Short communication:Intra- and inter-individual milk microbiota variability in healthy and infected water buffalo udder quarters

The concept that ruminant mammary gland quarters are anatomically and physiologically unrelated has been recently challenged by immunological evidence. How this interdependence reflects on individual quarter milk microbiota is unknown. The aim of the present study was to cover this gap by investigating the interdependence of quarters among the same mammary gland at the milk microbiota level using next-generation sequencing of the V4\u201316S rRNA gene. A total of 52 samples were included in this study and classified as healthy or affected by subclinical mastitis. Extraction of DNA, amplification of the V4\u201316S rRNA gene, and sequencing using Ion Torrent Personal Genome Machine (Thermo Fisher Scientific, Waltham, MA) were carried out. We found that the intra-individual variability was lower than the inter-individual one. The present findings further support at milk microbiota level the hypothesis of the interdependence of quarters, as previously demonstrated following immunological studies, suggesting that individual factors (e.g., immunity, genetics) may have a role in modulating milk microbiota

Effect of inactivated cultures of Lactobacillus rhamnosus on subclinical mastitis quarter milk microbiota

Water buffaloes mastitis represents a major issue in terms of animal health, cost of therapy, premature culling and decreased milk yeld. The emergence of antibiotic resistance has led to investigate strategies in order to avoid or minimize the antibiotic use, especially during subclinical mastitis disease (SM) (1). Lactobacillus rhamnosus is part of the normal gut microflora, having meanwhile an immunostimulatory activity (2). The aim of this study was to investigate the change of milk microbiota after the theraputic treatment with inactivated cultures of Lactobacillus rhamnosus of mammary gland quarters affected by subclinical mastitis. A number of 43 quarters were included in the study, and were treated with antibiotics, Lactobacillus rhamnosus  and PBS as negative control. Samples were collected at two time points, T0 and T5 (days) and V4 region of 16S rRNA gene was amplified by PCR and sequenced using Ion Torrent Personal Genome Machine. The microbiota structure of SM quarters showed no major changes after PBS treatment, while differed after antibiotic treatment where Staphylococcus decreased its relative abundance from 41% at T0 to 3% at T5. Lactobacillus rhamnosus induced a less dramatic change in milk microbiota, although the relative abundance of some genera were found to be modifidied, among which an increase of Pseudomonas from 1.5% at T0 up to 4% at T5. No differences were present between the microbiota structure of quarters treated with Lactobacillus rhamnosus and PBS. This study allowed to characterize the changes of microbiota in milk from animals with Lactobacillus rhamnosus and antibiotics. While changes in milk microbiota after antibiotic treatment were evident, changes after Lactobacillus rhamnosus were more limited. Further investigations are needed to evaluate alternative strategies to mastitis treatment.

The microbiota of water buffalo milk during mastitis

<div><p>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 <i>Micrococcus</i>, <i>Propionibacterium</i>, <i>5-7N15</i>, <i>Solibacillus</i>, <i>Staphylococcus</i>, <i>Aerococcus</i>, <i>Facklamia</i>, <i>Trichococcus</i>, <i>Turicibacter</i>, <i>02d06</i>, <i>SMB53</i>, <i>Clostridium</i>, <i>Acinetobacter</i>, <i>Psychrobacter</i> and <i>Pseudomonas</i>. Only two genera (<i>Acinetobacter</i> and <i>Pseudomonas</i>) 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 <i>Psychrobacter</i>, whose relative abundance decreased from 16.26% in the milk samples from healthy quarters to 3.2% in clinical mastitis. Other genera, such as <i>SMB53</i> and <i>Solibacillus</i>, 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.</p></div

Relative abundance of microbiota taxa at phylum level.

<p>Relative abundance of microbiota taxa at phylum level.</p

Water buffalo milk taxonomic profile at genus level.

<p>The microbial relative abundance at genus level between: H = Healthy samples; SM = Sub-Clinical mastitis samples; CM = Clinical mastitis samples; * indicates statistical significance (<i>p</i> ≤ 0.05).</p

Alpha diversity analysis.

<p>Rarefaction curves of samples with regards to quarter patho-physiological status (CM: clinical mastitis; H: healthy; SM: sub-clinical mastitis), as defined by the Shannon index. Statistical difference is present between H and CM groups (<i>p</i> = 0.03).</p

Beta diversity analysis.

<p>Unweighted Unifrac analysis including H (Healthy) and CM (Clinical mastitis) samples. Adonis: R = 0.17 p = 0.001 ANOSIM: R = 0.37 <i>p</i> = 0.001. Panel A: results including H, SM and CM quarters. Panel B: results including only H and CM. o = CM; + = SM; Δ = H</p

Water buffalo milk taxonomic profile at phylum level.

<p>Microbiota composition at the phylum level for the 16S rRNA. H = Healthy samples; SM = Sub-Clinical mastitis samples; CM = Clinical mastitis samples</p

Relative abundance frequencies at genus level.

<p>Grouping following SCC classes.</p

Water buffalo milk taxonomic profile at genus level.

<p><b>Microbiota composition at the genus level for the 16S rRNA gene</b> Microbiota composition at the genus level for the 16S rRNA gene. H = Healthy samples; SM = Sub-Clinical mastitis samples; CM = Clinical mastitis samples.</p