Characterization of the OTU172 (C. cuniculi). The OTU172 (unique change identified the day of ERE onset) was isolated from a caecotroph sample collected from a rabbit developing ERE. Its genome was sequenced and compared with representative sequences from the 98 genomes classified as Clostridiales within the GenBank RefSeq complete genome database (see “Materials and methods”). A The tree represents the best phylogenetic location of the OTU172 and species belonging to the Clostridium clade. The bootstrap value is stated when it was lower than 97%. The heatmap represents percentage of identity of the OTU172 with other species of the Clostridium clade calculated by implementation of cgANI. The analysis revealed that OTU172 represents a novel species within the genus Clostridium, which we named Clostridium cuniculi (cgANI < 95% compared to any member of the Clostridiales clade). Tree branches length represent the number of amino acid changes per site. The presence/absence in the different Clostridium species of potential virulence factors identified in C. cuniculi (discussed in the manuscript) is indicated. This include an operon shown in more detail in B, a hemolysin and a gene similar to the cytotoxin exoU. B The schematic representation of the operon detected in the genome of C. cuniculi and corresponding operons in genomes of C. botulinum A str. ATCC 3502 and C. perfringens SM101, plasmid pSM101B. The names above C. cuniculi ORFs correspond to the best matches against ORFs present in the other operons (E < 1e−15). ORFs that did not match the C. botulinum or C. perfringens operons were blast against the NCBI nr database. Its best match is indicated (E < 1e−101). If the coverage against the reference ORF was lower than 66%, the ORF is marked with an asterisk. Only ORFs with > 60 amino acids are shown. In yellow are indicated transcriptional regulators; in red, effector proteins; in blue, hemagglutinins with a similar organization in C. cuniculi and C. botulinum, and in brown putative holins. botA botulinum toxin A, NTNH nontoxic, nonhemagglutinin protein, HA hemagglutinin, IR inverse repeats.