Whole transcriptome analysis of peripheral blood identifies systemic innate immune responses and translation inhibition in subclinical Holstein cattle naturally infected with <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i>

Bovine paratuberculosis (PTB), caused by <i>Mycobacterium avium</i> subsp. <i>paratuberculosis</i> (MAP), is a chronic granulomatous intestinal disease that leads to substantial economic losses in the global dairy industry. Current diagnostic tests have limited sensitivity, as they can reliably detect only animals that have advanced stages of disease characterized by diffuse lesions and with the presence of clinical signs, but fail to identify those in earlier or subclinical stages, with focal or multifocal lesions in gut tissues. Previous studies have suggested that multifocal granulomas prevent lesions progression, but the molecular mechanisms involved in the establishment and maintenance of a chronic MAP infection are not fully understood. This study aimed to compare the whole transcriptomic profiles of peripheral blood (PB) samples from Holstein cattle with multifocal lesions and those without lesions in gut tissues. Total RNA was extracted from samples from PB samples collected from 11 cows with multifocal lesions and no clinical signs of PTB, and from 4 control cows without lesions that tested negative in several PTB diagnostic assays. RNA libraries were prepared using 250 ng of RNA with the Illumina NEBNext Ultra Directional RNA library preparation kit and sequenced on an Illumina NextSeq sequencer. On average, 34.08 million raw reads were sequenced from PB samples. In cows with multifocal lesions compared with control cows, 1,272 differentially expressed genes (DEGs) were identified in PB. Protein-to-protein interaction analysis revealed that several DEGs were highly interconnected and associated with molecular processes related to splicing and translation inhibition, as well as with the activation of a robust innate immune response in PB. Overall, this study provides new insights into MAP pathogenesis and identifies potential biomarkers and therapeutic targets.