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Objectives: In order to find the optimal inactivation conditions for Clostridium chauvoei culture, different factors were investigated and the immunogenicity of inactivated cultures was studied., Methods: C. chauvoei was cultured with different formalin percentages (0.3, 0.5 or 0.7% V/V), inactivation temperatures (37 °C or room temperature) and incubation times (one or two weeks). Sterility tests were performed and residual formaldehyde and pH were measured. Rabbits were immunized twice with inactivated cultures and sera were used for detection of immune response., Results: In the one-week experiment, 0.5 and 0.7% formalin inactivated the bacteria after one week, and the percentage of 0.3 inactivated after three weeks. The residual formaldehyde at weeks 1 and 8 was not significantly different. In the two-week experiment, cultures treated with 0.3 and 0.5% formalin were inactivated after four weeks, and those with 0.7% formalin were inactivated after three weeks. Residual formaldehyde at week 8 differed significantly from that of week 1. Residual formaldehyde was affected by incubation temperature since it was lower at 37 °C than in room temperature. Also, a significant effect was observed for formalin on pH, as higher formalin contents led to lower pH values of the cultures. ELISA showed the lowest antibody titer achieved by 0.7% formalin group. Antibody titer was not different between 0.3 and 0.5% formalin., Conclusions: The best condition for inactivation of C. chauvoei was considered as one-week incubation with 0.5% formalin at 37 °C, leading to a high antibody response., Competing Interests: Declaration of competing interest The authors declare that they have no known conflict of interests or personal relationships that could influence the study in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Some serovars of salmonella cause huge global diseases such as enteric fever and invasive non typhoidal Salmonella disease. Flagellin as a key antigenic component of salmonella, can induce humoral and cellular immunity responses. In this research, we performed an opsonophagocytic killing assay (OPKA) as an important mechanism of the host-defense system, for salmonella to study the activity of anti-sera of native FliC, truncated modified recombinant FliC (tmFliC) and full length recombinant FliC proteins (flFliC). Also, the potency of antibodies for inhibiting bacterial movement was evaluated by traditional and newly-designed motility inhibition assay methods. Results showed both recombinant FliC anti-sera and native FliC (nFliC) anti-serum had the ability to opsonize Salmonella typhimurim, which led to bacterial clearance by mice macrophages. Also, inhibition of bacterial motility was observed for all anti-sera. Anti-nFliC and anti-flFliC sera showed higher effects on Salmonella typhimurim motility than that of tmFliC. In traditional method, about 88%, 86% and 80% inhibition were observed by using 5% nFliC, anti-flFliC and anti-tmFliC sera, respectively. In the newly-designed method using SIM (Sulfide indole motility) medium, results confirmed the traditional method for motility inhibition. Our findings suggest that salmonella fliC as a protective antigen may disrupt the flagellum apparatus activity., Competing Interests: Declaration of competing interest The authors declared that they have no conflicts of interest., (Copyright © 2020. Published by Elsevier Ltd.)

Problems regarding purification efficacy in recombinant technologies is due to the protein structure. Experimental manipulation of genes and the subsequent proteins may overcome this issue. In order to improve production efficacy and maintain immunestimulatory effect of flagellin, the Toll-like Receptor 5 (TLR5) ligand and a potent adjuvant, we performed a bioinformatic study to find the best model for FliC manipulation. Truncated modified FliC (tmFliC) and full length FliC (flFliC) genes were cloned and expressed in pET-21a vector and protein purification was carried out using an improved His-Tag method. Polyclonal antibodies were generated against flFliC and tmFliC in New Zealand white rabbits. IgG response to the recombinant proteins was determined by ELISA. Cross-reactivity assay was performed by ELISA for all proteins and bacteria. Immunogenicity of tmFliC and flFliC was evaluated in chicken cells, and expression level of tumor necrotic factor-α (TNF-α) and interleukin-6 (IL-6) were relatively analyzed by Real-Time-PCR. Results showed high purification efficacy for tmFliC. Antibody titer of tmFliC was significantly higher than that of flFliC. In addition, the cross-reactivity assay for both proteins and Salmonella was positive which indicates similar epitopic regions. Stimulation of both FliCs significantly increased TNF-α and IL-6 expression in peripheral blood mononuclear cells (PBMCs) and splenocytes, with higher effect observed with flFliC. IL-8 protein level increased after 6 and 24 h stimulation with different concentrations of tmFliC and flFliC. These results suggest that the aimed gene modification in fliC gene produces a bioactive immunostimulant type of flagellin which upregulates TLR5 downstream genes as well as in flFliC., (Copyright © 2018. Published by Elsevier Ltd.)