Expressão gênica diferencial e análise protéica do leite de búfalas sadias e com mastite subclínica
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2018-02-27
Autores
Tanamati, Fernanda
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Universidade Estadual Paulista (Unesp)
Resumo
A mastite em búfalas leiteiras causa perdas econômicas devido à diminuição da produção de leite, além de ter um efeito negativo no bem-estar dos animais. Deste modo, foram realizados dois estudos: o objetivo do estudo I foi avaliar o nível de expressão dos genes da lactoferrina (LTF), fator de necrose tumoral alfa (TNF-α), interleucina-1 beta (IL-1β), interleucina-8 (IL-8), e toll-like receptors 2 (TLR-2) e 4 (TLR-4) em búfalas Murrah com e sem mastite subclínica. Amostras de leite de 12 búfalas com mastite e 12 de búfalas sem mastite foram coletadas, para a extração de RNA, síntese de cDNA e validação dos perfis de expressão por qRT-PCR. O ΔΔCt foi estimado utilizando contrastes ortogonais da expressão dos genes alvo corrigidos para a expressão dos genes housekeeping entre os tratamentos. A expressão dos genes TLR-2, TLR-4, TNF-α, IL-1β e IL-8 foi regulada positivamente em búfalos com mastite, enquanto que o gene LTF não apresentou expressão diferencial. O estudo desses genes da função imune que são ativos na glândula mamária é importante para o desenvolvimento de estratégias destinadas a preservar a saúde do úbere. O objetivo do estudo II foi avaliar as proteínas presentes no soro do leite de búfalas com e sem mastite subclínica, utilizando uma abordagem proteômica para identificar proteínas diferencialmente expressas e potencial biomarcador para a doença. Para isso, 16 amostras de leite de búfalas Murrah foram coletadas, sendo oito animais com e oito animais sem mastite subclínica. O método contagem espectral foi utilizado para quantificar a abundância relativa das proteínas individuais e os bancos de dados de proteínas anotadas para Bubalus bubalis e para Bos taurus foram utilizados na análise. Após integração das anotações genéticas das referências de búfalos e bovinos, foram identificadas 1.033 proteínas, das quais 156 proteínas foram diferencialmente reguladas entre animais sadios e infectados. Foram identificados 18 processos biológicos nos quais essas proteínas participam e a catelicidina-3 foi identificada como um potencial biomarcador da mastite subclínica. Os resultados são importantes para compreender melhor o comportamento da mastite na glândula mamária das búfalas e, portanto, podem auxiliar no diagnóstico precoce da doença.
Mastitis in dairy buffalo causes economic losses due to decreased milk production, along with having a negative effect on the animals’ welfare. Thus, two studies were performed: the objective of study I was to evaluate the expression levels of lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin- 8 (IL-8) and toll-like receptors 2 (TLR-2) and 4 (TLR-4) in Murrah buffaloes with and without subclinical mastitis. Milk samples were collected from 12 buffaloes with mastitis and 12 buffaloes without mastitis for RNA extraction, cDNA synthesis, and validation of expression profiles by qRT-PCR. The ΔΔCt was estimated using orthogonal contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both treatments. The expression of the TLR-2, TLR-4, TNF-α, IL-1β and IL-8 genes were upregulated in buffaloes with mastitis, while the LTF gene showed no differential expression. The study of these immune function genes that are active in the mammary gland is important for the development of strategies aimed at preserving the health of the udder. The objective of study II was to evaluate the proteins present in the milk whey from buffaloes with and without subclinical mastitis, using a proteomic approach to identify differentially expressed proteins and potential biomarkers for the disease. For this, 16 samples of Murrah buffalo milk were collected, comprised of eight animals with and eight animals without subclinical mastitis. The spectral counting method was used to quantify the relative abundance of the individual proteins, and the databases of proteins annotated for Bubalus bubalis and for Bos taurus were used in the analysis. After integrating the gene annotations from the buffalo and bovine references, a total of 1,033 proteins were identified, of which 156 proteins were differentially regulated between healthy and affected animals. 18 biological processes in which these proteins participate were identified, and cathelicidin-3 was identified as a potential biomarker of subclinical mastitis. These results are important to better understand the behavior of mastitis in the buffalo mammary gland, and in turn may aid in the early diagnosis.
Mastitis in dairy buffalo causes economic losses due to decreased milk production, along with having a negative effect on the animals’ welfare. Thus, two studies were performed: the objective of study I was to evaluate the expression levels of lactoferrin (LTF), tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β), interleukin- 8 (IL-8) and toll-like receptors 2 (TLR-2) and 4 (TLR-4) in Murrah buffaloes with and without subclinical mastitis. Milk samples were collected from 12 buffaloes with mastitis and 12 buffaloes without mastitis for RNA extraction, cDNA synthesis, and validation of expression profiles by qRT-PCR. The ΔΔCt was estimated using orthogonal contrasts of the target genes expression adjusted for the expression of the housekeeping genes between both treatments. The expression of the TLR-2, TLR-4, TNF-α, IL-1β and IL-8 genes were upregulated in buffaloes with mastitis, while the LTF gene showed no differential expression. The study of these immune function genes that are active in the mammary gland is important for the development of strategies aimed at preserving the health of the udder. The objective of study II was to evaluate the proteins present in the milk whey from buffaloes with and without subclinical mastitis, using a proteomic approach to identify differentially expressed proteins and potential biomarkers for the disease. For this, 16 samples of Murrah buffalo milk were collected, comprised of eight animals with and eight animals without subclinical mastitis. The spectral counting method was used to quantify the relative abundance of the individual proteins, and the databases of proteins annotated for Bubalus bubalis and for Bos taurus were used in the analysis. After integrating the gene annotations from the buffalo and bovine references, a total of 1,033 proteins were identified, of which 156 proteins were differentially regulated between healthy and affected animals. 18 biological processes in which these proteins participate were identified, and cathelicidin-3 was identified as a potential biomarker of subclinical mastitis. These results are important to better understand the behavior of mastitis in the buffalo mammary gland, and in turn may aid in the early diagnosis.
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Palavras-chave
Bubalus bubalis, glândula mamária, LC/MS-MS, proteômica, qRT-PCR, mammary gland, proteomic