Frontiers in veterinary science
In sickness and in health: profiling the bovine fecal eukaryome using optimized 18S metabarcoding with a mammal-blocking primer
By Zafar I, Sakurai R, Fukuda Y and 5 others.
Abstract
Introduction
Calf diarrhea, caused by the apicomplexan pathogen Cryptosporidium parvum (C. parvum), poses significant economic and production challenges to the livestock industry. While 16S rRNA sequencing has advanced understanding of the bovine bacterial gut microbiota, the eukaryotic community remains underexplored due to methodological constraints, notably host DNA contamination. This study aimed to optimize an 18S rRNA metabarcoding workflow using mammal-blocking primers for bovine fecal eukaryome profiling and to characterize eukaryotic community differences between healthy and naturally C. parvum-infected diarrheic calves.
Methods
We compared four 18S rRNA primer sets (V8f/1510R, TAReuk454FWD1/TAReukREV3, 528F/706R, and 616F/1132R) with two mammal-blocking primers using fecal samples from healthy (n = 28) and C. parvum-positive diarrheic calves (n = 24) across Japan. The optimal primer-blocker combination was selected based on host-read depletion and taxonomic recovery. Using this optimized workflow, we characterized and compared the fecal eukaryotic microbiota between healthy and C. parvum-infected calves via 18S rRNA amplicon sequencing.