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Aplicação da técnica de MLVA para tipificação molecular de Mycoplasma bovis isolados, em Portugal, de doenças respiratórias bovinas
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Mycoplasma bovis é um agente patogénico capaz de causar mastite, pneumonia, artrite e otite em bovinos. Uma das doenças mais graves é a BRDC (Bovine respiratory disease complex). Apesar da BRDC poder ser causada por vários agentes patogénicos, apenas para M. bovis não existe antibioterapia e vacinação eficazes. Com o objectivo de avaliar o contributo de M. bovis no desenvolvimento de doenças respiratórias em Portugal, foram sujeitas a análise 303 amostras post mortem colhidas em 2015 de bovinos com suspeitas de tuberculose bovina. Foram obtidos nove isolados de micoplasmas, pertencentes a diversas regiões, que foram caracterizados com recurso a testes bioquímicos e PCR, tendo sido identificados como pertencentes a estirpes de M. bovis. As frequências relativas calculadas mostraram que existem essencialmente dois focos a nível regional, nomeadamente para a região Norte (6,7 %) e para a região de Lisboa e Vale do Tejo (14,3 %). A nível de país para o ano de 2015 a frequência relativa foi de 3 %. Apenas dois casos de co-infecção com Mycoplasma bovis e Mycobacterium bovis foram verificados (0,7 %), ambos pertencentes à região Norte. De modo a evidenciar a possível relação epidemiológica de isolados de M. bovis, foi desenvolvida e adaptada a técnica de MLVA com base num painel de 12 loci. Para além dos nove isolados obtidos, foram também utilizados 58 isolados de M. bovis (67 no total) de 1997-2017 pertencentes ao biobanco do INIAV I.P., obtidos de surtos de PPCB (1993-1998) e de outros bovinos com suspeitas de pneumonia, mastite ou tuberculose bovina. Foi também incluído um isolado da Hungria e um da Nigéria. Após a estandardização do método MLVA, foram apenas considerados 11 loci, pois o locus MbovTR49-51 mostrou, consistentemente, um padrão de multibanda. Foram encontrados neste estudo 14 novos alelos, nunca antes observados, para sete dos loci, dos quais oito são considerados como intermédios. A análise da diversidade HGDI, mostrou uma variedade a nível de locus de 3 a 7 polimorfismos, tendo o HGDI variado entre 0,337 e 0,777. Em termos de poder discriminatório da técnica para o painel de 11 loci foram obtidos 55 perfis MLVA para um HGDI de 0,989, sendo o perfil predominante o perfil 00201044324 (MbovTR14-MbovTR29-MbovTR30-MbovTR31-MbovTR35-MbovTR40-41-MbovTR52-MbovTR59-MbovTR147-MbovTR148-MbovTR427), encontrado em cinco estirpes. Estes valores de diversidade mostram que a técnica de MLVA pode ser utilizada em isolados de M. bovis com um objectivo de diferenciação. Para um limite de corte a 55 %, o dendrograma mostrou a formação de seis clusters, dos quais quatro pertencem a estirpes portuguesas e os outros dois a estirpes, respectivamente, dos EUA e da Nigéria, indicando que parecem existir diferenças entre estirpes a nível continental, com estirpes da Europa, Africa e América do Norte pertencendo a clusters diferentes. A análise por árvore de extensão mínima (MST) mostra que existem dentro das estirpes analisadas nove complexos clonais. Estes resultados sugerem uma origem múltipla e uma grande diversidade dentro das estirpes portuguesas. Não foram demostradas relações temporais nem regionais dentro dos isolados analisados a nível de país, algo que poderá estar relacionado com o tipo de gestão praticado nas explorações portuguesas, que poderá estar a favorecer a disseminação de M. bovis em Portugal. As estirpes deste estudo foram também comparadas com estirpes de outros estudos para um painel de 8 loci. A MST mostrou a presença de onze complexos clonais em estirpes europeias, onde igualmente é verificada uma origem múltipla a nível de país para Portugal e Hungria, mas também existe uma linhagem comum entre estirpes europeias, onde são incluídas algumas estirpes portuguesas. Estes resultados sugerem que a facilidade de transacção de bovinos dentro da comunidade europeia possa estar implicada na disseminação de M. bovis nos últimos 20 anos na Europa.
Mycoplasma bovis is a pathogen capable of causing mastitis, pneumonia, arthritis and otitis in cattle. One of the most serious diseases is BRDC (Bovine respiratory disease complex). Although BRDC can be caused by several pathogens, only for M. bovis there is no effective antibiotic therapy and vaccination. In order to evaluate the contribution of M. bovis in the development of respiratory diseases in Portugal, 303 post mortem samples collected in 2015 from bovine animals with suspected bovine tuberculosis were analyzed. Nine mycoplasma isolates were obtained from several regions, and were characterized by biochemical tests and PCR, and identified as belonging to M. bovis strains. The relative frequencies calculated showed that there are essentially two foci at regional level, namely for the Northern region (6.7 %) and for the Lisboa e Vale do Tejo region (14.3 %). At country level for the year 2015 the relative frequency was 3 %. Only two cases of co-infection (0.7 %) with Mycoplasma bovis and Mycobacterium bovis were found, both belonging to the Northern region. In order to show the possible epidemiological relationship of M. bovis isolates, the MLVA technique was developed and adapted based on a panel of 12 loci. In addition to the nine isolates obtained, 58 M. bovis isolates from 1997-2017 (making 67 isolates in total) belonging to the INIAV I.P., biobank, isolated from PPCB outbreaks (1993-1998) and from other cattle with suspected pneumonia, mastitis or bovine tuberculosis, were included in the analysis. An isolate from Hungary and one from Nigeria were also included. After standardization of the MLVA method, only 11 loci were selected, since the MbovTR49-51 locus consistently showed a multiband pattern. In this study 14 new alleles, never observed before, were found for seven of the loci, of which eight are considered as intermediates repeats. Analysis of the HGDI diversity showed a locus-wide variety of 3 to 7 polymorphisms, with HGDI varying between 0.337 and 0.777. In terms of discriminatory power of the technique, for the 11 loci panel, 55 MLVA profiles were obtained corresponding to a HGDI of 0.989, with the predominant profile being the profile 00201044324 (MbovTR14-MbovTR29-MbovTR30-MbovTR31-MbovTR35-MbovTR40-41-MbovTR52-MbovTR59-MbovTR147-MbovTR148-MbovTR427) found in five strains. These diversity values show that the MLVA technique can be used in M. bovis isolates for the purpose of differentiation. For a cut-off value of 55 %, the dendrogram showed the formation of six clusters, four of which belong to Portuguese strains and the other two to strains, respectively, from the USA and from Nigeria, indicating that seem to exist differences between strains at continental level, with European, African and North American strains belonging to distinct clusters. The analysis by minimum extension tree (MST) shows that there are nine clonal complexes within the analyzed strains. These results suggest a multiple origin and a great diversity within M. bovis Portuguese strains. No temporal or regional relationships have been found within isolates, which may be related to the type of management practiced in Portuguese farms, which may favor the dissemination of M. bovis. The strains of this study were also compared with strains from other studies using a panel of 8 loci. MST showed the presence of eleven clonal complexes in European strains, where a multiple country origin is also verified for Portugal and Hungary, but there is also a common lineage between European strains, where some Portuguese strains are included. These results suggest that the ease of transaction of cattle within the European community may be implicated in the spread of M. bovis in the last 20 years in Europe.
Mycoplasma bovis is a pathogen capable of causing mastitis, pneumonia, arthritis and otitis in cattle. One of the most serious diseases is BRDC (Bovine respiratory disease complex). Although BRDC can be caused by several pathogens, only for M. bovis there is no effective antibiotic therapy and vaccination. In order to evaluate the contribution of M. bovis in the development of respiratory diseases in Portugal, 303 post mortem samples collected in 2015 from bovine animals with suspected bovine tuberculosis were analyzed. Nine mycoplasma isolates were obtained from several regions, and were characterized by biochemical tests and PCR, and identified as belonging to M. bovis strains. The relative frequencies calculated showed that there are essentially two foci at regional level, namely for the Northern region (6.7 %) and for the Lisboa e Vale do Tejo region (14.3 %). At country level for the year 2015 the relative frequency was 3 %. Only two cases of co-infection (0.7 %) with Mycoplasma bovis and Mycobacterium bovis were found, both belonging to the Northern region. In order to show the possible epidemiological relationship of M. bovis isolates, the MLVA technique was developed and adapted based on a panel of 12 loci. In addition to the nine isolates obtained, 58 M. bovis isolates from 1997-2017 (making 67 isolates in total) belonging to the INIAV I.P., biobank, isolated from PPCB outbreaks (1993-1998) and from other cattle with suspected pneumonia, mastitis or bovine tuberculosis, were included in the analysis. An isolate from Hungary and one from Nigeria were also included. After standardization of the MLVA method, only 11 loci were selected, since the MbovTR49-51 locus consistently showed a multiband pattern. In this study 14 new alleles, never observed before, were found for seven of the loci, of which eight are considered as intermediates repeats. Analysis of the HGDI diversity showed a locus-wide variety of 3 to 7 polymorphisms, with HGDI varying between 0.337 and 0.777. In terms of discriminatory power of the technique, for the 11 loci panel, 55 MLVA profiles were obtained corresponding to a HGDI of 0.989, with the predominant profile being the profile 00201044324 (MbovTR14-MbovTR29-MbovTR30-MbovTR31-MbovTR35-MbovTR40-41-MbovTR52-MbovTR59-MbovTR147-MbovTR148-MbovTR427) found in five strains. These diversity values show that the MLVA technique can be used in M. bovis isolates for the purpose of differentiation. For a cut-off value of 55 %, the dendrogram showed the formation of six clusters, four of which belong to Portuguese strains and the other two to strains, respectively, from the USA and from Nigeria, indicating that seem to exist differences between strains at continental level, with European, African and North American strains belonging to distinct clusters. The analysis by minimum extension tree (MST) shows that there are nine clonal complexes within the analyzed strains. These results suggest a multiple origin and a great diversity within M. bovis Portuguese strains. No temporal or regional relationships have been found within isolates, which may be related to the type of management practiced in Portuguese farms, which may favor the dissemination of M. bovis. The strains of this study were also compared with strains from other studies using a panel of 8 loci. MST showed the presence of eleven clonal complexes in European strains, where a multiple country origin is also verified for Portugal and Hungary, but there is also a common lineage between European strains, where some Portuguese strains are included. These results suggest that the ease of transaction of cattle within the European community may be implicated in the spread of M. bovis in the last 20 years in Europe.
Descrição
Tese de mestrado, Microbiologia Aplicada, Universidade de Lisboa, Faculdade de Ciências, 2017
Palavras-chave
Mycoplasma bovina BRDC MLVA Epidemiologia molecular Bovinos Teses de mestrado - 2017