Microbiome of the upper and lower respiratory tract in healthy and bovine respiratory disease-affected calves

Bovine Respiratory Disease (BRD) is one of the main health issues in beef calves. It is a multifactorial disease in which management, environmental and immunity factors predispose to the colonization by several pathogenic agents, both viral and bacterial. By means of classical bacteriological methods, most frequently identifed pathogens in BRD are: M. haemolytica, P. multocida, H. somni and M. bovis. Better understanding BRD etiology is a necessary step to apply prevention measures. The 16S metabarcoding approach has been successfully applied to characterize the microbiome of the bovine upper respiratory tract, but data about lower respiratory airways are still lacking. Aim of this study was to characterize the microbiome of the upper and lower respiratory tract in Piedmontese calves and to investigate correlation between the bacterial communities and the clinical status of the animals. A total of 20 post-weaned Piedmontese calves from fve farms, not drug-treated in the prior week, were included in the study. Each animal was examined for clinical signs of BRD (cough, nasal discharge, abnormal lung sound). Moreover, nasal swabs (NS) and trans-tracheal wash (TTW) samples were collected from each animal with sterile kits and were stored at -80°C until processing. DNA was extracted using DNAzol, quantifed with the Qubit DNA HS kit and processed according to the Illumina 16S metabarcoding protocol. Library pool was sequenced on the Illumina MiSeq platform with a 2x300bp paired-end protocol. Primer and quality trimming was performed with bbduk2 and bbmerge software, and fnally trimmed reads were analyzed using the QIIME pipeline. Nine out of 20 calves showed clinical signs of BRD. A total of 26 samples (11 NS and 15 TTW) were submitted to Illumina sequencing (samples with a DNA concentration lower than 5 ng/µl were excluded). From NS and TTW samples, 523,549 and 1,104,222 reads were obtained, respectively. Median number of identifed OTUs was 944 (487 - 2,471) from NS, and 129 (41 - 690) from TTW samples. In NS samples, 29 phyla and 284 genera were identifed, while in TTW samples phyla and genera were 20 and 173, respectively. Mycoplasma was the most abundant genus both in TTW fluid and in NS samples with a relative abundance of 76.79% and 27.32%, respectively. Moreover, in TTW fluids samples, the second most represented genus was Pasteurella (7.4%), while in NS was Moraxella (16.95%). Bacterial community profle and diversity between NS and TTW samples signifcantly differed (p<0.05). Moreover, when NS and TTW were compared in relation to the presence of clinical signs, no statistical difference was found (p>0.05). NS microbiome signifcantly differed in relation to the farm of origin (p<0.05). Most abundant phyla identifed in trans-tracheal wash (TTW) samples and nasal swabs (NS) of 20 calves. Figure 2. Most abundant genera identifed in trans-tracheal wash (TTW) samples and in nasal swabs (NS) of 20 calves. The results of this study confrm that the microbiome of the bovine upper respiratory tract is represented by a large number of different microbial populations and suggest that the external environment can influence its composition. However, more microbiomes should be analyzed to confrm this hypothesis and to evaluate how this interaction could impact on the clinical status of the herds. Moreover, the metabarcoding approach applied on TTW samples led to the identifcation of a complex bacterial population colonizing the lung. In agreement with human studies, the bovine lower respiratory tract showed a low microbial diversity and the community composition partly reflected the upper one, even though with different relative abundances. Further studies are needed to better defne the composition of the lung microbiome with a fner resolution at the taxonomic level in order to elucidate its role in BRD.