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

Abstract

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.

Keywords: 16S sequencing; Bovine respiratory disease; diagnostics; lung microbiome.

Fig. 1

Number of reads (classified as bacterial) within post-mortem lung and mediastinal lymph node tissues corresponding to each sample type; calves which died from the BRD (n = 38), and healthy Holstein-Friesian calves (with (n = 12) and without (n = 8) lung lesions). Fatal BRD - RVL = samples from calves with the bovine respiratory disease complex collected at regional veterinary laboratories. Healthy – lesions present = samples from healthy Holstein-Friesian calves with lung lesions. Healthy – no lesions = samples from healthy Holstein-Friesian calves without lung lesions. Boxplot = Tukey boxplot (lowest datum (whisker) is within a 1.5 interquartile range of the lower quartile, highest datum (whisker) is within a 1.5 interquartile range of the upper quartile, outliers (individual points) fall above and below these whiskers)

Fig. 2

Percentage relative abundance of bacteria in post-mortem lung (L) and corresponding lymph node (LN) samples from calves which died from bovine respiratory disease. Adjacent profiles of L and LN are from the same animal. Athlone = sample collected at Athlone regional veterinary laboratory (RVL). Kilkenny = sample collected at Kilkenny RVL. Sligo = sample collected at Sligo RVL. Grey bars represent infrequently occurring OTUs (see Additional file 6 for full details of all bacterial OTU classifications)

Fig. 3

Percentage relative abundance of bacteria in post-mortem lung (L) and corresponding lymph node (LN) samples from clinically healthy H-F calves with lung lesions (n = 12) and without lung lesions (n = 8). Adjacent profiles of L and LN are from the same animal. The first seven calves received antibiotic treatment during their lifetimes. Grey bars represent infrequently occurring OTUs (see Additional file 6 for full details of all bacterial OTU classifications)