The impact of the topical exposure to Cytinus hypocistis extract on fungi of medical relevancy and in Staphylococcus aureus as a potential alternative treatment for bovine mastitis
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2022-07-19
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A mastite bovina é definida como uma inflamação intramamária. Pode ser causada por vários fatores como lesões físicas, mas dentre estes a infeção bacteriana é o mais comum. Esta doença tem repercussões não só no leite, mas também no bem-estar do animal. Esta é das doenças que provocam mais perdas económicas no setor leiteiro. Geralmente, é tratada utilizando antibióticos, mas a sua eficácia tem vindo a diminuir devido ao emergente aparecimento de cada vez mais resistentes. Então existe um crescente interesse em formas alternativas de tratamento para a mastite bovina.
A planta Cytinus hypocistis (L.) L. presente em várias regiões Mediterrâneas apresenta múltiplas propriedades de interesse como atividade antimicrobiana devido ao seu elevando teor em taninos, especificamente gallotaninos.
Assim o principal objetivo deste trabalho foi analisar o efeito que uma formulação tópica enriquecida com extrato de C. hypocistis (EF) tem na microbiota das orelhas de murganho de modo a avaliar o potencial deste extrato como tratamento alternativo para a mastite.
Para isto foram utilizadas 3 EF com concentrações diferentes de extrato: CH1, 3.1 mg de extrato/g de creme; CH2 6.2 mg/g; e CH3, 12.4 mg/g. trinta amostras foram recolhidas dos pavilhões auriculares de 30 murganhos fêmea FBV/n, e inoculadas em meio Potato Dextrose Agar (PDA). O procedimento experimental foi aprovado pela Comissão de Ética Animal- DGAV number 0421/000/000/2014, 24/09/2014 (020172).
As espécies bacterianas e fúngicas foram isoladas e identificadas utilizando testes API e microscopia com lactofenol com azul de algodão, respetivamente. Adicionalmente, furam realizados um antibiograma e a deteção de fatores de virulência da bactéria.
Para avaliar o potencial antifúngico do extrato, foram ainda determinadas as percentagens de inibição (IP%) do extrato no crescimento de Aspergillus fumigatus. A analise estatística utilizou o programa SPSS® versão 25.0 e um teste t Student com p<0,05 de significância foi utlizado para avaliar a diferença entre as percentagens de inibição médias para as duas concentrações testadas.
Furam identificados 4 géneros fúngicos: Penicillium (16.7%), Mucor (16.7%), Cladosporium (3.33%), Aspergillus (3.33%). No total, 14 (46.7%) dos animais apresentaram fungos nas suas amostras. Após o tratamento, apenas uma amostra apresentou a presença de fungos, este não foi identificado. As colónias bacterianas de Staphylococcus aureus de ambos os períodos de
amostragem, mostraram-se resistentes a penicilina G e ceftazidime, e apresentaram os genes para as Staphylococcus enterotoxinas G e I, para a hemolisina-α e para a toxina da síndrome de choque toxico-1.
A concentração de 0.115 mg/mL apresentou a maior IP% (67%) 3 dias após a inoculação. As IP% para esta concentração registadas foram sempre maiores ao longo do procedimento comparativamente a concentração de 0.230 mg/mL. Mas, a diferença entre as médias de IP% para as duas concentrações de estrato não foi significativa (t=0.232; p=0.828).
Estes resultados sugerem a existência de uma grande biodiversidade de microrganismos nas no pavilhão auricular do murganho e que a exposição ao extrato de levou a inibição do crescimento de A. fumigatus. No entanto, não foi possível formular qualquer suposição acerca do efeito deste extrato in vivo e no tratamento de mastite bovino.
Bovine mastitis is defined as an intramammary inflammation. It can be caused by several factors like physical injuries but usually, the cause is infectious, which is most commonly bacterial in origin. It affects not only the milk production and quality but also the animal’s welfare. Bovine mastitis is seen as one of the most financially damaging diseases in the dairy sector. The treatment employs the use of antibiotics however, the efficiency of these drugs has been decreasing due to the emergence of antibiotic-resistant species. Therefore, there is a strong demand for alternative treatments for bovine mastitis. Cytinus hypocistis (L.) L. is a holoparasitic plant occurring in the Mediterranean region. Its tannin-rich extract has been found to have multiple beneficial properties, accredited to the presence of gallotannins. The main objective of the present work was to study the effect of a C. hypocistis enriched topical formulation (EF) in mice’s ears microbial isolates using routine mycological techniques, to access the potential of this extract as an alternative treatment for bovine mastitis. For this study, three EF were prepared with different concentrations of the C. hypocistis extract: CH1, 3.1 mg of extract/g of cream; CH2, 6.2 mg /g; and CH3, 12.4 mg/g. Thirty samples were collected from each female mice’s outer ears (strain FBV/n) and inoculated in Potato Dextrose Agar (PDA). The experiment procedure was approved by an Animal Ethics Committee-DGAV number 0421/000/000/2014, 24/09/2014 (020172). The bacterial and fungal species isolated were identified using API test and microscopy with Lactophenol with Cotton Blue staining, respectively. An antibiogram and identification of virulence factors were also performed on the bacterial species. To access the specific antifungal effect of the extract, the inhibition percentage (IP%) method was calculated using the growth of Aspergillus fumigatus. The statistical analysis was performed using the program SPSS® version 25.0. The t Student test with p˂0.05 of significance was used to analyse the differences between the mean IP% for each extract concentration. Four fungal genera were isolated: Penicillium (16.7%), Mucor (16.7%), Cladosporium (3.33%), Aspergillus (3.33%). In total, fungi were isolated in 14 (46.7%) animals. After the treatment, only one fungal colony was isolated, and the genus was not identified. The bacterial colonies were identified as Staphylococcus aureus for both the collection instances. These showed resistance to penicillium G and ceftazidime, and presented the genes for Staphylococcus enterotoxins G and I, hemolysin-α and toxic shock syndrome toxin-1. The higher IP% was for the concentration of 0.115 mg/mL on the third day after inoculation, being higher than 67%. The IP% for this concentration were always higher compared to the ones for the concentration of 0.230 mg/mL, on each day of assessment. However, the differences between the means of the IP% for both the concentrations were found to be not significant (t=0.232; p=0.828). The results obtained suggest a high fungi biodiversity in the mice’s ears and that the extract inhibits the growth of A. fumigatus in vitro. However, no assumptions could be made about the in vivo effects of this extract or about the potential to treatment mastitis.
Bovine mastitis is defined as an intramammary inflammation. It can be caused by several factors like physical injuries but usually, the cause is infectious, which is most commonly bacterial in origin. It affects not only the milk production and quality but also the animal’s welfare. Bovine mastitis is seen as one of the most financially damaging diseases in the dairy sector. The treatment employs the use of antibiotics however, the efficiency of these drugs has been decreasing due to the emergence of antibiotic-resistant species. Therefore, there is a strong demand for alternative treatments for bovine mastitis. Cytinus hypocistis (L.) L. is a holoparasitic plant occurring in the Mediterranean region. Its tannin-rich extract has been found to have multiple beneficial properties, accredited to the presence of gallotannins. The main objective of the present work was to study the effect of a C. hypocistis enriched topical formulation (EF) in mice’s ears microbial isolates using routine mycological techniques, to access the potential of this extract as an alternative treatment for bovine mastitis. For this study, three EF were prepared with different concentrations of the C. hypocistis extract: CH1, 3.1 mg of extract/g of cream; CH2, 6.2 mg /g; and CH3, 12.4 mg/g. Thirty samples were collected from each female mice’s outer ears (strain FBV/n) and inoculated in Potato Dextrose Agar (PDA). The experiment procedure was approved by an Animal Ethics Committee-DGAV number 0421/000/000/2014, 24/09/2014 (020172). The bacterial and fungal species isolated were identified using API test and microscopy with Lactophenol with Cotton Blue staining, respectively. An antibiogram and identification of virulence factors were also performed on the bacterial species. To access the specific antifungal effect of the extract, the inhibition percentage (IP%) method was calculated using the growth of Aspergillus fumigatus. The statistical analysis was performed using the program SPSS® version 25.0. The t Student test with p˂0.05 of significance was used to analyse the differences between the mean IP% for each extract concentration. Four fungal genera were isolated: Penicillium (16.7%), Mucor (16.7%), Cladosporium (3.33%), Aspergillus (3.33%). In total, fungi were isolated in 14 (46.7%) animals. After the treatment, only one fungal colony was isolated, and the genus was not identified. The bacterial colonies were identified as Staphylococcus aureus for both the collection instances. These showed resistance to penicillium G and ceftazidime, and presented the genes for Staphylococcus enterotoxins G and I, hemolysin-α and toxic shock syndrome toxin-1. The higher IP% was for the concentration of 0.115 mg/mL on the third day after inoculation, being higher than 67%. The IP% for this concentration were always higher compared to the ones for the concentration of 0.230 mg/mL, on each day of assessment. However, the differences between the means of the IP% for both the concentrations were found to be not significant (t=0.232; p=0.828). The results obtained suggest a high fungi biodiversity in the mice’s ears and that the extract inhibits the growth of A. fumigatus in vitro. However, no assumptions could be made about the in vivo effects of this extract or about the potential to treatment mastitis.
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Mestrado em Engenharia Zootécnica