Abstract
The aim of this study was to define the microbiota of water buffalo milk during sub-clinical and clinical mastitis, as compared to healthy status, by using high-throughput sequencing of the 16S rRNA gene. A total of 137 quarter samples were included in the experimental design: 27 samples derived from healthy, culture negative quarters, with a Somatic Cell Count (SCC) of less than 200,000 cells/ml; 27 samples from quarters with clinical mastitis; 83 samples were collected from quarters with subclinical mastitis, with a SCC number greater of 200,000 cells/ml and/or culture positive for udder pathogens, without clinical signs of mastitis. Bacterial DNA was purified and the 16S rRNA genes were individually amplified and sequenced. Significant differences were found in milk samples from healthy quarters and those with sub-clinical and clinical mastitis. The microbiota diversity of milk from healthy quarters was richer as compared to samples with sub-clinical mastitis, whose microbiota diversity was in turn richer as compared to those from clinical mastitis. The core microbiota of water buffalo milk, defined as the asset of microorganisms shared by all healthy milk samples, includes 15 genera, namely Micrococcus, Propionibacterium, 5-7N15, Solibacillus, Staphylococcus, Aerococcus, Facklamia, Trichococcus, Turicibacter, 02d06, SMB53, Clostridium, Acinetobacter, Psychrobacter and Pseudomonas. Only two genera (Acinetobacter and Pseudomonas) were present in all the samples from sub-clinical mastitis, and no genus was shared across all in clinical mastitis milk samples. The presence of mastitis was found to be related to the change in the relative abundance of genera, such as Psychrobacter, whose relative abundance decreased from 16.26% in the milk samples from healthy quarters to 3.2% in clinical mastitis. Other genera, such as SMB53 and Solibacillus, were decreased as well. Discriminant analysis presents the evidence that the microbial community of healthy and clinical mastitis could be discriminated on the background of their microbiota profiles.
Highlights
The development of culture-independent techniques by means of high-throughput DNA sequencing has just begun to unravel the impact of large community of micro-organisms, the so called microbiota, on human and animal health [1]
Microbial diversity was analysed after classification of quarter milk as follows: 27 samples were collected from healthy quarters with no clinical signs of mastitis during the present lactation, with two consecutive Somatic Cell Counts (SCC) values lower than 200,000 cells/ml and aerobic culture negative for udder pathogens (H); 27 samples with clinical mastitis (CM) were collected from quarters showing signs of clinical mastitis and aerobic culture positive
Among sub-clinical mastitis (SM) affected quarters, bacteria that are potentially associated with mastitis were recovered in 67 samples (81%), whereas the others 15 were negative after microbiological culture with SCC > 200.000 cells/ml
Summary
The development of culture-independent techniques by means of high-throughput DNA sequencing has just begun to unravel the impact of large community of micro-organisms, the so called microbiota, on human and animal health [1]. Microbiota establishes mutual relationship with its hosts and the resulting cross-talk extends beyond the balance between tolerance to commensal micro-organisms and developing protection against pathogens [2]. Metagenomic techniques have revealed how “healthy” microbiota, e.g. the microbial community belonging to healthy individuals, includes potential pathogens. Recent studies on gut microbiota have provided the evidence that the onset of a disease can be the result of a change in the interaction with other microorganisms [3]. A new concept of pathobiome, which can be defined as the microbiota environment integrating pathogenic agents, is taking shape and has been recently discussed and thoughtfully reviewed [4]. A metagenomic approach has been applied to the relationship between resident microbiomes and the development of reproductive diseases [10,11,12,13,14]
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