Development and Application of Real-Time PCR Assay for Detection of Salmonella Abortusequi

Abstract

Salmonella enterica subsp. enterica serovar Abortusequi is a major pathogen in horse and donkey herds, causing abortion in pregnant equids and resulting in enormous economic losses. A rapid and reliable method is urgently needed to detect S. Abortusequi in herds where the disease is suspected. To achieve this goal, a TaqManbased real-time PCR assay targeting the gene for the flagellin protein phase 2 antigen FljB was developed. This real-time PCR assay had high specificity, sensitivity, and reproducibility. The detection limit of the assay was 30 copies/mL of standard plasmid and 10 CFU/mL of bacterial DNA. Furthermore, 540 clinical samples, including 162 tissue, 192 plasma, and 186 vaginal swab samples collected between 2018 and 2021 in China, were tested to assess the performance of the developed assay. Compared to the gold standard method of bacterial isolation, the real-time PCR assay exhibited 100% positive agreement for all tissue, plasma and vaginal swab tests. Additionally, this assay detected DNA from S. Abortusequi from 56.7% (34/60) culture-negative tissue and 22.9% (41/179) culture-negative vaginal swab samples from infected equids. Receiver operating characteristic analysis demonstrated that the results of the developed real-time PCR assays were in significant agreement with those of the culture method. The real-time PCR assay can be completed within 45 min of extraction of DNA from samples. Our results show that this assay could serve as a reliable tool for the rapid detection of S. Abortusequi in tissue, plasma, and vaginal swab clinical samples.