Development and application of an indirect ELISA and nested PCR for the epidemiological analysis of Klebsiella pneumoniae among pigs in China

Zengshuai Wu; Na Li; Ziheng Li; Jianlong Wang; Mengmeng Liu; Mengzhu Qi; Shaopeng Wei; Tong Wu; Yu Guo; Junhui Zhu; Hexiang Jiang; Ruixue Xue; Changjiang Sun; Xin Feng; Jingmin Gu; Wenyu Han; Fengyang Li; Liancheng Lei

doi:10.3389/fmicb.2023.1329609

Development and application of an indirect ELISA and nested PCR for the epidemiological analysis of Klebsiella pneumoniae among pigs in China

Front Microbiol. 2024 Jan 8:14:1329609. doi: 10.3389/fmicb.2023.1329609. eCollection 2023.

Authors

Zengshuai Wu^#¹, Na Li^#¹, Ziheng Li¹, Jianlong Wang², Mengmeng Liu³, Mengzhu Qi⁴, Shaopeng Wei¹, Tong Wu¹, Yu Guo², Junhui Zhu¹, Hexiang Jiang¹, Ruixue Xue⁵, Changjiang Sun¹, Xin Feng¹, Jingmin Gu¹, Wenyu Han¹, Fengyang Li¹, Liancheng Lei^{1

6}

Affiliations

¹ State Key Laboratory for Zoonotic Diseases, College of Veterinary Medicine, Jilin University, Changchun, China.
² Animal Disease Control Center of Inner Mongolia, Hohhot, China.
³ Department of First Hospital, Jilin University, Changchun, China.
⁴ College of Veterinary Medicine, Shandong Agricultural University, Taian, China.
⁵ Animal Disease Control Center of Shandong, Jinan, China.
⁶ College of Animal Science, Yangtze University, Jingzhou, China.

^# Contributed equally.

Abstract

Introduction: Klebsiella pneumoniae (K. pneumoniae) is an important opportunistic and zoonotic pathogen which is associated with many diseases in humans and animals. However, the pathogenicity of K. pneumoniae has been neglected and the prevalence of K. pneumoniae is poorly studied due to the lack of rapid and sensitive diagnosis techniques.

Methods: In this study, we infected mice and pigs with K. pneumoniae strain from a human patient. An indirect ELISA was established using the KHE protein as the coating protein for the detection of K. pneumoniae specific antibody in clinical samples. A nested PCR method to detect nuclei acids of K. pneumoniae was also developed.

Results: We showed that infection with K. pneumoniae strain from a human patient led to mild lung injury of pigs. For the ELISA, the optimal coating concentration of KHE protein was 10 µg/mL. The optimal dilutions of serum samples and secondary antibody were 1:100 and 1:2500, respectively. The analytical sensitivity was 1:800, with no cross-reaction between the coated antigen and porcine serum positive for antibodies against other bacteria. The intra-assay and inter-assay reproducibility coefficients of variation are less than 10%. Detection of 920 clinical porcine serum samples revealed a high K. pneumoniae infection rate by established indirect ELISA (27.28%) and nested PCR (19.13%). Moreover, correlation analysis demonstrated infection rate is positively correlated with gross population, Gross Domestic Product (GDP), and domestic tourists.

Discussion: In conclusion, K. pneumoniae is highly prevalent among pigs in China. Our study highlights the role of K. pneumoniae in pig health, which provides a reference for the prevention and control of diseases associated with K. pneumoniae.

Keywords: ELISA; Klebsiella pneumoniae; khe; nested PCR; pig serum.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Key Project of Chinese National Programs for Research and Development (2022YFD1800905), and the National Natural Science Foundation of China (32102670, 32102686).