Exploring the functional microbiome of pigs within the porcine respiratory disease complex: viral-bacterial co-infections and virulence factor profiling

Respiratory infections are among the most impacting on pigs' health and economic productivity. Despite this, detailed insights into the microbial community of the lower respiratory tract (LRT) are currently lacking, mainly because of difficulties in the processing of respiratory samples. In this study, we characterized the microbiota of the LRT of finisher pigs aged 3-5 months with respiratory symptoms for both the viral and bacterial components, using a previously validated metagenomic diagnostic assay and a full-length 16S rRNA gene sequencing approach, respectively. Functional characterization was carried out using metagenomic shotgun sequencing, revealing the presence of specific virulence factors (VFs). Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and swine Influenza A Virus (swIAV) were the most prevalent viruses, being detected in 30% and 23% of the tested samples, respectively. <i>Mesomycoplasma hyopneumoniae</i>, <i>Glaesserella parasuis</i>, and <i>Pasteurella multocida</i> were the three most abundant bacterial taxa based on both sequencing approaches, while other detected bacterial taxa consisted mainly of <i>Streptococcus</i>, <i>Clostridium</i>, and <i>Rothia</i> species. Detected virulence factors belonged mainly to <i>Mesomycoplasma</i> and <i>Pasteurella</i> and consisted of adhesion factors such as <i>p102</i>, <i>p97</i>, <i>p146</i>, <i>mhp108, mhp107</i> and the hemolysin-encoding gene <i>hly</i>A for <i>Mesomycoplasma</i>, and adhesin-encoding <i>ptf</i>A and endoxtoxin-related gene <i>lpxC</i> for <i>Pasteurella</i>. Our data show how the microbial community of the lower respiratory tract in pigs with respiratory symptoms includes key viral (PRRSV, swIAV) and bacterial pathogens (<i>M. hyopneumoniae</i>, <i>G. parasuis</i>, and <i>P. multocida</i>), along with specific virulence factors likely contributing to disease.IMPORTANCEThe obtained results offer insights into the composition of the swine respiratory tract microflora, opening new perspectives on its correlation with viral infections, functional characteristics, and overall health conditions. Moreover, the present study provides technical advancement on the possibility of extracting and amplifying bacterial DNA from low-biomass respiratory samples, with the resulting possibility of identifying virulence factors and better understanding their contribution to the disease state. These discoveries pave the way for future studies aimed at improving diagnostic accuracy and treatment strategies for respiratory disease in both veterinary and human medicine.