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Salmonella Pullorum (S. Pullorum) and Salmonella Gallinarum (S. Gallinarum) are two biovars of Salmonella enterica serovar Gallinarum, responsible for pullorum disease and fowl typhoid, which are the most prevalent and pathogenic forms of salmonellosis in poultry in developing countries. Traditional differentiation methods for S. Pullorum and S. Gallinarum are based on distinct clinical manifestations and biochemical traits, given their indistinguishable nature via serological assays alone. Molecular differentiation methods such as allele-specific PCR and dual PCR combined with gel electrophoresis or enzyme digestion have also been used to discriminate S. Pullorum and S. Gallinarum, but the detection efficiency is not high. This investigation introduces a Fluorescence Resonance Energy Transfer (FRET) PCR assay targeting the pegB gene, exclusively found in specific Salmonella serovars such as S. Pullorum and S. Gallinarum, and exhibiting conserved single-nucleotide polymorphisms across these two biovars. High-resolution melting curve analysis demonstrates distinct dissolution profiles, facilitating the precise discrimination of S. Pullorum and S. Gallinarum. This FRET-PCR assay exhibits a detection limit of 10 copies per reaction and has been rigorously validated utilizing 17 reference strains and 39 clinical isolates. The innovation presented herein provides a valuable tool for the rapid differentiation of S. Pullorum and S. Gallinarum, thereby enhancing diagnostic efficiency and molecular surveillance of poultry Salmonella. The developed pegB-targeting FRET-PCR assay presents a promising alternative to current cumbersome and time-consuming diagnostic modalities, offering significant potential for expedited identification and control of Salmonella in poultry and mitigating economic losses associated with Salmonella contamination in poultry production. [ABSTRACT FROM AUTHOR]

Salmonella Pullorum (S. Pullorum) and Salmonella Gallinarum (S. Gallinarum) are the biovars of Salmonella enterica serovar Gallinarum that are responsible for pullorum disease and fowl typhoid, respectively, in poultry. Traditional serological methods fail to quickly differentiate between these biovars due to their identical O antigenic factors (O9 and O12). Although single nucleotide polymorphism (SNP)-based methods have been used to distinguish between the biovars, they often lack the required accuracy and effectiveness. In this study, we developed a PCR high resolution melt (PCR-HRM) assay, which targeted a SNP at position 665 of the fimH gene, for rapid differentiation between S. Pullorum and S. Gallinarum. Our method showed 100% specificity and was able to detect as little as 0.033 pg of S. Pullorum DNA and 0.027 pg of S. Gallinarum DNA. The PCR-HRM results for 547 clinical isolates were in complete agreement with traditional serological methods. This PCR-HRM assay significantly reduced identification time and provided high throughput, efficient testing. This makes it a practical and reliable tool for accurate differentiation between S. Pullorum and S. Gallinarum in clinical settings. [ABSTRACT FROM AUTHOR]

Salmonella‐contaminated raw food is an important source of human food poisoning. Traditional methods for detecting Salmonella in meat involve many time‐consuming and laborious steps, preventing timely and effective control of the pathogen. In this study, a multiplex polymerase chain reaction (PCR) assay that targets serovar‐specific genes such as A7P63_0910 (Salmonella Indiana), sdfI (Salmonella Enteritidis), and STM4497 (Salmonella Typhimurium) for three meat‐associated Salmonella serovars was established. This multiplex PCR has been validated by extensive testing on reference strains and clinical isolates belonging to different Salmonella serovars (n = 348) and non‐Salmonella bacteria (n = 12). The detection limit is 100 CFU per reaction for bacterial culture, being equivalent to 10 pg per reaction for bacterial genomic DNA. Detection of Salmonella in raw meat samples by this multiplex PCR revealed that the prevalence of S. Indiana, S. Enteritidis, and S. Typhimurium is 7.7%, 5.7%, and 2.0%, respectively. The multiplex PCR established in this study can be used as a novel and convenient tool for simultaneous detection of three major meat‐associated Salmonella serovars, including the newly emerging and highly drug‐resistant S. Indiana. Practical applications: In this study, a multiplex polymerase chain reaction (PCR) assay that targets serovar‐specific genes such as A7P63_0910 (Salmonella Indiana), sdfI (Salmonella Enteritidis), and STM4497 (Salmonella Typhimurium) for three meat‐associated Salmonella serovars was established. This multiplex PCR is suitable for the simultaneous detection and differentiation of three major meat‐associated Salmonella serovars, and has been validated by extensive testing of reference strains and clinical isolates. The multiplex PCR assay can provide rapid and specific screening for top three Salmonella serovars in meat samples. [ABSTRACT FROM AUTHOR]

Infectious bronchitis virus (IBV) restricts cell tropism. Except for the Beaudette strain, other IBVs cannot infect mammalian cell lines. The limited cell tropism of other IBVs has hindered IBV vaccine development and research on the mechanisms of IBV infection. A novel Vero cell-adapted strain, HV80, has been previously reported. In this study, we constructed recombinants expressing the chimeric S glycoprotein, S1 or S2 subunit of strain H120 and demonstrated that mutations on S2 subunit are associated with the strain HV80 Vero cell adaptation. R687P or P687R substitution recombinants were constructed with the genome backbone of strains HV80 or H120. We found that the RRRR 690 /S motif at the S2′ cleavage site is crucial to the Vero cell adaptation of strain HV80. Another six amino acid substitutions in the S2 subunit of the recombinants showed that the Q855H mutation induced syncytium formation. A transient transfection assay demonstrated the S glycoprotein with the PRRR 690 /S motif at the S2′ cleavage site induced low-level cell–cell fusion, while H855Q substitution hindered cell–cell fusion and blocked cleavage event with S20 product. This study provides a basis for the construction of IBV recombinants capable of replicating in Vero cells, thus contributing to the advancement in the development of genetically engineered cell-based IBV vaccines. • S2 subunit mutations are associated with the strain HV80 Vero cell adaptation. • The RRRR 690 /S motif at the S2′ cleavage site triggers the infection. • The Q855H substitution enhances infection efficiency through cell-cell fusion. • The H855Q substitution also blocks the cleavage event with S20 product. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Typhimurium (ST) is a predominant serovar causing foodborne illnesses worldwide. Traditional detection methods often face challenges, including the need for specialized equipment, skilled operators, and lengthy procedures. To address these limitations, we developed a rapid, sensitive, and specific ST detection method by integrating loop-mediated isothermal amplification (LAMP) with the clustered regularly interspaced short palindromic repeats and associated protein 12b (CRISPR/Cas12b) system, all within a single tube. Our results indicate that the LAMP-CRISPR/Cas12b reaction can be completed isothermally in under 1 h without requiring specialized instruments. The platform's limit of detection (LoD) is 12.5 copies per reaction. Additionally, the system demonstrated 100% inclusivity and exclusivity when tested against 30 reference strains, highlighting its specificity. In practical applications, the LoDs for ST in pure nucleic acid and contaminated fecal samples were 2.32 and 23.2 CFU/mL, respectively, with higher sensitivity observed in pure nucleic acid samples. Overall, our findings underscore the potential of the one-tube LAMP-CRISPR/Cas12b platform as a rapid, sensitive, and specific tool for ST detection, particularly in resource-limited settings., Importance: Here, we have provided a novel one-step method for Salmonella Typhimurium detection in one pot by integrating the LAMP assay with the CRISPR/Cas12b system, offering significant advantages in terms of simplicity, speed, and accuracy., Competing Interests: The authors declare no conflict of interest.

The immune performance, SNPs and expression levels of candidate genes (IL1-β, Nramp1, TLR4, MyD88, NF-κB and NLRC5) were analyzed in carrier chickens of a Chinese indigenous breed infected with Salmonella enterica Serovar Pullorum at different persistence periods (12, 19 and 24 weeks of age). Carrier birds at 19 weeks of age presented significant difference in most immune parameters, as compared to carriers at 12 and 24 weeks of age, while no significant difference in most immune parameters was observed between carriers at 12 and 24 weeks of age. The genotype distributions of IL1-β and TLR4 presented significant differences between carriers and healthy birds. The expression levels of most candidate genes in carriers at 19 weeks of age were significantly higher than that in carriers at 12, 24 weeks of age and healthy birds and reached 1% level of significance between carriers at 19 weeks of age and healthy birds. The expression patterns of all genes, but IL-1fl and NLRC5 between carriers at 12 and 24 weeks of age in all tissues were similar. Compared with carriers at 12 weeks of age, IL1-β was significantly down-regulated, but NLRC5 was significantly up-regulated in carriers at 24 weeks of age. Our study demonstrated that immune performance of carrier birds was severely impaired at age of sexual maturation and NLRC5 might play as a negative mediator of NF-κB pathway involved in immune response to asymptomatic infection by S. Pullorum. The TLR4/MyD88/NF-κB pathway might be suitable for study on S. Pullorum infection in Chinese indigenous breeds.