Construction and Comparison of Molecular Identification Methods for Mannheimia haemolytica

Mannheimia haemolytica is a primary pathogen causing bacterial pneumonia in cattle and sheep. Multiple PCR targets exist for its identification, but no standardized primer pairs have been widely adopted. This study evaluated the universality of six reported primers (including rpt2) using clinical M. haemolytica isolates. Genus specificity was tested against standard strains of common ruminant pathogenic/commensal bacteria and other Mannheimia species. Four primer sets (gcp, artJ-lktC, lktD, sodA) failed to distinguish M. haemolytica from other Mannheimia species. The rpt2 primer showed suboptimal universality, with 3 of 22 tested isolates yielding negative results. The rpoB primer had favorable universality and specificity, but its 136 bp amplicon was unsuitable for constructing multiplex PCR with M. haemolytica serotyping primers. We identified design flaws in the original rpt2 primer and developed a novel rpt2-N primer. Evaluation confirmed its strong universality, specificity, and sensitivity, as well as compatibility with serotyping primers for multiplex PCR assays. Field testing on three goat farms in the Chongqing region showed markedly higher M. haemolytica positivity rates after sharp temperature drops and transportation. Serotype A2 was confirmed as the dominant serotype in goats in Chongqing. This study systematically assessed multiple PCR identification primers, confirmed the feasibility of rpoB, and resolved the universality limitation of rpt2, providing technical support for rapid and accurate M. haemolytica detection.