The bovine respiratory disease complex (BRDC) is a global issue affecting dairy and beef farms and is of major concern due to the high morbidity and mortality rates in calves, as well as decreased production it causes, resulting in significant economic losses. Mannheimia haemolytica is one of the secondary pathogens associated with BRDC. M. haemolytica is classified into 12 serotypes based on capsular antigens. In addition to the prevalent serotypes A1, A2, and A6, strains belonging to other serotypes also cause respiratory diseases in cattle and other ruminants, necessitating a method for their rapid and easy identification. In this study, we organized the capsule biosynthesis genes based on genome information from all serotype strains and designed 11 PCR primer pairs targeting serotype-specific genes, which could individually identify serotypes A14/A16, which possess homologous genes, as well as all other serotypes. Additionally, we developed two multiplex PCR kits that include these serotype-specific and M. haemolytica species-specific primers. Specificity testing using reference strains confirmed that these kits can simultaneously and clearly identify both the species and their serotypes. The PCR-based system described here could be a valuable tool for subtyping M. haemolytica strains in epidemiological studies and surveillance efforts in cattle and other reservoir animals. This study also carefully compared and discussed the differences between the capsule synthesis genes of A8 and A14 from previously published and those obtained in this study
