Most cases of chicken salmonellosis are caused by Salmonella enterica serovar Gallinarum biovars Gallinarum and Pullorum, which lead to a significant morbidity and fatality rate. Although the conventional Kaufmann-White scheme is the reliable method for the serotyping of Salmonella, it does not distinguish between closely related biotypes like S. Pullorum and S. Gallinarum. Herein, we conducted a single one-step multiplex PCR assay that can identify and distinguish between S. Pullorum and S. Gallinarum in an accurate manner. This PCR method was based on three genes, including torT for S. Pullorum identification, I137_14430 for S. Gallinarum identification, and stn as the genus-level reference gene for Salmonella. By comparing S. Pullorum to S. Gallinarum and other serovars of Salmonella, in silico study revealed that only the former has a deletion of 126 bp-region in the carboxyl terminus of torT. The I137_14430 gene does not exist in S. Gallinarum. However, it is present in all other Salmonella serotypes. The multiplex PCR approach utilizes unique sets of primers that are intended to specifically target these three different genes. The established PCR method was capable of distinguishing between the biovars Pullorum and Gallinarum from the 29 distinct Salmonella serotypes as well as the 50 distinct pathogens that are not Salmonella, showing excellent specificity and exclusivity. The minimal amount of bacterial cells required for PCR detection was 100 CFU, while the lowest level of genomic DNA required was 27.5 pg/μL for both S. Pullorum and S. Gallinarum. After being implemented on the clinical Salmonella isolates collected from a poultry farm, the PCR test was capable of distinguishing the two biovars Pullorum and Gallinarum from the other Salmonella strains. The findings of the PCR assay were in line with those of the traditional serotyping and biochemical identification methods. This new multiplex PCR could be used as a novel tool to reinforce the clinical diagnosis and differentiation of S. Pullorum and S. Gallinarum, particularly in high-throughput screening situations, providing the opportunity for early screening of infections and, as a result, more effective management of the illness among flocks.

The flagellin (FliC) of Salmonella typhimurium is a potential vaccine adjuvant as it can activate innate immunity and promote acquired immune responses. Macrophages are an important component of the innate immune system. The mechanism of flagellin’s adjuvant activity has been shown to be related to its ability to activate macrophages. However, few studies have comprehensively investigated the effects of Salmonella flagellin in macrophages using transcriptome sequencing. In this study, RNA-Seq was used to analyze the expression patterns of RAW264.7 macrophages induced by FliC to identify novel transcriptomic signatures in macrophages. A total of 2204 differentially expressed genes were found in the FliC-treated group compared with the control. Gene ontology and KEGG pathway analyses identified the top significantly regulated functional classification and canonical pathways, which were mainly related to immune responses and regulation. Inflammatory cytokines (IL-6, IL-1β, TNF-α, etc.) and chemokines (CXCL2, CXCL10, CCL2, etc.) were highly expressed in RAW264.7 cells following stimulation. Notably, flagellin significantly increased the expression of interferon (IFN)-β. In addition, previously unidentified IFN regulatory factors (IRFs) and IFN-stimulated genes (ISGs) were also significantly upregulated. The results of RNA-Seq were verified, and furthermore, we demonstrated that flagellin increased the expression of IFN-β and IFN-related genes (IRFs and ISGs) in bone marrow-derived dendritic cells and macrophages. These results suggested that Salmonella flagellin can activate IFN-β-related immune responses in macrophages, which provides new insight into the immune mechanisms of flagellin adjuvant.