Abstract
Inflammation and infection of bovine mammary glands, commonly known as mastitis, imposes significant losses each year in the dairy industry worldwide. While several different bacterial species have been identified as causative agents of mastitis, many clinical mastitis cases remain culture negative, even after enrichment for bacterial growth. To understand the basis for this increasingly common phenomenon, the composition of bacterial communities from milk samples was analyzed using culture independent pyrosequencing of amplicons of 16S ribosomal RNA genes (16S rDNA). Comparisons were made of the microbial community composition of culture negative milk samples from mastitic quarters with that of non-mastitic quarters from the same animals. Genomic DNA from culture-negative clinical and healthy quarter sample pairs was isolated, and amplicon libraries were prepared using indexed primers specific to the V1–V2 region of bacterial 16S rRNA genes and sequenced using the Roche 454 GS FLX with titanium chemistry. Evaluation of the taxonomic composition of these samples revealed significant differences in the microbiota in milk from mastitic and healthy quarters. Statistical analysis identified seven bacterial genera that may be mainly responsible for the observed microbial community differences between mastitic and healthy quarters. Collectively, these results provide evidence that cases of culture negative mastitis can be associated with bacterial species that may be present below culture detection thresholds used here. The application of culture-independent bacterial community profiling represents a powerful approach to understand long-standing questions in animal health and disease.
Highlights
Bovine mastitis resulting from an infectious agent is a significant disease in the dairy industry
Samples were subjected to Roche 454 pyrosequencing with Titanium chemistry, an approach that has been shown to be of sufficient accuracy for identification of bacterial genera based on their 16S rDNA hypervariable regions and offers greater sample depth at a much lower cost than Sanger sequencing [8]
26 pairs of samples were identified for which culture negative pairs of mastitis and healthy quarter samples were available for screening
Summary
Bovine mastitis resulting from an infectious agent is a significant disease in the dairy industry. The lack of identification of microorganisms in culture negative, clinical mastitis cases may have multiple explanations including our inability to culture the bacteria responsible for disease, their presence below current detection thresholds, the absence of the bacteria at the time culture is initiated, or that the mastitis may be caused by non-bacterial microorganisms. We targeted the V1–V2 hypervariable regions of 16S rDNA This region has been shown to accurately differentiate between bacterial genera and has been used in 16S rRNA gene studies of samples from mammalian hosts [5,6,7]. Samples were subjected to Roche 454 pyrosequencing with Titanium chemistry, an approach that has been shown to be of sufficient accuracy for identification of bacterial genera based on their 16S rDNA hypervariable regions and offers greater sample depth at a much lower cost than Sanger sequencing [8]
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