Construction of multiplex PCR assay based on the citE2 gene to identity Salmonella Pullorum and its effector SteE in pathogenicity and immunity

Abstract

The thesis is devoted to providing development and scientific justification of the prevention and accurate detection of S. Pullorum, and clarify the mechanism of persistent S. Pullorum infection in chickens. S. Pullorum is a host-specific pathogen, causes severe economic losses to the chicken farms, and also can induce an anti-inflammatory response in chickens. A rapid and accurate method can help us make corresponding prevention and control measures in clinical and food samples. Additionally, S. Pullorum effectors with anti-inflammatory function can be a unique drug candidate for targeting salmonellosis. In order to confirm the pathogenic bacteria species and its drug susceptibility that causes severe diarrhea (pullorum disease) and systemic fatal diseases in chickens. The diseased chickens with suspected S. Pullorum from a large-scale chicken farms were subjected to bacterial culture identification and drug sensitivity test. S. Pullorum were the main pathogenic bacteria of the diseased chickens. Drug susceptibility test confirmed that the isolated S. Pullorum were resistant to amoxicillin, sulfamethazine, tetracycline and ciprofloxacin, but sensitive to ceftriaxone, ceftiofur and kanamycin. The citE2 gene and the interval sequence of SPS4_00301-SPS4_00311 gene existed in all Salmonella serovars, but the regions of difference in citE2 was found only in S. Pullorum. The developed multiplex PCR system based on the two molecular markers can be as low as 6.25 pg/μL and 104 CFU/mL for genomic DNA and S. Pullorum cells, respectively. The developed multiplex PCR assay distinguished S. Pullorum from 33 different Salmonella serotypes and 13 non-target species. The detection of egg samples artificially contaminated with S. Pullorum, S. Enteritidis and naturally contaminated 69 anal swab samples showed that results were consistent with the culture method. In order to the analysis of the association of the type III secretion system encoded steE with S. Pullorum virulence, the steE deletion of S. Pullorum (ΔsteE) strain was constructed using the λ-Red recombination system. We performed in vitro and in vivo analysis using HD-11 cells and chickens, infected with wild-type S. Pullorum (WT) or ΔsteE strain, respectively. The results of virulence assay showed that the LD50 of ΔsteE strain was 22.8 times higher than that of WT and ΔsteE strains in chickens. The colonization experiment of bacteria showed that the overall change trend of the number of WT and ΔsteE strains were similar in chicken livers, spleens, heart, bursas, and cecums, which increased first and then decreased. Compared to that in the WT strain, ΔsteE strain reduced invasion and proliferation of bacteria in the infected HD-11 cells. In addition, we analyzed the mRNA expression levels of effector genes and cytokines by qRT-PCR. SteE was associated with the regulation of various effector genes and inflammatory cytokines in HD-11 cells during S. Pullorum infection. Compared with those of the WT or ΔsteE+steE strain-infected group, the mRNA transcript levels of IL-12 and IFN-γ were significantly higher, whereas the IL-10 mRNA expression was significant decreased in the ΔsteE strain-infected liver and bursa, the IL-4 mRNA expression displayed dramatically decreased transcriptional level in the ΔsteE strain-spleen, cecum, and heart, and the IL-10 mRNA expression was significantly lower in the ΔsteE strain-infected spleen and cecum. The chickens infected with the ΔsteE strain showed weak pathological lesions in the liver and spleen histopathological lesions compared to those in the WT strain. To elucidate the mechanism via which steE regulators the Th1/Th2 cytokines expression in the case of S. Pullorum infection, we used S. Pullorum as a model pathogen to evaluate the role of steE in Salmonella-induced inflammation. We demonstrated that steE diminished the expression of Th1-related cytokines (IFN-γ and IL-12) and promoted the expression of Th2-related cytokines (IL-4 and IL-10) in HD-11 cells and chicken models of S. Pullorum infection. SOCS3 silencing suppressed the function of steE in HD-11 cells, led to the imbalance of Th1/Th2-related cytokines. SteE promoted SOCS3 expression by activating STAT3 in HD-11 cells. Moreover, steE inhibited NF-B P65 expression and blocked its translocation to the nucleus by promoting SOCS3 expression. In summary, the combined application of bacterial culture identification, drug sensitivity test analysis and the developed multiplex PCR system can accurately understand the distribution, drug resistance and detection of S. Pullorum, provide data support and a valuable tool for the purification of salmonellosis. In addition, steE enhanced the persistent infection and virulence of S. Pullorum by regulating inflammation response in the case of S. Pullorum infection. Finally, our results also illustrated for the first time that steE regulated the expression of Th1/Th2 cytokines via modulation of the STAT3/SOCS3 and NF-κB axis that might be associated with the Th1/Th2 cells differentiation and could, therefore, be a novel therapeutic strategy against salmonellosis. Based on the materials of the dissertation, methodological recommendations «Construction of multiplex PCR assay based on the citE2 gene to identity Salmonella Pullorum and its effector SteE in pathogenicity and immunity» was developed and approved by the Academic Council of SNAU (Protocol № 5, dated 29.12.2022). We recommend using the materials of the dissertation work when studying the courses "Veterinary microbiology", "Veterinary immunology" for masters of the Faculty of Veterinary Medicine of Sumy NAU. And for the courses "Veterinary microbiology" and "Veterinary immunology" for masters of the Henan Institute of Science and Technology (HIST).