Etiological and Pathological Investigation of Bacterial Pneumonia in Forest Musk Deer (Moschus berezovskii) Using a Novel Multiplex PCR Assay.

Pneumonia is a major cause of mortality in forest musk deer (M. berezovskii) breeding, leading to significant economic losses. Conventional pathogen detection methods, such as bacterial isolation and culture, are time-consuming and costly. This study aimed to develop a rapid, specific, and sensitive multiplex PCR assay for the simultaneous detection of four common bacterial pathogens. Specific primers were designed to target genes: gyrB for Escherichia coli, PhoE for Klebsiella pneumoniae, LasI for Pseudomonas aeruginosa, and Kmt1 for Pasteurella multocida. The multiplex PCR system was optimized by adjusting the annealing temperature and primer concentrations, and its specificity, sensitivity, and reproducibility were evaluated. The technique successfully detected all four pathogens simultaneously, demonstrating high specificity and good reproducibility. The detection limits were 9.2 × 104 CFU/mL for E. coli, 1.3 × 105 CFU/mL for K. pneumoniae, 1.5 × 105 CFU/mL for P. aeruginosa, and 3.7 × 104 CFU/mL for P. multocida. Using this method, a total of 47 lung tissue samples from dead forest musk deer across four regions in China were analyzed, and results were compared with bacterial isolation and 16S rRNA sequencing. A high rate of mixed infections (74.5%) was observed, with E. coli showing the highest detection rate (80.8%), followed by K. pneumoniae (65.9%), while P. aeruginosa (40.4%) and P. multocida (40.4%) were less prevalent. Moreover, histopathological analysis confirmed typical pneumonia lesions. This method provides a reliable method for rapid detection of major bacterial pathogens in musk deer, supporting clinical diagnosis and epidemiological studies.