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Updated: May 10, 2026

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing
Published on: March 19, 2018
Faecal microbial diversity in a cattle herd infected by Mycobacterium avium subsp. paratuberculosis: a possible
Wisal A Elmagzoub1,2, Sanaa M Idris1,3, Marwa H E Elnaiem4
1Department of Animal Health and Safety of Animal Products, Institute for Studies and Promotion of Animal Exports, University of Khartoum, Shambat, 13314, Khartoum North, Sudan.
Abstract:
Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, or paratuberculosis (PTB) in ruminants, besides having zoonotic potential. It possibly changes the gut microbiome, but no conclusive data are available yet. This study aimed at investigating the influence of MAP on the faecal microbiome of cattle naturally infected with PTB. In a follow up period of 10 months, PTB status was investigated in a herd of dairy cattle with history of clinical cases. Each animal was tested for MAP infection using serum and milk ELISA for MAP anti-bodies and IS900 real-time PCR and recombinase polymerase amplification assays for MAP DNA in the faeces and milk monthly for 4 successive months, then a last one after 6 months. The faecal samples were subjected to 16S rDNA metagenomic analysis using Oxford Nanopore Sequencing Technology. The microbial content was compared between animal groups based on MAP positivity rate and production status. All animals were MAP positive by one or more tests, but two animals were consistently negative for MAP DNA in the faeces. In all animals, the phyla firmicutes and bacteroidetes were highly enriched with a small contribution of proteobacteria, and increased abundance of the families Oscillospiraceae, Planococcaceae, and Streptococcacaceae was noted. Animals with high MAP positivity rate showed comparable faecal microbial content, although MAP faecal positivity had no significant effect (p > 0.05) on the microbiome. Generally, richness and evenness indices decreased with increasing positivity rate. A significantly different microbial content was found between dry cows and heifers (p < 0.05). Particularly, Oscillospiraceae and Rikenellaceae were enriched in heifers, while Planococcaceae and Streptococcaceae were overrepresented in dry cows. Furthermore, abundance of 72 genera was significantly different between these two groups (p < 0.05). Changes in faecal microbiome composition were notably associated with increasing MAP shedding in the faeces. The present findings suggest a combined influence of the production status and MAP on the cattle faecal microbiome. This possibly correlates with the fate of the infection, the concern in disease control, again remains for further investigations.
Insights
Mycobacterium avium subsp. paratuberculosis (MAP) infection in cattle alters the gut microbiome, with richness decreasing as MAP positivity increases. Production status also significantly impacts fecal microbial composition.
Area of Science:
- Veterinary Microbiology
- Ruminant Health
- Gut Microbiome Research
Background:
- Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease (PTB) in ruminants and has potential zoonotic implications.
- The impact of MAP on the gut microbiome remains incompletely understood, lacking conclusive data.
Purpose of the Study:
- To investigate the influence of MAP on the fecal microbiome of cattle naturally infected with PTB.
- To compare microbial content based on MAP positivity and animal production status.
Main Methods:
- Longitudinal study over 10 months in a dairy herd with clinical PTB cases.
- MAP infection assessed via ELISA and molecular assays (PCR, RPA) in serum, milk, and feces.
- Fecal samples analyzed using 16S rDNA metagenomics with Oxford Nanopore Sequencing.
Main Results:
- All animals tested positive for MAP by at least one method; two were consistently MAP DNA negative in feces.
- Firmicutes and Bacteroidetes were the dominant phyla; Oscillospiraceae, Planococcaceae, and Streptococcaceae families were abundant.
- Fecal microbiome richness and evenness decreased with higher MAP positivity rates.
- Significant differences in microbial composition were observed between dry cows and heifers, with distinct family and genus abundances.
Conclusions:
- Cattle fecal microbiome is influenced by both MAP infection status and production status (dry cows vs. heifers).
- Increasing MAP shedding correlates with changes in fecal microbiome composition.
- Further research is needed to understand the implications for disease control and infection progression.
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