Caracterização da microbiota da glândula mamária bovina saudável e com mastite subclínica e seleção de bactérias potencialmente probióticas isoladas deste ecossistema

Abstract

Mastitis is the most impactful disease on milk production in cattle. Due to strategic importance of dairy livestock in food production, control of this disease is of utmost importance. To address international pressure to reduce the use of antimicrobials in animal production, development of alternative methods of treatment and prevention of mastitis should be implemented to replace the traditional antibiotic therapy. Due to increasing use of probiotic bacteria in the control of different infectious diseases, bacteriotherapy offers a possible strategy for control of mastitis. The lactic acid bacteria are members of the natural microbiota of bovine mammary gland, having a long history of safe use, and present several proven beneficial health effects on the host, and therefore are strong candidates for selection of probiotics for the mammary gland. The objectives of this study were to characterize the microbiota of mammary gland in four bovine cattle (Gir, Guzerat, Girolando 1/2 and Holstein) and isolate and characterize BAL from raw milk and intramammary swab samples sources. The microbiota of the mammary gland of 200 cows (131 healthy and 69 with subclinical mastitis) showed composition dominated by Firmicutes and Proteobacteria. In general, a low percentage of OTUs was shared between different breeds. Our data indicate that the environmental effect on the composition of the microbial community seems to be strong. The change in microbiota composition with the installation of subclinical mastitis (reduced abundance of Acinetobacter and Pseudomonas and increased of Staphylococcus) indicate that this disorder is associated with dysbiosis. With the exception of Holstein, that seems to have subclinical mastitis condition characterized by increased abundance of Lactococcus, the relative abundance of BAL decreased in CCS> 200.000 cel/mL animals. This indicates that BAL may be related to healthier udders. Therefore, samples of 115 cows were subjected to the isolation of BAL. 192 isolates were obtained from BAL belonging to 30 species, of which 82 were selected for evaluation of its potential probiotic. In general, the BAL inhabiting the mammary gland had low autoagregation, hydrophobicity of the cell surface, co-aggregation with pathogens and biofilm production capacity. Moreover, it seems to be typical that these BAL did not exhibit the EPS production capacity and had a high percentage of antagonism against mastitis pathogens with high H2O2 production. Two BAL strains were able to adhere and internalize in MDA-MB-231 mammary epithelial cells and had potential protective effect against mastitis. WcoGIRO48L1* (Weissella confusa GIR48L1*) significantly reduced adhesion of Staphylococcus aureus ATCC 29213, adhesion of Escherichia coli ATCC 25723 and of Tnfa production. On the other hand, LactGIRO4S8* (L. lactis GIRO4S8*) was able to significantly reduce adhesion and invasion of S. aureus ATCC 29213 and adhesion of E. coli ATCC 25723, and increased IL8 expression. Both strains were able to reduce IL1β production. Our results contribute for production of new diagnostic and treatment tools for mastitis in the future.