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Background  Bovine respiratory disease (BRD) is caused by growth of single or multiple species of pathogenic bacteria in lung tissue following stress and/or viral infection. Next generation sequencing of 16S ribosomal RNA gene PCR amplicons (NGS 16S amplicon analysis) is a powerful culture-independent open reference method that has recently been used to increase understanding of BRD-associated bacteria in the upper respiratory tract of BRD cattle. However, it has not yet been used to examine the microbiome of the bovine lower respiratory tract. The objective of this study was to use NGS 16S amplicon analysis to identify bacteria in post-mortem lung and lymph node tissue samples harvested from fatal BRD cases and clinically healthy animals. Cranial lobe and corresponding mediastinal lymph node post-mortem tissue samples were collected from calves diagnosed as BRD cases by veterinary laboratory pathologists and from clinically healthy calves. NGS 16S amplicon libraries, targeting the V3-V4 region of the bacterial 16S rRNA gene were prepared and sequenced on an Illumina MiSeq. Quantitative insights into microbial ecology (QIIME) was used to determine operational taxonomic units (OTUs) which corresponded to the 16S rRNA gene sequences.    Results    Leptotrichiaceae, Mycoplasma, Pasteurellaceae , and  Fusobacterium  were the most abundant OTUs identified in the lungs and lymph nodes of the calves which died from BRD.  Leptotrichiaceae, Fusobacterium ,  Mycoplasma, Trueperella  and  Bacteroides  had greater relative abundances in post-mortem lung samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions.  Leptotrichiaceae, Mycoplasma  and  Pasteurellaceae  showed higher relative abundances in post-mortem lymph node samples collected from fatal cases of BRD in dairy calves, compared with clinically healthy calves without lung lesions. Two  Leptotrichiaceae  sequence contigs were subsequently assembled from bacterial DNA-enriched shotgun sequences.    Conclusions  The microbiomes of the cranial lung lobe and mediastinal lymph node from calves which died from BRD and from clinically healthy H-F calves have been characterised. Contigs corresponding to the abundant  Leptotrichiaceae  OTU were sequenced and found not to be identical to any known bacterial genus. This suggests that we have identified a novel bacterial species associated with BRD.    Electronic supplementary material  The online version of this article (doi:10.1186/s12917-017-1035-2) contains supplementary material, which is available to authorized users.

Illumina MiSeq 16S amplicon sequence analysis of bovine respiratory disease associated bacteria in lung and mediastinal lymph node tissue