospital Clínico Universitario de la ciudad de Valencia, provincia de Valencia. De éstos aislados, 126 fueron identificados por métodos convencionales y moleculares como MTBC, correspondientes a 106 pacientes. La cepa control Mycobacterium tuberculosis ATCC 25177 también fue identificada como tal mediante este método. La técnica AFLPs permitió agrupar a las cepas en doce patrones (P1 a P8, P10, P12, P13, P14), de los cuales los más prevalentes fueron los patrones P1 y P2 con 77 (61,1%) y 27 (21,4%) aislados respectivamente, lo que supone el 82,5% del total de los mismos. Le siguieron en frecuencia el patrón P5 con 5 (3,9%) aislados, los patrones P3, P4 y P6 agruparon a 3 aislados cada uno (2,4%), los patrones P8 y P12 con 2 aislados (1,6%) y finalmente los patrones P7, P10, P13 y P14 con 1 aislado cada uno (0,8%). La cepa control M. tuberculosis ATCC 25177, mostró un perfil de restricción que no permitió su inclusión en ninguno de los patrones descritos. El poder discriminatorio del método (HGDI) fue de 0,5812 frente a 0.9843 de la técnica MIRU-15, que agrupó a 69 cepas (54,8%) en 20 complejos clonales y 57 patrones únicos (45,2%). Para el caso de España, las cepas estuvieron relacionadas en su mayoría con el linaje 4 o Euro-Americano que incluye: Cammeroon (1,59%), Haarlem (36,51%), S (31,75%), y LAM (19,05%); el linaje 6 o West Africa I (9,53%), el linaje 1 o EIA (1,59%), Para el caso de Ecuador las cepas estaban relacionadas con el linaje 4: Haarlem (42,86%), S (33,33%), y LAM (22,22%) y el linaje 2 Beijing (1,59%) originario de Asia. La técnica MIRU-VNTR (15 loci) demostró ser un sistema estable, reproducible y con un poder discriminatorio alto en comparación con AFLPs lo que permitiría emplearlo para realizar estudios poblacionales prospectivos con la finalidad de contribuir a los programas de Salud Pública para el control de la Tuberculosis (TB). / [CAT] En els últims anys s'han desenvolupat diverses tècniques de genotipificació per aïllats de Mycobacterium tuberculosis complex (MTBC) que han demostrat tenir un alt poder discriminatori. En aquest estudi, després de la identificació de les soques seleccionades al nivell d'espècie mitjançant la tècnica comercial GenoType MTBC, s'ha avaluat la utilitat de la tècnica simplificada del Polimorfisme de longitud de fragments amplificats (AFLPs) i la tècnica d'Unitats repetitives Intercalades micobacterianes (Miru-15). Es van analitzar un total de 131 aïllats clínics dels quals 68 aïllats van ser recollectats a Equador, provinents tant del Laboratori Clínic de l'Hospital Alli Causai situat a la ciutat d'Ambato, província de Tungurahua com del Laboratori de Bacteriologia de l'Hospital Carlos Andrade Marín ubicat a la ciutat cabdal Quito, província de Pichincha. Els 63 aïllats restants van ser recollectats a Espanya i pertanyien a la collecció de microorganismes dels Serveis de Microbiologia del Consorci Hospital General Universitari i Hospital Clínic Universitari de la ciutat de València, província de València. D'aquests aïllats, 126 van ser identificats per mètodes convencionals i moleculars com MTBC, corresponents a 106 pacients. La soca control Mycobacterium tuberculosi ATCC 25177 també va ser identificada com a tal mitjançant aquest mètode. La tècnica AFLPs va permetre agrupar les soques en dotze patrons (P1 a P8, P10, P12, P13, P14), dels quals els més prevalents van ser els patrons P1 i P2 amb 77 (61,1%) i 27 (21, 4%) aïllats respectivament, fet que suposa el 82,5% del total dels mateixos. El van seguir en freqüència el patró P5 amb 5 (3,9%) aïllats, els patrons P3, P4 i P6 van agrupar a 3 aïllats cadascun (2,4%), els patrons P8 i P12 amb 2 aïllats (1,6%) i finalment els patrons P7, P10, P13 i P14 amb 1 aïllat cadascun (0,8%). La soca control M. tuberculosis ATCC 25177, va mostrar un perfil de restricció que no va permetre la seva inclusió en cap dels patrons descrits. El poder discriminatori del mètode (HGDI) va ser de 0,5812 enfront de 0,9843 de la tècnica MIRU-15, que va agrupar a 69 soques (54,8%) en 20 complexos clonals i 57 patrons únics (45,2%). Per al cas d'Espanya, les soques van estar relacionades majoritàriament amb el llinatge 4 o Euro-Americà que inclou: Cammeroon (1,59%), Haarlem (36,51%), S (31,75%), i LAM (19,05%); el llinatge 6 o West Africa I (9,53%), el llinatge 1 o EIA (1,59%), Pel cas de l'Equador les soques estaven relacionades amb el llinatge 4: Haarlem (42,86%), S ( 33,33%), i LAM (22,22%) i el llinatge 2 Beijing (1,59%) originari d'Àsia. La tècnica Miru-VNTR (15 loci) va demostrar ser un sistema estable, reproduïble i amb un poder discriminatori alt en comparació amb AFLPs, el que permetria emprar-lo per realitzar estudis poblacionals prospectius amb la finalitat de contribuir als programes de salut pública per al control de la Tuberculosi (TB). / Jiménez Arias, AP. (2016). Genotipificación de Mycobacterium tuberculosis complex mediante herramientas moleculares [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/62681 / TESIS

Foi estabelecida uma parceria entre o Departamento de Medicina Veterinária Preventiva e Saúde Animal (VPS) da FMVZ-USP, a Coordenadoria de Defesa Agropecuária do Estado de São Paulo e o Serviço de Inspeção Federal (SIF) para organizar um sistema capaz de detectar focos de tuberculose bovina no Estado, com base nas rotinas de inspeção de carcaças em abatedouros, cujos objetivos foram: 1) conhecer a diversidade genética de isolados de Mycobacterium bovis em bovinos no Estado de São Paulo; 2) estudar a distribuição espacial desses focos; 3) estudar a tipologia das unidades de criação de bovinos caracterizadas como focos de tuberculose; 4) verificar se é possível, com a atual infra-estrutura existente em São Paulo, operar um sistema de vigilância para detecção de focos de tuberculose bovina. Assim, foi estruturado um sistema de coleta, envolvendo as redes SISP (Sistema de Inspeção do Estado de São Paulo) e SIF, que realizou as coletas de materiais e informações de maio de 2002 a janeiro de 2004. Todo o material seguiu para o VPS, onde foram processados. As propriedades caracterizadas como focos foram rastreadas e delas foi coletada outro conjunto de informações. Seguem os resultados alcançados: 1) foram identificados 33 diferentes espoligotipos dentre os 248 isolados de M. bovis de bovinos no Estado de São Paulo. Os isolados do espoligotipo SB0295 foram re-discriminados em 13 novos perfis genéticos de M. bovis pela técnica MIRU-VNTR; 2) dentre os dois espoligotipos mais prevalentes estudados (SB0295 e SB0121), apenas o SB0295 apresentou-se de forma agrupada nas análises espaciais; 3) foram geradas várias informações sobre a tipologia e o manejo das unidades de criação de bovinos caracterizadas como focos de tuberculose; 4) a atual infra-estrutura existente no Estado de São Paulo foi capaz de operar um sistema de detecção de focos de tuberculose bovina / A partnership between the Department of Preventive Veterinary Medicine and Animal Health (VPS) of the FMVZ-USP, the Coordination of Agriculture and Animal Defense of the State of São Paulo, and the Federal Inspection Service (SIF) was established to organize a work system for detection of bovine tuberculosis focus in the state, based on routine methods of carcass inspection in the abattoir, with the following objectives: 1) to determine the genetic diversity of the isolates of Mycobacterium bovis from bovines in the state of São Paulo; 2) to study the spatial distribution of the focuses; 3) to study the typology of the bovine breading units (farms), which were characterized as tuberculosis focus; 4) to verify the possibility of operating a surveillance system for detection of bovine tuberculosis focus based on the current network in the state of São Paulo. Thus, it was performed a system for data collection involving the current systems SISP (System of Inspection of the State of São Paulo) and SIF, who performed the collection of biological samples and information from May 2002 to January 2004. All samples were addressed to the VPS, where they were processed. Farms characterized as focus were traced to obtain new information. The results obtained in this study follow: 1) A total of 33 different spoligotypes were determined out of 248 bovine isolates of M. bovis in the state of São Paulo. The spoligotype SB0295 isolates were re-discriminated into 13 new M. bovis genetic profiles by the MIRU-VNTR technique; 2) From the two most prevalent spoligotypes analyzed in this study (SB0295 e SB0121), only SB0295 showed a cluster presentation by the spatial analyses; 3) Several information about typology and bovine breeding unit management were generated regarding the status of tuberculosis focus; 4) the current network in the state of São Paulo was capable of operating a system for detection of bovine tuberculosis focus

Técnicas de genotipificação do Mycobacterium bovis baseadas na amplificação do DNA via PCR como MIRU-VNTR constituem importantes ferramentas na investigação epidemiológica da tuberculose bovina (TB) em áreas de baixa prevalência, como no Estado de Mato Grosso. Isto posto e diante de limitadas informações a respeito dos genótipos grassantes no Estado, foi realizado monitoramento nas linhas de inspeção sanitária pós-abate em 35 matadouros-frigoríficos sob inspeção oficial para detectar lesões granulomatosas sugestivas de TB e proceder a coleta e análise de amostras. Foram processadas e analisadas 167 amostras mediante isolamento bacteriano e técnicas moleculares. Através do método de genotipagem MIRU-VNTR, 49 isolados de M. bovis foram analisados aplicando-se 24 pares de primers diferentes. Desses, 20 marcadores de VNTR mostraram polimorfismo genético. O locus QUB26 apresentou alto poder discriminatório (h=0,66) enquanto ETRC (0,54), Mtub21 (0,5), QUB11b (0,33) e Mtub34 (0,31) mostraram moderada diversidade alélica. Os outros 15 loci (Mtub 04, 29 e 39; MIRU 04, 16, 23, 24, 26, 27, 31, 39 e 40; ETR A; ETR B; QUB 4156) revelaram baixo poder discriminatório (h=0,02 - 0,27). Quatro loci (MIRU 2, 10 e 20; Mtub30) não apresentaram diversidade alélica. Foram observados 28 perfis genéticos (V1-V28) nos isolados de M. bovis obtidos em 35 propriedades rurais. Dentre essas, 30 (85,71%) apresentaram somente 1 tipo de perfil genético. Apenas 19 amostras (38,77%) apresentaram perfil MIRU-VNTR único revelando a existência de isolados com o mesmo perfil MIRU-VNTR entre diferentes regiões geográficas no Estado. O isolamento, seguido da identificação molecular e discriminação genotípica por MIRU-VNTR em isolados de M. bovis constituíram fontes de relevantes informações auxiliares no desenvolvimento de estratégias de erradicação da TB na região de estudo. / Mycobacterium bovis genotyping techniques based on PCR amplification as MIRU-VNTR are important tools in the epidemiological investigation of bovine tuberculosis in areas of low prevalence, such as in the state of Mato Grosso. Based on limited information about the genotypes in state, post-slaughter sanitary inspection lines were monitored in 35 slaughterhouses under official inspection to detect granulomatous lesions suggestive of TB and to collect and analyze samples. 167 Samples were processed and analyzed by bacterial isolation and molecular techniques. Through the MIRU-VNTR genotyping method, 49 M. bovis isolates were analyzed by applying 24 different primer pairs. Of these, 20 VNTR markers showed genetic polymorphism. The QUB26 locus presented high discriminatory power (h = 0.66) while ETRC (0.54), Mtub21 (0.5), QUB11b (0.33) and Mtub34 (0.31) showed moderate allelic diversity. The other 15 loci (Mtub 04, 29 e 39; MIRU 04, 16, 23, 24, 26, 27, 31, 39 e 40; ETR A; ETR B; QUB 4156) showed low discriminatory power (h = 0.02 - 0.27). Four loci (MIRU2, MIRU10, MIRU20 and Mtub30) did not present allelic diversity. Twenty-eight genotypes (V1-V28) were observed in the M. bovis isolates obtained from 35 rural properties. Among these, 30 (85.71%) presented only 1 genotype type. Only 19 samples (38.77%) presented a single MIRU-VNTR profile revealing the existence of isolates with the same MIRU-VNTR profile among different geographic regions in the State. Isolation, followed by molecular identification and genotypic discrimination by MIRU-VNTR in M. bovis isolates were sources of relevant auxiliary information in the development of strategies to eradicate TB in the study region.

Foi estabelecida uma parceria entre o Departamento de Medicina Veterinária Preventiva e Saúde Animal (VPS) da FMVZ-USP, a Coordenadoria de Defesa Agropecuária do Estado de São Paulo e o Serviço de Inspeção Federal (SIF) para organizar um sistema capaz de detectar focos de tuberculose bovina no Estado, com base nas rotinas de inspeção de carcaças em abatedouros, cujos objetivos foram: 1) conhecer a diversidade genética de isolados de Mycobacterium bovis em bovinos no Estado de São Paulo; 2) estudar a distribuição espacial desses focos; 3) estudar a tipologia das unidades de criação de bovinos caracterizadas como focos de tuberculose; 4) verificar se é possível, com a atual infra-estrutura existente em São Paulo, operar um sistema de vigilância para detecção de focos de tuberculose bovina. Assim, foi estruturado um sistema de coleta, envolvendo as redes SISP (Sistema de Inspeção do Estado de São Paulo) e SIF, que realizou as coletas de materiais e informações de maio de 2002 a janeiro de 2004. Todo o material seguiu para o VPS, onde foram processados. As propriedades caracterizadas como focos foram rastreadas e delas foi coletada outro conjunto de informações. Seguem os resultados alcançados: 1) foram identificados 33 diferentes espoligotipos dentre os 248 isolados de M. bovis de bovinos no Estado de São Paulo. Os isolados do espoligotipo SB0295 foram re-discriminados em 13 novos perfis genéticos de M. bovis pela técnica MIRU-VNTR; 2) dentre os dois espoligotipos mais prevalentes estudados (SB0295 e SB0121), apenas o SB0295 apresentou-se de forma agrupada nas análises espaciais; 3) foram geradas várias informações sobre a tipologia e o manejo das unidades de criação de bovinos caracterizadas como focos de tuberculose; 4) a atual infra-estrutura existente no Estado de São Paulo foi capaz de operar um sistema de detecção de focos de tuberculose bovina / A partnership between the Department of Preventive Veterinary Medicine and Animal Health (VPS) of the FMVZ-USP, the Coordination of Agriculture and Animal Defense of the State of São Paulo, and the Federal Inspection Service (SIF) was established to organize a work system for detection of bovine tuberculosis focus in the state, based on routine methods of carcass inspection in the abattoir, with the following objectives: 1) to determine the genetic diversity of the isolates of Mycobacterium bovis from bovines in the state of São Paulo; 2) to study the spatial distribution of the focuses; 3) to study the typology of the bovine breading units (farms), which were characterized as tuberculosis focus; 4) to verify the possibility of operating a surveillance system for detection of bovine tuberculosis focus based on the current network in the state of São Paulo. Thus, it was performed a system for data collection involving the current systems SISP (System of Inspection of the State of São Paulo) and SIF, who performed the collection of biological samples and information from May 2002 to January 2004. All samples were addressed to the VPS, where they were processed. Farms characterized as focus were traced to obtain new information. The results obtained in this study follow: 1) A total of 33 different spoligotypes were determined out of 248 bovine isolates of M. bovis in the state of São Paulo. The spoligotype SB0295 isolates were re-discriminated into 13 new M. bovis genetic profiles by the MIRU-VNTR technique; 2) From the two most prevalent spoligotypes analyzed in this study (SB0295 e SB0121), only SB0295 showed a cluster presentation by the spatial analyses; 3) Several information about typology and bovine breeding unit management were generated regarding the status of tuberculosis focus; 4) the current network in the state of São Paulo was capable of operating a system for detection of bovine tuberculosis focus