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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. [ABSTRACT FROM AUTHOR]