Distribution and genetic diversity of Mannheimia species, including recently described taxa, in cattle in Japan.

The genus Mannheimia comprises Gram-negative bacteria that primarily inhabit the upper respiratory tract of ruminants. Currently, 10 species are recognized within the genus Mannheimia, among which M. haemolytica has been extensively studied due to its association with bovine respiratory disease (BRD). In contrast, information on other species, particularly the four species proposed since 2021 (M. pernigra, M. bovis, M. cairinae, and M. indoligenes), remains extremely limited. In this study, we retrospectively analyzed 210 Mannheimia isolates obtained from nasal swabs of cattle in Japan between 2010 and 2020. Based on 16S rRNA gene sequence comparisons, 173 isolates showed ≥98.65% sequence similarity to their respective type strains and were classified into six species: M. haemolytica (n = 98), M. varigena (n = 30), M. pernigra (n = 21), M. indoligenes (n = 21), M. granulomatis (n = 2), and M. ruminalis (n = 1). The remaining 37 isolates showed <98.65% sequence similarity and were classified as related to four species: M. bovis (n = 19), M. indoligenes (n = 15), M. varigena (n = 2), and M. ruminalis (n = 1). A newly developed PCR assay revealed that all isolates, except for one M. ruminalis isolate, one M. ruminalis-related isolate, and four M. bovis-related isolates, carried the lktA gene encoding leukotoxin, a major virulence factor. These findings indicate that multiple Mannheimia species are widely distributed among cattle in Japan. Notably, some isolates showing <98.65% 16S rRNA gene sequence similarity to known type strains may represent novel taxa, warranting taxonomic re-evaluation and phylogenetic reassessment within Mannheimia. Moreover, because many of these isolates harbor key virulence-associated genes linked to BRD, further studies are required to clarify the pathogenic potential of each species as opportunistic pathogens and their contribution to BRD.