Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis

and are two coagulase-variable staphylococcal species that can be isolated from bovine milk and are difficult to differentiate. The objectives of this study were to characterize isolates of bovine milk origin from a collection that had previously been characterized as coagulase-positive based on phenotypic species identification methods and to develop a PCR-based method for differentiating , , and Isolates ( = 62) were selected from a previous study in which milk samples were collected from cows on 15 dairy herds. Isolates were coagulase tested and identified to the species level using housekeeping gene sequencing. A multiplex PCR to differentiate , , and was developed. Pulsed-field gel electrophoresis was conducted to strain type the isolates. Based on gene sequencing, 44/62 of the isolates were determined to be either ( = 43) or ( = 1). Overall, 88% (37/42) of coagulase-positive isolates were found to be coagulase positive at 4 h. The herd-level prevalence of coagulase-positive ranged from 0 to 2.17%. Strain typing identified 23 different strains. Six strains were identified more than once and from multiple cows within the herd. Three strains were isolated from cows at more than one time point, with 41 to 264 days between samplings. These data suggest that is likely more prevalent on dairy farms than Also, some isolates in this study appeared to be contagious and associated with persistent infections.