Characterization of Microbial Communities and Bacterial Markers of PRRSV-positive and -negative Pigs

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to pose significant challenges to swine health and productivity. The objective of this study was to characterize nasal microbial communities of weaning pigs and identify bacterial markers associated with PRRSV-infection. Nasal swabs were collected from weaning pigs (approximately 21 days old) across five farms in the Midwest region, including two PRRSV-negative (Farm 1 & 2) and three PRRSV-positive (Farm 3 to 5) sites. Samples were collected and placed in RNAse/DNAse-free buffer, transported to the laboratory for DNA extraction. Microbial profiling was conducted using 16S rRNA sequencing targeting V3-V4 region (Illumina Miseq, Ilumina, San Diego, CA). Concurrently, blood samples were collected from the same pigs for PRRSV detection via PCR and antibody titter via ELISA X3 assay. The PRRSV infection status was confirmed by sequencing using ORF5 region. Microbial communities analysis were analyzed using QIIME2 (version 2024.10.1). Alpha diversity among groups was compared with Kruskal-Wallis tests, and beta diversity was evaluated using Bray Curtis dissimilarity and PERMANOVA. Bacterial markers associated with infection status were identified using linear discriminant analysis (LDA) Effect Size. The results indicated that PRRSV RNA was not detected in Farms 1 and 2 as expected, while Farms 3 to 5 tested positive with Cycle threshold values of 22.8, 24.8, and 24, respectively. Sequencing data identified multiple PRRSV strains among the positive farms, including 1-4-1 L1C.3, 1-4-4 L1C.5, and 1-7-4 L1A for Farms 3 to 5, respectively. PRRSV-positive pigs exhibited significantly lower alpha diversity than that of PRRS-negative pigs (Shannon index; P = 0.006). Beta diversity differed significantly between PRRSV infection status (P = 0.001). Among the top 15 genera, PRRSV-negative pigs were associated to Streptococcus, Glaesserella, Segatella, Filobacterium, Megasphaera, Prevotellaceae_Incertae_Sedis, Faecalibacterium, Anaerovibrio, and Agathobacter. In contrast, PRRSV-positive pigs associated with Mesomycoplasma, Clostridium, Trueperella, Bacteroides, Pasteurella, and Terrisporobacter (LDA score ≥ 3.7; P ≤ 0.01). These results indicate distinct microbial shifts with PRRSV infection and suggest bacterial markers for the disease status. Understanding these microbial changes may provide insights into the potential strategies for secondary bacterial management and PPRSV mitigation.