<em>Staphylococcus aureus</em> is a well-known pathogen that infects humans and animals. However, information on the fatal respiratory infection in rabbits caused by<em> S. aureus</em> is still limited. In the present study, a <em>S. aureus</em> isolate designated ND01 was recovered from lung samples of rabbits that died of fatal respiratory infection, and the ND01 was characterised by intranasal infection of rabbits, multi-locus sequencing typing, screening virulence genes and testing antimicrobial susceptibility. Clinical signs of matted forepaws and pathological lesions of haemorrhagic tracheitis and necrotising haemorrhagic pneumonia were observed in the ND01 infected rabbits, which were identical to those of naturally infected ones. The sequence type of the ND01 was defined as ST3320 and the ND01 was further grouped into the clonal complex 398. Notably, the ND01 was <em>pvl-positive</em> <em>S. aureus</em> and carried the human-associated scn gene. Moreover, the ND01 was methicillin-susceptible <em>S. aureus</em> and was susceptible to 6 of 10 tested antibiotics. This study described the characteristics of the ND01 causing fatal respiratory infection in rabbits. The results are helpful to further the understanding of the pathogenicity of S. aureus ST3320 clone in rabbits. The results also highlighted that operators must be on the alert for the colonisation of <em>pvl-positive</em> <em>S. aureus</em> in rabbits and potential transmission events between rabbits and humans.
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