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Home / Equine Research Bank / PLoS One / Direct and culture-enriched 16S rRNA sequencing of cecal con...
PloS one2023; 18(4); e0284193; doi: 10.1371/journal.pone.0284193

Direct and culture-enriched 16S rRNA sequencing of cecal content of healthy horses and horses with typhlocolitis.

Abstract: Next generation sequencing has demonstrated that alpha diversity of the fecal microbiota is significantly altered in horses with typhlocolitis. The objective of this study was to evaluate the bacterial composition of the cecum content of horses with and without typhlocolitis through direct and culture-enriched 16S gene sequencing of six healthy horses and six horses with acute typhlocolitis; a case-control study design. Cecal content was collected after euthanasia. An aliquot was used for direct 16S gene sequencing. Another was serially diluted with brain heart infusion (BHI) and plated onto five different agar media. All culture medias, except for MacConkey, were incubated anaerobically. Bacterial colonies were harvested in bulk and used for DNA extraction, 16S PCR amplification, and sequenced using the Illumina MiSeq platform. Predominant phyla in healthy and diseased horses were Firmicutes, followed by Bacteroidetes in all cultured medias, except for MacConkey agar, in which Proteobacteria was the dominant phylum. Greater bacterial richness was identified in sequenced cecal contents as compared to cultured plates (P < 0.05). Culture-enriched molecular profiling combined with 16S rRNA gene sequencing offer an alternative method for the study of the gut microbiota of horses. For direct cecum content 16S gene amplification, the alpha diversity indices were lower in diarrheic horses compared to healthy horses (P < 0.05). A higher relative abundance of Fusobacteriota was found in 2/6 samples from diarrheic horses. The role of Fusobacteriota in equine colitis deserves investigation.
Copyright: © 2023 Zakia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Publication Date: 2023-04-13 PubMed ID: 37053174PubMed Central: PMC10101396DOI: 10.1371/journal.pone.0284193Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article explores the differences in the bacterial compositions of a horse’s cecum content with and without typhlocolitis using direct and culture-enriched 16S gene sequencing. It found significant alterations in microbiota diversity in horses afflicted with the disease and identified a larger bacterial richness in sequenced cecal content compared to culture plates.

Research Context and Goal

  • This research aimed to understand the bacterial composition of the cecum content in horses suffering from and without typhlocolitis. Typhlocolitis is a serious disease affecting the cecum and colon in horses, and recent studies hint at the involvement of altered gut microbiome.
  • Previous studies using next-generation sequencing demonstrated a significant change in the fecal microbiota’s alpha diversity (the biodiversity within a particular area or ecosystem) in horses with typhlocolitis. By leveraging 16S gene sequencing, this study sought to analyze these changes more in-depth.

Methodology

  • A case-control study was set using six healthy horses and six horses with acute typhlocolitis. The content of their cecum was collected post-euthanasia for analysis.
  • The researchers directly sequenced the 16S gene from one aliquot. Another aliquot was diluted using brain heart infusion and cultured onto five different agar media.
  • Molecular profiling and subsequent sequencing were performed on harvested bacterium colonies using the Illumina MiSeq platform. Cultures were grown anaerobically, except those on MacConkey agar, which were grown aerobically.

Findings

  • The dominant bacterial phyla in both healthy and diseased horses included Firmicutes followed by Bacteroidetes, except for the culture grown on MacConkey agar where Proteobacteria was prevalent.
  • The sequencing results showed a greater bacterial richness in the cecum contents as compared to the cultured plates, significantly indicating the richness of gut microbiota that could not be reflected entirely in the cultures.
  • For direct gene amplification from the cecum content, the alpha diversity indices were lower in diarrheic horses compared to healthy horses, implying dysbiosis (microbial imbalance) in diseased horses.
  • Interestingly, a higher relative abundance of Fusobacteriota was found in two out of six diarrheic horse samples, suggesting the potential role of this bacterial phyla in equine typhlocolitis which merits further investigation.
  • The results indicate that culture-enriched molecular profiling combined with 16S rRNA gene sequencing could provide a constructive way to study gut microbiota in horses.

Cite This Article

APA
Zakia LS, Gomez DE, Caddey BB, Boerlin P, Surette MG, Arroyo LG. (2023). Direct and culture-enriched 16S rRNA sequencing of cecal content of healthy horses and horses with typhlocolitis. PLoS One, 18(4), e0284193. https://doi.org/10.1371/journal.pone.0284193

Publication

PloS one
ISSN: 1932-6203
NlmUniqueID: 101285081
Country: United States
Language: English
Volume: 18
Issue: 4
Pages: e0284193
PII: e0284193

Researcher Affiliations

Zakia, Luiza S
  • Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Gomez, Diego E
  • Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Caddey, Benjamin B
  • Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada.
Boerlin, Patrick
  • Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.
Surette, Michael G
  • Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
Arroyo, Luis G
  • Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada.

MeSH Terms

  • Horses / genetics
  • Animals
  • RNA, Ribosomal, 16S / genetics
  • Agar
  • Case-Control Studies
  • Bacteria / genetics
  • Cecum / microbiology

Conflict of Interest Statement

The authors have declared that no competing interests exist.

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