Bovine Faecal Extracellular Vesicles: A novel non-invasive tool for understanding gut physiology and pathophysiology in calves

Dairy calf gut health is linked with the development and future production. Fecal extracellular vesicles (fEVs) have emerged as a non-invasive tool in elucidating gut physiology and pathophysiology. Being a complex matrix, enrichment of extracellular vesicles (EVs) from ruminant/pre-ruminant feces is difficult. Nevertheless, if enriched, they have great potential as a gut health diagnostic and monitoring tool in dairy calves. Therefore, this study aimed to devise a protocol to enrich and characterize fEVs from pre-weaned calves. We developed a fEV enrichment method by combination of differential centrifugation and double size exclusion chromatography and then characterized the fEVs from the healthy calves. The study also assessed sample storage conditions, and the results indicated that storing pre-processed fecal samples at −80°C effectively preserves EVs without introducing additional nanoparticles. Finally, fEVs from 10 d old healthy and Cryptosporidium spp. positive calves were enriched and a comparative analysis of fEV characteristics between the 2 groups was performed. Characterization results on EVs specific protein biomarkers, size profile, total protein content, zeta potential, and morphology clearly established the enrichment of fEVs with the developed protocol. Cryptosporidium spp. positive and negative calves fEV analysis revealed a significant decrease in average nanoparticle size and AU:zeta potential values in AU:Cryptosporidium spp. infected calves. Furthermore, the enriched fEV carried protein and nucleic acid cargo which could be further analyzed for other biomarkers to predict the gut physiology and pathophysiology of calves. In conclusion, our study has successfully optimized a protocol to enrich high purity grade EVs from calf feces and displayed potential diagnostic application as a non-invasive tool.