Research MSc thesis

Comparison of bulk tank milk and water microbiota at Brazilian dairy farms during the dry and wet seasons

The bulk tank milk (BTM) microbiota is highly diverse, with multiple contamination sources contributing to its complexity. It has been suggested that the continuous use of water during the cleaning process and the frequentl poor quality of water in dairy farms may negatively impact the microbiological quality of raw milk. This master dissertation therefore investigated the relationship between the bacterial communities of BTM microbiota and the water used in Brazilian dairy farms in the dry and wet seasons.  The association between BTM microbiota and quality parameters (somatic cell count, total bacterial count, and differentiated bacterial counts) was also studied. In addition, the relationship between BTM microbiota and water quality indices (physicochemical and microbiological studies) was investigated.

The milk (n = 22) and water (n = 22) samplings from 11 Brazilians dairy farms were performed in the dry (May, 2017) and wet (September, 2017) seasons. The microbiome analyses of the water and BTM were performed through next-generation sequencing analysis, utilizing the hypervariable regions of the 16S rRNA gene (V3-V4) using standard Illumina protocols, followed by bioinformatic data analyses. The BTM somatic cell and total bacteria counts, antimicrobial residues screening, and psychotropic microorganisms, Streptococcus agalactiae, non-agalactiae Streptococcus, Staphylococcus aureus, non-aureus Staphylococcus and Mammaliicoccus, coliforms, Klebsiella spp., Pseudomonas spp., and Bacillus cereus counts were also determined. The tap water from the “point-of-use” were also collected to investigate the physicochemical (total solids, pH, turbidity and hardness) and microbiological (aerobic mesophilic heterotrophic bacteria, total coliforms and Escherichia coli counts) analyses.

In this study, eight (72.73%) and one (9.09%) of the BTM samples collected in the dry and wet seasons, respectively, showed somatic cell counts above the threshold held within Brazilian legislation (5 x 105 cells/mL), although all dairy farms had BTM total bacteria counts lower than the legal thresholds (3 x 105 cfu/mL). Antimicrobial residue screening tests were negative in all BTM samples. All water physicochemical outcomes were within the current Brazilian standards, except for two water samples that had high turbidity. Nonetheless, 66.33% of the tap water samples had aerobic mesophilic heterotrophic bacteria counts higher than the legal thresholds. Furthermore, E. coli was present at detectable levels in 45.45% and 63.63% of the tap water samples in the dry and wet seasons, respectively. Indeed, just 9.09% of the water samples were compliant (absence of coliforms in 100 mL) with total coliform standards in both samplings time.

Overall, the water microbiota had no effect on the BTM bacterial community, despite the fact that a considerable proportion of the water samples used in the dairy farms did not meet the recommended bacteriological quality standards. However, the usage of amplicon-based high-throughput sequencing is subject to bias due to the conversion of obtained values to relative abundance. Therefore, after further analysis, searching for any association of water quality potability parameters and BTM microbiota relative abundance and absolute values, non-compliant water (e.g., heterotrophic bacteria plate count and E. coli counts) negatively impact BTM microbiological quality.

Furthermore, a negative correlation was found between the number of bacterial species (diversity) on BTM and somatic cell counts. In this study, a higher BTM somatic cell count was associated with a higher abundance of Streptococcus spp. and a tendency toward a higher abundance of Staphylococcus spp. Besides that, a higher abundance of Bacillus spp. and a tendency toward a higher abundance of Lactococcus spp. was associated with a higher BTM total bacterial counts, and consequently could be used as an indicator of milk quality. A large variation in BTM microbiota membership between sampling times was observed, which could impact the microbial signatures of traditional artisanal cheeses. No effect of season on tap water microbiota was found.

Therefore, the results of this dissertation underscore the importance of ensuring a safe source of water on dairy farms as a way to mitigate the possible contamination of unpasteurized milk and the associated risks of waterborne diseases in Brazil.

Ana Claudia Dumont Oliveira graduated as a veterinarian in 2012 from Federal University of Minas Gerais, Brazil. Since then, she has worked as a veterinary adviser for udder health and mastitis. She is also a co-founder of the company MilkCare. She completed her master’s degree in 2018 at the same institution under the supervisor of Prof. dr. Monica Maria Oliveira Pinho Cerqueira, co-supervised by Prof. dr. Fernando Nogueira Souza. Currently, she is a Ph.D. Student at the University of São Paulo (Brazil) under the supervision of Fernando Nogueira de Souza working with bovine mammary gland immunology and vaccine development. She is now doing part of her Ph.D. (1 year internship) with M-teamUGent in Belgium.







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