In sickness and in health: profiling the bovine fecal eukaryome using optimized 18S metabarcoding with a mammal-blocking primer.
5 affiliations
- 1Laboratory of Sustainable Animal Environment, Graduate School of Agricultural Science, Tohoku University, Osaki, Miyagi, Japan.
- 2Department of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.
- 3Vets Support WA, Itoshima, Fukuoka, Japan.
- 4Department of Veterinary Medicine, Faculty of Veterinary Medicine, Okayama University of Science, Imabari, Ehime, Japan.
- 5Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka, Japan.
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.Results
The V8 primer-blocker demonstrated the most consistent host suppression (30-50% reduction), unmasking diverse eukaryotic taxa while improving evenness and preserving community richness. Cryptosporidium dominated the diarrheic group and was confirmed as C. parvum via nested GP60 PCR. Infected calves were associated with lower eukaryotic richness, with reduced Chao1 richness (p < 0.00001) and Shannon diversity (p < 0.041), and distinct community composition (Bray-Curtis: R ² = 0.145, p = 0.001; Jaccard: R ² = 0.060, p = 0.001). After accounting for geographic variation, clinical status remained significantly associated with community composition (PERMANOVA, R ² = 0.129, p = 0.0001), whereas geography was not significant (p = 0.180). LEfSe identified Cryptosporidium (LDA score = 5.6) as the strongest discriminator in the Crypto group, while Kazachstania (4.35), Giardia (4.0), and Aspergillus (0.8) were enriched in healthy calves, reflecting complex interkingdom interactions.Conclusion
The optimized V8-mammal-blocking strategy provides an effective framework for bovine fecal eukaryome profiling by reducing host interference and improving recovery of clinically relevant microeukaryotes. C. parvum infection is associated with significant restructuring of the fecal eukaryotic community. This workflow strengthens livestock disease surveillance, facilitates future epidemiological studies, and advances our understanding of complex host-parasite and microeukaryome interactions.
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BibTeX
@article{zafar2026sickness,
title = {{In sickness and in health: profiling the bovine fecal eukaryome using optimized 18S metabarcoding with a mammal-blocking primer.}},
author = {Iqra Zafar and Riku Sakurai and Yasuhiro Fukuda and Megumi Itoh and Atsushi Fujikura and Akira Goto and Kazuhiro Bandai and Kentaro Kato},
journal = {Frontiers in veterinary science},
year = {2026},
month = oct,
doi = {10.3389/fvets.2026.1948426},
url = {https://europepmc.org/article/MED/42923057},
note = {CC BY},
}
RIS
TY - JOUR
TI - In sickness and in health: profiling the bovine fecal eukaryome using optimized 18S metabarcoding with a mammal-blocking primer.
AU - Iqra Zafar
AU - Riku Sakurai
AU - Yasuhiro Fukuda
AU - Megumi Itoh
AU - Atsushi Fujikura
AU - Akira Goto
AU - Kazuhiro Bandai
AU - Kentaro Kato
PY - 2026
DA - 2026/10/08
JO - Frontiers in veterinary science
DO - 10.3389/fvets.2026.1948426
UR - https://europepmc.org/article/MED/42923057
AB - 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.Results The V8 primer-blocker demonstrated the most consistent host suppression (30-50% reduction), unmasking diverse eukaryotic taxa while improving evenness and preserving community richness. Cryptosporidium dominated the diarrheic group and was confirmed as C. parvum via nested GP60 PCR. Infected calves were associated with lower eukaryotic richness, with reduced Chao1 richness (p < 0.00001) and Shannon diversity (p < 0.041), and distinct community composition (Bray-Curtis: R ² = 0.145, p = 0.001; Jaccard: R ² = 0.060, p = 0.001). After accounting for geographic variation, clinical status remained significantly associated with community composition (PERMANOVA, R ² = 0.129, p = 0.0001), whereas geography was not significant (p = 0.180). LEfSe identified Cryptosporidium (LDA score = 5.6) as the strongest discriminator in the Crypto group, while Kazachstania (4.35), Giardia (4.0), and Aspergillus (0.8) were enriched in healthy calves, reflecting complex interkingdom interactions.Conclusion The optimized V8-mammal-blocking strategy provides an effective framework for bovine fecal eukaryome profiling by reducing host interference and improving recovery of clinically relevant microeukaryotes. C. parvum infection is associated with significant restructuring of the fecal eukaryotic community. This workflow strengthens livestock disease surveillance, facilitates future epidemiological studies, and advances our understanding of complex host-parasite and microeukaryome interactions.
SN - 2297-1769
ER -