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Salmonellaspp. are important zoonotic pathogens that are responsible for severe diseases in both animals and humans. Salmonella entericasubsp. entericaserovar Gallinarum biovar Gallinarum (S. Gallinarum) and biovar Pullorum (S. Pullorum) are typical infectious pathogens detected in the chicken industry that have caused great economic losses. To facilitate their detection and prevent contamination, we developed a rapid multiple PCR method, which can simultaneously detect Salmonellaspp. and further identify the biovars S. Pullorum/Gallinarum. This PCR detection method is based on the cigRgene, which is conserved among Salmonellaspp. but has a 42-bp deletion in S. Pullorum/Gallinarum. The specificity and sensitivity of the PCR assay was evaluated with 41 different strains: 34 Salmonellastrains, including 5 S. Pullorum/Gallinarum strains, and 7 non-Salmonellastrains. The lower limit of detection was 8.15 pg of S. Pullorum (S06004) genomic DNA and 20 cfu in PCR, which shows a great sensitivity. In addition, this method was applied to detect or identify Salmonellafrom processing chicken liver and egg samples, and the results corresponded to those obtained from serotype analysis using the conventional slide agglutination test. Overall, the new cigR-based PCR assay is efficient and practical for Salmonelladetection and S. Pullorum/Gallinarum identification and will greatly reduce the workload of epidemiologic investigation.

Salmonella entericasubsp. entericaserovar Gallinarum biovar Gallinarum (S. Gallinarum) and biovar Pullorum (S. Pullorum) are gram-negative bacteria, members of the most important infectious pathogens, and have caused common problems in the poultry industry, especially in the developing countries. O- and H-antigen specific anti-sera are commonly for slide and tube agglutination tests to identify Salmonellaserovars. However, it is both labor intensive and time consuming, so there is an urgent need for a new technique for the rapid detection of the major Salmonellaserovars. In this study, we developed a 1-step PCR assay to identify the serovar Gallinarum. This PCR-based assay was based on the SPUL_2693 gene, which was located in SPI-19 and found by comparing the genomes of the S. Pullorum and S. Gallinarum in the whole data of NCBI. The specificity of this gene was evaluated by bioinformatics analysis, and the results showed that the SPUL_2693 gene exists in all serovar Gallinarum. The specificity and sensitivity of this PCR assay were evaluated in our study. The developed PCR assay was able to distinguish the serovar Gallinarum from 27 different Salmonellaserovars and 5 different non-Salmonellapathogens. The minimum limit of genomic DNA of S.Pullorum for PCR detection was 2.143 pg/μL, and the minimum limit number of cells was 6 CFU. This PCR assay was also applied to analyze Salmonellastrains isolated from a chicken farm in this study. The PCR assay properly identified the serovar Gallinarum from other Salmonellaserovars, and the results were in agreement with the results of a traditional serotyping assay. In general, the newly developed PCR-based assay can be used to accurately judge the presence of the serovar Gallinarum and can be combined with traditional serotyping assays, especially in the case of large quantities of samples.