A multiplex polymerase chain reaction assay for the identification of Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis

The objective of this study was to design a multiplex PCR assay to identify Mannheimia haemolytica, Mannheimia glucosida and Mannheimia ruminalis. The multiplex PCR included primer sets HP, amplifying a DNA region from an unknown hypothetical protein, Lkt and Lkt2, amplifying different regions of the leukotoxinD gene, and 16S to amplify universal bacterial sequences of the 16S rRNA gene. Based on positive amplification, isolates were delineated as M. haemolytica (HP, Lkt, 16S), M. glucosida (HP, Lkt, Lkt2, 16S), or M. ruminalis (HP, 16S). The validity of the assay was examined against 22 reference strains within the family Pasteurellaceae and 17 field isolates (nasal) that had been collected previously from feedlot cattle and tentatively identified as M. haemolytica based on morphology and substrate utilization. Additionally, 200 feedlot cattle were screened for M. haemolytica using multiplex PCR. Forty-four isolates from 25 animals were identified as M. haemolytica. The PCR assay positively identified all M. haemolytica, as confirmed by phenotypic tests and clustering based upon cellular fatty acid methyl ester (FAME) profiles. Selected nasal isolates that exhibited evidence of haemolysis, but were M. haemolytica-negative based on PCR, were also confirmed negative by phenotypic and FAME analyses. The multiplex PCR assay required no additional phenotypic tests for confirmation of M. haemolytica, within the group of bacteria tested.