Characterization of eukaryotic communities in small ruminant bile shows substantial extra-intestinal location of Cryptosporidium parvum.

Next-generation sequencing has expanded the study of microbial communities, yet the eukaryotic fraction of host-associated microbiomes remains poorly characterized, particularly in non-intestinal matrices. This study aimed to assess eukaryotic community diversity in bile from small ruminants. A total of 400 bile samples from sheep and goats collected at a Portuguese slaughterhouse were analyzed using 18S rRNA PCR and long-read Oxford Nanopore sequencing on 80 pooled samples. Overall, 12.5% (10/80) of pools were found to have eukaryotic elements, and Cryptosporidium-assigned reads were detected in 30% (3/10) of these, all originating from adult sheep. These findings provide molecular evidence of C. parvum in bile, extending its known tissue distribution beyond the gastrointestinal tract, suggesting potential chronic or subclinical biliary carriage and expanding its recognized tissue tropism. This work highlights the utility of long-read 18S rRNA metabarcoding for detecting protozoan parasites in unconventional biological matrices and characterizing eukaryotic diversity in livestock systems.