{
  "abstract": "Introduction\n\nCalf 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\n\nWe 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\n\nThe 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\n\nThe 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.",
  "abstract_shown": true,
  "affiliations": [
    "Laboratory of Sustainable Animal Environment, Graduate School of Agricultural Science, Tohoku University, Osaki, Miyagi, Japan.",
    "Department of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, Japan.",
    "Vets Support WA, Itoshima, Fukuoka, Japan.",
    "Department of Veterinary Medicine, Faculty of Veterinary Medicine, Okayama University of Science, Imabari, Ehime, Japan.",
    "Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka, Japan."
  ],
  "article_type": "research article",
  "arxiv": null,
  "author_total": 8,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Iqra Zafar"
    },
    {
      "affs": [
        0
      ],
      "name": "Riku Sakurai"
    },
    {
      "affs": [
        0
      ],
      "name": "Yasuhiro Fukuda"
    },
    {
      "affs": [
        1
      ],
      "name": "Megumi Itoh"
    },
    {
      "affs": [
        2
      ],
      "name": "Atsushi Fujikura"
    },
    {
      "affs": [
        3
      ],
      "name": "Akira Goto"
    },
    {
      "affs": [
        4
      ],
      "name": "Kazuhiro Bandai"
    },
    {
      "affs": [
        0
      ],
      "name": "Kentaro Kato"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/5ejdaxb5m4xdc.bib",
  "cited_by": 0,
  "doi": "10.3389/fvets.2026.1948426",
  "fulltext_url": "https://europepmc.org/articles/PMC13686857",
  "id": "5ejdaxb5m4xdc",
  "issns": [
    "2297-1769"
  ],
  "landing_url": "https://europepmc.org/article/MED/42923057",
  "language": "eng",
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "2405|Parasitic Infections and Diagnostics"
    ]
  ],
  "openalex": "W7220987945",
  "pdf_url": "https://europepmc.org/articles/PMC13686857?pdf=render",
  "pmcid": "PMC13686857",
  "pmid": "42923057",
  "published": "2026-10-08",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/5ejdaxb5m4xdc.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:20:36Z",
      "native_id": "MED:42923057",
      "source": "europepmc",
      "url": "https://europepmc.org/article/MED/42923057"
    }
  ],
  "subjects": [
    {
      "field": "biology",
      "origin": "openalex:2405|Parasitic Infections and Diagnostics",
      "subfield": "microbiology"
    }
  ],
  "title": "In sickness and in health: profiling the bovine fecal eukaryome using optimized 18S metabarcoding with a mammal-blocking primer.",
  "title_text": "In sickness and in health: profiling the bovine fecal eukaryome using optimized 18S metabarcoding with a mammal-blocking primer.",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:20:36Z",
  "url": "https://research.draconitic.com/paper/5ejdaxb5m4xdc",
  "venue": "Frontiers in veterinary science",
  "versions": []
}