Avaliação microbiológica e molecular de isolados de Mycoplasma spp. a partir do leite de mastite clínica bovina
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Data
2018-11-28
Autores
Salina, Anelise
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Universidade Estadual Paulista (Unesp)
Resumo
O gênero Mycoplasma inclui mais de 200 espécies responsáveis por causar doenças nos animais. Apresenta células de tamanho reduzido e em formatos variáveis. Ocasiona mastite em bovinos, podendo também, estar relacionado a outras manifestações como artrite e pneumonia em bezerros e novilhas. Neste estudo, objetivou-se o isolamento de espécies de micoplasmas a partir de amostras de leite de mastite clínica bovina, bem como, a detecção de Mycoplasma (M.) bovis pelas técnicas moleculares de PCR convencional e PCR em tempo real. Um total de 1166 amostras de leite de casos de mastite clínica, provenientes de nove propriedades leiteiras da região da bacia leiteira ABCW – Paraná e de uma propriedade situada na região de São Pedro – São Paulo foram avaliadas para a presença do DNA de Mollicutes. Desse total, em 100 amostras de leite que foram positivas à PCR para a classe Mollicutes, realizou-se a PCR para detecção de M. bovis obtendo-se positividade de 13% (13/100). Na análise pela PCR em tempo real obteve-se 11% (11/100) de amostras positivas para M. bovis. No cultivo microbiológico, obteve-se 6% (6/100) de crescimento ao se utilizar o meio Hayflick e, 11% (11/100) de crescimento ao se utilizar o meio SP4 (incluindo as positivas ao primeiro). A partir desses isolados em caldo PPLO, nove deles foram positivos à PCR convencional para M. bovis e dois, negativos. Na análise filogenética dos isolados, a partir dos resultados do sequenciamento, obteve-se 100% de M. bovis para todos os isolados. Frente aos resultados obtidos, ressalta-se a importância de estudos mais aprofundados para a elucidação de outras espécies de micoplasmas envolvidas na mastite bovina, e reforça a participação de M. bovis na sua etiologia.
The Mycoplasma genus includes more than 200 species responsible for causing diseases in animals. It presents cells of reduced size and in variable shapes. It may cause mastitis in cattle, and may also be related to other manifestations such as arthritis and pneumonia in calves and heifers. The objective of this study was to isolate mycoplasma species from bovine clinical mastitis milk samples, as well as the detection by molecular techniques of conventional PCR and real-time PCR of M. bovis. A total of 1166 milk samples from clinical mastitis cases from nine dairy farms in the ABCW - Paraná dairy region and from a property located in the São Pedro region - São Paulo were evaluated for the presence of Mollicutes DNA. From this total, 100 milk samples that were PCR positive for the Mollicutes class, the PCR was performed to detect the M. bovis, obtaining 13% positivity (13/100). Real-time PCR analysis yielded 11% (11/100) of positive samples for M. bovis. In the microbiological culture, 6% (6/100) of growth was obtained when using the Hayflick medium and 11% (11/100) growth when using SP4 medium (including positive ones in the first one). From these isolates in PPLO broth, nine of them were positive to conventional PCR for M. bovis and two, negative. In the phylogenetic analysis of the isolates, from the results of the sequencing, 100% of M. bovis was obtained for all the isolates. In view of the results obtained, the importance of further studies for the elucidation of other species of mycoplasmas involved in bovine mastitis is emphasized, and reinforces the participation of M. bovis in its etiology.
The Mycoplasma genus includes more than 200 species responsible for causing diseases in animals. It presents cells of reduced size and in variable shapes. It may cause mastitis in cattle, and may also be related to other manifestations such as arthritis and pneumonia in calves and heifers. The objective of this study was to isolate mycoplasma species from bovine clinical mastitis milk samples, as well as the detection by molecular techniques of conventional PCR and real-time PCR of M. bovis. A total of 1166 milk samples from clinical mastitis cases from nine dairy farms in the ABCW - Paraná dairy region and from a property located in the São Pedro region - São Paulo were evaluated for the presence of Mollicutes DNA. From this total, 100 milk samples that were PCR positive for the Mollicutes class, the PCR was performed to detect the M. bovis, obtaining 13% positivity (13/100). Real-time PCR analysis yielded 11% (11/100) of positive samples for M. bovis. In the microbiological culture, 6% (6/100) of growth was obtained when using the Hayflick medium and 11% (11/100) growth when using SP4 medium (including positive ones in the first one). From these isolates in PPLO broth, nine of them were positive to conventional PCR for M. bovis and two, negative. In the phylogenetic analysis of the isolates, from the results of the sequencing, 100% of M. bovis was obtained for all the isolates. In view of the results obtained, the importance of further studies for the elucidation of other species of mycoplasmas involved in bovine mastitis is emphasized, and reinforces the participation of M. bovis in its etiology.
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Palavras-chave
Micoplasmose, PCR, Mastite bovina, Mollicutes, Leite, Mycoplasmosis, PCR, Bovine mastitis, Mollicutes, Milk