Detection and quantification of Klebsiella pneumoniae in American bullfrog (Rana catesbeiana) by Taqman MGB probe real-time fluorescence quantitative PCR

Klebsiella pneumoniae is an important pathogen in American bullfrog farming. It causes serious diseases, resulting in significant losses. However, the spread and replication of K. pneumoniae in infected bullfrog tissues are poorly understood. This study aimed to investigate the variation in K. pneumoniae loads in the organs of infected bullfrogs to clarify the effect of K. pneumoniae replication. In this work, a TaqMan minor groove binder probe fluorescence real-time quantitative polymerase chain reaction (qPCR) assay was developed to rapidly detect and quantify K. pneumoniae.The detection limit of the qPCR method was as low as 2.9 copies/mu L, which was 100 times more sensitive than that of the conventional PCR. In addition, comparison of K. pneumoniae in tissue mixing and mucus showed similar positive rates, K. pneumoniae in bullfrogs could be preliminarily detected and monitored by anal mucus detection under noninvasive conditions. The changes in bacterial load detected by qPCR in different tissues of bullfrogs infected with K. pneumoniae showed that the bacterial concentrations increased tremendously 3 days after infection, and the spleen was the major site for K. pneumoniae replication. The determination of the changes in tissue bacterial load provided useful data for elucidating the pathogenesis of K. pneumoniae in bullfrogs, while the mucus assay could be an effective tool for the early surveillance and prevention of K. pneumoniae in bullfrogs.