Retrospective investigation of 43 necropsy cases of Tyzzer disease in foals and partial genome sequence of Clostridium piliforme by shotgun metagenomics

Clostridium piliforme is an obligate intracellular filamentous bacterium that causes Tyzzer disease (TD) in many animals. The disease manifests as severe, multifocal necrotizing hepatitis, with a high fatality rate in foals. Through retrospective investigation, we detected C. piliforme in 43 equine necropsy cases from 2012 to 2024. Positive cases were diagnosed from February to July, peaking in May. The age of affected foals ranged from 4 days to 2 months. Histologically, all cases had necrotizing hepatitis with multifocal, coalescing pinpoint, tan or reddish foci. Since only a partial 16S rRNA gene sequence was available for the horse strain of C. piliforme, we used shotgun metagenomics to obtain its genome sequence from the liver of a necropsied foal with TD. The sequences obtained were compared against the NCBI NT/NR database with the highest number of reads and contigs aligning to Clostridium species. A complete 16S rRNA gene was obtained, showing the highest identity to a 16S rRNA gene of the horse strain of C. piliforme (99.05 %), followed by 98.02–96.71 % identities to rabbit and rodent strains of C. piliforme, indicating cross-species variation. Additional identified genes included alveolysin, exo-α-sialidase, flagellar and spore formation/vegetation, providing the first genetic evidence of virulence factors for C. piliforme. Furthermore, presence of genes encoding multidrug export and multidrug resistance proteins suggested C. piliforme could develop resistance to beta-lactams and fluoroquinolones. This study provides the first partial genome sequence of C. piliforme using a shotgun metagenomics hepatic sampling approach on a foal with TD.