The application of 16S rRNA gene to characterise bovine milk microbiome diversity in Australia

Abstract

Bovine mastitis is inflammation of the mammary gland, with significant economic and welfare implications to the dairy industry worldwide. The broad objectives of the research presented in this thesis were (a) investigate the prevalence of mastitis-associated bacteria and their antimicrobial resistances (AMR) profile in Australian dairy herds, (b) characterise the milk microbiota of healthy and mastitic milk samples, and (c) investigate the effect of different mastitis pathogens on the healthy milk microbiota.

Mastitis can be caused by a variety of pathogenic bacteria; However, there is a paucity of up-to-date data on the prevalence of different mastitis-inducing bacterial species and their AMR profiles in Australian dairy farms. Also, all previous studies were limited to studying affected herds from few locations in Australia. Therefore, this study provided a unique opportunity to investigate the prevalence of mastitis-associated bacteria and their AMR profile in dairy herds from three different regions in Australia (Northern Queensland, Southern Queensland, and Victoria).

Culture-independent microbiota analysis, by sequencing of amplicons of 16S ribosomal RNA genes (16S rRNA) provides information about bacteria communities (i.e., microbiota) present in the udder’s milk. Changes to the structure of milk microbiota (i.e., dysbiosis) have been proposed as a determinant of mastitis in apparently healthy cattle. However, a reverse-causality pathway could also be postulated, where the exposure to a mastitis-causing pathogen would result in the observed imbalance in the milk microbiota in mastitic milk. The motivation behind the work described here was the paucity of literature on the structure of microbiota in the milk of apparently healthy and mastitis cows in Australian dairy herds. Therefore, the main aims of this thesis were to characterise milk microbiota of apparently healthy and mastitic quarters, and to quantify the association between mastitis-causing pathogens and milk microbiota structure in dairy herds from Queensland and Victoria.

To achieve the thesis aims, milk samples were collected from dairy herds located in Northern Queensland, Southern Queensland, and Victoria. Mastitic milk samples were collected from clinical cases. Healthy milk samples were collected from healthy quarters from infected cows (internal control) and from healthy quarters of apparently healthy cows (external control). Herd selection was based on convenience, farmers’ willingness to participate, and the herd’s rolling average bulk milk tank somatic cell counts (BMTSCC) divided into three categories (≤150 × 1000 cells/mL, 150-300 × 1000 cells/mL, and >300 ×1000 cells/mL). Milk samples from both healthy and clinical mastitic quarters were cultured, and DNA from all milk samples was extracted for 16S rRNA gene sequencing. This data was used for the characterisation of milk microbiota structure (communities, abundance, and diversity), and to quantify the association between exposure to a mastitis-causing pathogen and milk microbiota abundance and diversity. The intention of this work was to address the knowledge gap in the literature as outlined above.

Staphylococci (S. aureus and non-aureus staphylococci (NAS) species) were the most prevalent bacteria detected in the milk samples. Contagious mastitis bacteria were more prevalent in Victoria compared to Queensland dairy herds. NAS species and Enterobacteriaceae were less prevalent in herds with BMTSCC >300,000 cells/mL compared with herds with low BMTSCC ≤150,000 cells/mL. Enterobacteriaceae and Streptococcus spp. showed high resistance rates to 1 and 2 antimicrobial classes tested in this study. More than one third of the Enterobacteriaceae were resistant to at least three antimicrobials. The findings of this study aid in making informed decisions in mastitis control and antimicrobial stewardship programs aimed to reduce the prevalence of mastitis and antimicrobial resistance in dairy herds.

The relative abundance and diversity, of taxa, and the core members of the milk microbiota were compared between internal and external control samples using the the MicrobiomeAnalyst online analysis platform. High abundance of bacterial families, Corynebacteriaceae, Oxalobacteraceae, Planococcaceae and Ruminococcaceae were detected in both internal and external control milk samples with no significant difference. However, the relative abundance of Streptococcaceae in internal control samples was significantly higher compared to external control samples. Corynebacteriaceae, Oxalobacteraceae, and Ruminococcaceae bacterial families were identified as core healthy microbiota in this study. Bacterial diversity was not significantly different between internal and external control samples. However, differences in the abundance of some bacterial taxa were noted in herds with different BMTSCCs and from different geographical regions.

The microbiota composition from clinical mastitis and healthy quarterss also varied greatly. The prevalence of families Corynebacteriaceae, Bacteroidaceae, Oxalobacteraceae, Ruminococcaceae, Staphylococcaceae, and Planococcaceae were significantly lower in mastitis compared to healthy quarters. However, family Streptococcaceae was more abundant in mastitis samples. The analysis of the effect of different mastitis associated bacteria on healthy milk microbiota showed that Streptococcus uberis had a distinct bacterial profile compared to healthy samples, it was dominated by Streptococcaceae. A similar finding was observed with Escherichia coli infected quarters, which were dominated by Enterobacteriaceae. No major differences in milk microbiota were found in Staphylococcus chromogenes and Staphylococcus aureus associated mastitis when compared to healthy milk samples. This study showed the importance of identifying the causative bacteria to understand the changes of the milk microbiota in the development of mastitis.