Live-attenuated Salmonella enterica serotype Choleraesuis vaccine with regulated delayed fur mutation confer protection against Streptococcus suis in mice

Live-attenuated Salmonella enterica serotype Choleraesuis vaccine with regulated delayed fur mutation confer protection against Streptococcus suis in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Live-attenuated Salmonella enterica serotype Choleraesuis vaccine with regulated delayed fur mutation confer protection against Streptococcus suis in mice Yu-an Li, Yunyun Chen New, Yuan zhao Du, Weiwei Guo, Dianfeng Chu, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.13690/v4 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 May, 2020 Read the published version in BMC Veterinary Research → Version 4 posted 4 You are reading this latest preprint version Show more versions Abstract Background: Recombinant Salmonella enterica serotype Choleraesuis ( S . Choleraesuis) vaccine vector could be used to deliver heterologous antigens to prevent and control pig diseases. We have previously shown that a live-attenuated S . Choleraesuis vaccine candidate strain rSC0011 (ΔP crp527 ::TT araC P BAD crp Δ pmi-2426 Δ relA199 :: araC P BAD lacI TT Δ asdA33 , Δ, deletion, TT, terminator) delivering SaoA, a conserved surface protein in most of S . suis serotypes, provided excellent protection against S. suis challenge, but occasionally lead to morbidity (enteritidis) in vaccinated mice (approximately 1 in every 10 mice). Thus, alternated attenuation method was sought to reduce the reactogenicity of strain rSC0011. Herein , we described another recombinant attenuated S. Choleraesuis vector, rSC0012 (ΔP fur88 :: TT araC P BAD fur Δ pmi-2426 Δ relA199 :: araC P BAD lacI TT Δ asdA33 ) with regulated delayed fur mutation to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. Results: The strain rSC0012 strain with the ΔP fur88 ::TT araC P BAD fur mutation induced less production of inflammatory cytokines than strain rSC0011 with the ΔP crp527 ::TT araC P BAD crp mutation in mice. When delivering the same pS-SaoA plasmid, the intraperitoneal LD 50 of rSC0012 was 18.2 times higher than that of rSC0011 in 3-week-old BALB/C mice. rSC0012 with either pS-SaoA or pYA3493 was cleared from spleen and liver tissues 7 days earlier than rSC0011 with same vectors after oral inoculation. The strain rSC0012 synthesizing SaoA induced high titers of anti-SaoA antibodies in both systemic (IgG in serum) and mucosal (IgA in vaginal washes) sites, as well as increased level of IL-4, the facilitator of Th2-type T cell immune response in mice. The recombinant vaccine rSC0012(pS-SaoA) conferred high percentage of protection against S. suis or S . Choleraesuis challenge in BALB/C mice. Conclusions: The live-attenuated Salmonella enterica serotype Choleraesuis vaccine rSC0012(pS-SaoA) with regulated delayed fur mutation provides a foundation for the development of a safe and effective vaccine against S . Choleraesuis and S. suis . Animal Science General Microbiology Salmonella Choleraesuis virulence immunogenicity Fur inflammatory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Streptococcus suis is a pandemic pathogen responsible for a wide range of invasive diseases such as pneumonia, meningitis and bacteraemia in both humans and pigs[ 1 ,2]. S. suis type 2 (SS2) is the most frequently and virulent isolated from both humans and pigs among all serotypes reported to date [1,3]. The surface-anchored protein (Sao) is a highly conserved membrane-anchored protein and proved to be a immunogenic vaccine candidate[4]. However, Sao formulated with Emulsigen-Plus® provides only partial protection to mice against SS2 infection [3]. In our previous study, a recombinant attenuated Salmonella enterica serotype Choleraesuis vaccine strain rSC0016 carrying saoA gene, provided full protection to mice against SS2 challenge[5]. From the above, an effective delivery system such as live Salmonella enterica serotype Choleraesuis play a crucial role to the effectiveness of Sao. The use of intracellular Salmonella enterica as a vehicle to deliver heterologous protective antigens against pathogens is an attractive strategy. Curtiss et al. developed the RDAS (Regulated Delayed Attenuated Strategies), which enable live salmonella vaccine effectively colonize lymphoid tissues during the invasion stage because of its wild-type aggressiveness and then be full attenuated by silencing the virulence factor, while stimulate both strong cellular and humoral immunity in the immunized mice[6]. Several ways were used to implement this strategy (RDAS). One way is the reverse synthesis of lipopolysaccharide O-antigen by pmi mutation[7]. Another way is to replace the upstream regulatory and promoter sequences of virulence genes with a tightly regulated araC P BAD activator–promoter [8]. This strategy has been successfully used for S . Typhimurium and S . Typhi[6, 7, 8]. With this strategy, we construct a regulated delayed S. Choleraesuis vaccine strain rSC0011 with ΔP crp527 ::TT araC P BAD crp and pmi mutations[9]. rSC0011 delivering S. suis antigens were effective to induce protective immunity against SS2 in mice, but it occasionally caused enteritidis. We sought to improve our S . Choleraesuis candidate vector vaccine by using alternative mutation or introducing new mutation to decreasing its potential to induce enteritidis and enhance immunogenicity. Fur is an important regulatory protein in Salmonella . In the presence of iron, Fur acts as a repressor of iron-controlled genes and mounts an adaptive acid tolerance response [8]. Synthesis of Fur in a vaccine strain during growth confers acid tolerance and maintains iron homeostasis. A decrease of Fur synthesis in Salmonella leads to acid sensitivity and iron acquisition [ 9 ]. Curtiss et al. reported that a S. Typhimurium strain with an arabinose regulated delayed fur mutation is highly immunogenic [10]. In these consideration, an arabinose regulated delayed fur mutation (ΔP fur88 :: TT araC P BAD fur ) was introduced into a S . Choleraesuis vaccine strain with multiple preexist mutations (Δ pmi-2426 Δ relA199::araC P BAD lacI TT Δ asdA33 ) to generate strain rSC0012. A plasmid pS-SaoA[5], encoding saoA from SS 2, was transformed into this strain. We evaluated the virulence, immunogenicity and protection against challenge with virulent SS 2 or S . Choleraesuis C78-3. Results Construction and characterization of the S. Choleraesuis vaccine strain rSC0012 Fur is a ferric uptake regulator that is involved not only in iron metabolism, uptake, and transport, but also invasion and survival of S. Typhimurium in the hosts [11-13]. The absence of Fur attenuates S. Typhimurium [6,14]. To improve the safety and increase the immunogenicity of S. Choleraesuis vector, a new strain, rSC0012, was generated with an arabinose regulated fur , ΔP fur88 :: TT araC P BAD fur (Figure 1A). The phenotypes of the mutations ΔP fur88 ::TT araC P BAD fur and Δ relA :: araC P BAD lacI TT were confirmed by western blot analysis (Figure 1B). The level of Fur synthesis decreased with arabinose dilution (Figure 1B). The presence of mutation Δ relA :: araC P BAD lacI TT in rSC0012(pS-SaoA) were confirmed by the increased synthesis of SaoA (Figure 1B) due to the derepression of P trc promoter on plasmid in rSC0012, which resulted from reduced LacI production whose production was controlled by arabinose. The pmi gene encodes 6-phosphomannose isomerase that interconverts fructose-6-phosphate and mannose-6-phosphatein in Salmonella [7]. Because mannose is required for O-antigen synthesis, the Δ pmi mutation enables the strain rSC0012 to display a smooth LPS pattern in nutrient broth in the presence of mannose and a rough pattern in the absence of mannose (Figure 1C). The Δ asdA mutation enables the strain rSC0012 to have an obligate requirement for DAP [18], which can be complemented with a vector harboring the asd gene then eliminates the need for antibiotic resistance genes for plasmid maintenance[19]. The growth rates of rSC0012 with DAP, rSC0012(pS-SaoA), and rSC0012(pYA3493) were similar (Figure 1D). rSC0012 could grow only with DAP (Figure 1D). Antigen synthesis and plasmid stability in S. Choleraesuis rSC0012 Stable maintenance of plasmids and the production of heterologous antigens are critical to ensure efficacy of recombinant live vaccines. The SaoA protein is a highly conserved surface protective antigen among S. suis serotypes [2, 5]. Using live attenuated S. Choleraesuis vector delivering SaoA antigen from S. suis will allow to develop a bivalent vaccine against both S . Choleraesuis and S . suis . The stabilities of pS-SaoA and pYA3493 in rSC0012 were evaluated by continuous culturing for 50 generations. The stabilities of both Asd + plasmids, pS-SaoA and pYA3493, were 100% in rSC0012 (data not shown). All rSC0012 colonies examined (100 clones/generation) by endonuclease digestion possessed the Asd+ plasmid pS-SaoA or pYA3493. The 34-kDa SaoA protein was detected in cells obtained from both the first and 50th generations of rSC0012(pS-SaoA) (data not shown), indicating the stability of plasmid and stable synthesis of SaoA. Distribution of secreted SaoA in S. Choleraesuis rSC0012 The production levels of SaoA in various subcellular fractions from pS-SaoA-carrying strains, rSC0011, rSC0012, and rSC0018, were determined (Figure 2A-B). rSC0018 is a derivative of C500 [5, 20], a licensed live S. Choleraesuis vaccine attenuated by chemical methods in China, with an asdA mutation. The results showed that the level of the SaoA protein produced in the cytoplasm by strain rSC0012(pS-SaoA) was significantly lower than strain rSC0011(pS-SaoA) (Figure 2B; #, P <0.05), but significantly higher in supernatant when compared with rSC0011(pS-SaoA) and rSC0018(pS-SaoA) (Figure 2B; **, P <0.01). The SaoA protein produced in the periplasm fraction of rSC0012(pS-SaoA) was significantly higher than strain rSC0018(pS-SaoA)(Figure 2A-B). Lower virulence of S. Choleraesuis rSC0012 in vivo To evaluate the virulence of strains with mutations ΔP fur88 :: TT araC P BAD fur or ΔP crp527 ::TT araC P BAD crp , the LD 50 values of rSC0012 and rSC0011 were tested in 3-week-old BALB/c mice, which represents young mice. rSC0018 was used as an attenuation control. All strains carried the expression plasmid pS-SaoA. The results revealed that the LD 50 s of rSC0011(pS-SaoA), rSC0012(pS-SaoA), and rSC0018(pS-SaoA) were at least 10 9 CFU by oral inoculation; whereas, the LD 50 of wild-type C78-3 was 9.5×10 2 CFU (Table 2). Following intraperitoneal infection, the LD 50 of rSC0012(pS-SaoA) was 38.89-fold higher than that of rSC0011(pS-SaoA) and 3.2-fold higher than that of rSC0018(pS-SaoA) in mice. These results indicated that the virulence of rSC0012(pS-SaoA) harboring ΔP fur88 :: TT araC P BAD fur was significantly lower than that of rSC0011(pS-SaoA) harboringΔP crp527 ::TT araC P BAD crp by intraperitoneal route in young mice. Tissue d istribution of S. Choleraesuis strains in BALB/c mice Fur and Crp are important regulatory proteins in Salmonella . Inactivation of the fur and crp genes, attenuates the organism [6,21]. To quantitatively the colonization of the S. Choleraesuis strains containing regulated delayed fur or crp mutations, 3-week-old BALB/c mice were orally inoculated with rSC0011(pYA3493), rSC0012(pYA3493), rSC0018(pYA3493), rSC0011(pS-SaoA), rSC0012(pS-SaoA), rSC0018(pS-SaoA), or wild-type C78-3 (Figure 3A–F). The mice inoculated with wild-type S. Choleraesuis C78-3 died 3–5 days after inoculation, whereas the mice infected orally with vaccine strains rSC0011, rSC0012, rSC0018 containing either plasmid pS-SaoA or pYA3493 survived. The bacteria titers of wild-type strain C78-3 in Peyer’s patches, spleen, and liver were significantly higher than those of vaccine strains rSC0011, rSC0012, rSC0018 containing either plasmid pS-SaoA or pYA3493 at 3 days after inoculation (Figure 3A–C). The titers of bacteria in Peyer’s patches were similar for strains rSC0012(pYA3493), rSC0012(pS-SaoA), rSC0011(pYA3493), and rSC0011(pS-SaoA) at 3-21 days post-inoculation(Figure 3A). At 3d, 7 d, 14 d, 21 d and 28 d, the numbers of rSC0012(pS-SaoA) were significantly higher than those of attenuated vaccine strains rSC0018(pS-SaoA). Same for two control vector, the numbers of rSC0012(pYA3493) were significantly higher than those of attenuated vaccine strains rSC0018(p YA3493) and there was no significant difference with same strain with expression or control vector. respectively ( P < 0.01; Fig. 3A). These results indicating that the colonization abilities of strains rSC0012(pS-SaoA) and rSC0012(pYA3493) in Peyer’s patches were higher than that of rSC0018 with either pYA3493 or pS-SaoA. In spleen, the titers of rSC0012(pYA3493) and rSC0012(pS-SaoA) were similar to those of vaccine strains rSC0018(pYA3493) and rSC0018(pS-SaoA) at 7, 14, 21, 28 days after inoculation (Figure 3B). The titers of strains rSC0011(pYA3493) and rSC0011(pS-SaoA) were significantly higher than those of rSC0012(pYA3493), rSC0012(pS-SaoA), rSC0018(pYA3493), and rSC0018(pS-SaoA), at 3, 7, 14, and 21 days, respectively (Figure 3B). In liver, the titers of rSC0012(pYA3493) and rSC0012(pS-SaoA) were similar to those of vaccine strains rSC0018(pYA3493) and rSC0018(pS-SaoA) at 3, 14, 21, 28 days after inoculation (Figure 3C). At 3d, 7 d, 14 d, 21 d and 28 d post-inoculation, the titers of rSC0012(pS-SaoA) were significantly lower than those of rSC0011(pS-SaoA), same for two control vector, the numbers of rSC0012(pYA3493) were significantly lower than those of strains rSC0011(pYA3493).respectively( P < 0.01; Fig. 3C), whereas the strain rSC0012(pYA3493) was the fastest to be cleared in liver (Figure 3C). These results indicated that the ΔP fur88 :: TT araC P BAD fur mutation impaired the colonization in the liver of S . Choleraesuis vaccine strains. In a summary, the ΔP fur88 :: TT araC P BAD fur mutation reduced the colonization ability of S. Choleraesuis vaccine strains in mice spleen and liver but not in the Peyer’s patches. A ntibody responses in mice immunized with S. Choleraesuis strains All of the mice immunized with strains containing pS-SaoA developed anti-SaoA antibodies (Figure 4A). rSC0012(pS-SaoA) induced significantly higher anti-SaoA IgG titer than did rSC0011(pS-SaoA) 3 weeks after the immunization in 3-week-old mice (Figure 4A; *, P<0.05). Although the anti-SaoA IgG titer of rSC0012(pS-SaoA) were slightly higher than that of rSC0011(pS-SaoA) 5 weeks after immunization, they were not significantly different (Figures 4A). Compared to mice immunized with rSC0018(pS-SaoA), higher serum IgG titers against SaoA were detected in mice immunized with both rSC0011(pS-SaoA) and rSC0012(pS-SaoA) at 3 weeks and 5 weeks postimmunization (Figure 4A, *, P<0.05, **, P<0.01). After boosting, higher titers of anti-SaoA IgG were observed in mice immunized with all three strains containing pS-SaoA (Figure 4A, #, P<0.05). All three attenuated recombinant Salmonella strains induced significant OMP titers after the first immunization in mice (Figure 4B). A significant boosting of serum antibody responses to OMPs was observed after the second immunization, (Figure 4B; #, P<0.05). Mucosal IgA anti-SaoA responses were detected at week 3 in mice immunized with all three attenuated strains containing pS-SaoA. rSC0018(pS-SaoA) induced lower titers of anti-SaoA IgA than rSC0012(pS-SaoA) or rSC0011(pS-SaoA) did in mice (Figure 4C, *, P<0.05, **, P<0.01). Anti-SaoA IgA levels detected in the rSC0012(pS-SaoA) immunized group were significantly higher than those induced in the rSC0011(pS-SaoA) immunized group at 3 and 5 weeks after the immunization (Figure 4C, *, P<0.05, **, P<0.01). These results indicated that strain rSC0012(pS-SaoA) harboring ΔP fur88 :: TT araC P BAD fur elicited a stronger immune response than strain rSC0011(pS-SaoA) harboring ΔP crp527 ::TT araC P BAD crp did, especially elicit a stronger mucosal immune response. IFN-γ or IL-4 production induced by S. Choleraesuis strains To further evaluate the effect of the ΔP fur88 :: TT araC P BAD fur and ΔP crp527 ::TT araC P BAD crp mutations in a strain with multiple preexist mutations on Th1/Th2 immune responses, the levels of IFN-γ and IL-4 in the spleen tissues of mice 7 days and 14 days after booster immunization were measured. The results showed that the titers of IFN-γ and IL-4 induced in mice immunized with rSC0012, and rSC0011 with either pYA3493 or pS-SaoA were significantly higher than those induced in mice inoculated by strain rSC0018 with either pYA3493 or pS-SaoA at both 7 and 14 days after booster (Figure 5A, B; *, P< 0.01,**, P< 0.05). Although rSC0011(pS-SaoA) induced a slightly higher level of IFN-γ in mice than rSC0012(pS-SaoA) did, no significant difference was observed in 3-week-old mice at both 7 and 14 days after booster (Figure 5A-C). However, rSC0011(pYA3493) elicited a higher level of IFN-γ than rSC0012(pYA3493) did at 7 days after booster. The titers of IL-4 induced by rSC0012 with either pS-SaoA or pYA3493 in spleen tissues were significantly higher than those induced by rSC0011 with either pS-SaoA or pYA3493 in 3-week-old mice (Figure 5B; *, P <0.05, **, P <0.01) at 7 and 14 days after booster. Of note, rSC0012(pS-SaoA) induced a dominant Th2 immune response (IFN-γ < IL-4, IFN/IL-4 IL-4, IFN/IL-4>1) in 3-week-old mice (Figure 5C, *, P <0.05, **, P< 0.01). These results indicated the isogenic strain with two different mutations, ΔP fur88 ::TT araC P BAD fur or ΔP crp527 ::TT araC P BAD crp , affected different branches of immune responses. In additional, the titers of IFN-γ and IL-4 induced by the three attenuated strains harboring pS-SaoA in immunized mice were higher than those induced by the same strains harboring the emptor vector pYA3493 (Figure 5A–C; #, P <0.05, ##, P <0.01), suggesting that the protein, SaoA, might augment the immune responses in mice. Induction of inflammation in mice The inflammatory properties of intestinal tissue were investigated in mice after immuned vaccine strains and wild type strain,C78-3.The expression of cytokine genes, TNFα, IL-1β, IL-6 and IL-8, were assessed by quantitative real -time PCR in gut tissue samples at 6 hours and 12 hours postinfection. All strains with mutation ΔP fur88 ::TT araC P BAD fur showed significantly lower transcription levels of cytokine genes IL-1β, IL-6, IL-8 and TNFα than the wild -type strain, C78-3 at both 6 h and 12 h postinfection (Fig 6A-D,*, P <0.05, **, P< 0.01). The C78-3 with mutation ΔP fur88 ::TT araC P BAD fur induced significantly lower transcription levels of cytokine genes IL-1β, IL-6,IL-8 and TNFα than the same strain with ΔP crp527 ::TT araC P BAD crp mutation at both 6 hours and 12 hours postinfection (Fig 6 C and D, &, P <0.05, &&, P< 0.01). At 6 hours postinfection, strain rSC0012( pS-SaoA) showed significantly lower transcription levels of cytokine genes IL-1β , IL-6, IL-8 and TNFα than strain rSC0011( pS-SaoA) (Fig 6A) , a similar trend was seen with IL-1β , IL-6, IL-8 and TNFα at 12 h, although the differences were not significant for IL-6 and TNFα (Fig 6B,#, P <0.05, ##, P< 0.01). These results suggest that the addition of the ΔP fur88 ::TT araC P BAD fur mutations could decreased the inflammatory potential of strains rSC0012( pS-SaoA). Comparison of the protective immunity induced by S. Choleraesuis strains To evaluate the protective immunity conferred by rSC0012(pS-SaoA), the mice in each immunized group were challenged orally with 50×LD 50 of the virulent S . Choleraesuis C78-3 strain or 20×LD 50 of the virulent SS2 strain at 14 days post-boost immunization. After challenge with C78-3, the results revealed 100% protection in mice immunized with either strain rSC0011(pS-SaoA) or strain rSC0012(pS-SaoA), suggesting full protection. Mice immunized with the rSC0018(pS-SaoA) strains resulted in 20% survival. In contrast, all the mice in the PBS group succumbed to the challenge after 4 days. There were no significant difference between the groups immunized with rSC0012(pS-SaoA) and rSC0011(pS-SaoA), though both displayed significantly higher levels of protection than the group immunized with rSC0018(pS-SaoA) (Figure 7A). After the SS2 challenge, immunization with the rSC0011(pS-SaoA), rSC0012(pS-SaoA) and rSC0018(pS-SaoA) strains resulted in 80% survival, 95% survival and 16.7% survival with the lethal SS2 challenge, respectively. In contrast, all the mice in the PBS group succumbed to the challenge after 2 days. Discussion The ultimate goal of an engineered live vaccine strain relies on achieving the proper balance between immunogenicity and attenuation [22,23]. Achieving that goal will restrict unacceptable reactogenicity to avoid over-excitation of inflammatory responses, but sufficient metabolic activity should be maintained to enable the live vaccine to reach deep lymphatic tissues and induce protective immunity[24]. The development of bacterial vaccine relies on a combination of defined mutations[23,25,26]. In order to enhancing the immunogenicity of vaccine strains or to disarm them, multiple independent defined mutations were introduced into Salmonella to generate new recombinant attenuated vaccine strains[6,25,23,27]. By coalescent proper mutations, vaccine strains can be befittingly designed to avoid unacceptable reactogenicity and enhanced immunogenicity [21]. Our previous live attenuated S . Choleraesuis vaccine vector rSC0011 occasionally caused enteritidis in mice[9]. One of the ways to address this problem is by incorporating a sopB mutation[5,25]. In this paper, another way to improve the S . Choleraesuis vector was reported. Fur is an important regulatory proteins of Salmonella , which has been implicated in the acid tolerance response since fur mutants are acid sensitive and cause altered expression of several acid shock proteins [28,29]. A S. Typhimurium strain with an arabinose regulated fur mutation is adequately attenuated and highly immunogenic [6,30]. However, S . Typhimurium belong to group B, while S . Choleraesuis group C. Whether the ΔP fur88 ::TT araC P BAD fur mutation that results in the proper balance between attenuation and immunogenicity of S . Typhimurium is also appropriate for attenuated S . Choleraesuis vaccine has not been reported previously. We corroborated the S . Choleraesuis strain rSC0012 (ΔP fur88 ::TT araC P BAD fur Δ pmi Δ relA :: araC P BAD lacI TT Δ asdA ) displayed a regulated decrease of Fur production in the absence of arabinose. In a previous publication, S . Typhimurium strain with a single ΔP fur88 ::TT araC P BAD fur mutation has shown higher virulent than S . Typhimurium with a single ΔP crp527 ::TT araC P BAD crp mutation by an oral immunization. Unlike report from Curtiss et al in S . Typhimurium [6], our studies did not showed that the ΔP fur88 ::TT araC P BAD fur mutation has higher virulent than ΔP crp527 ::TT araC P BAD crp mutation in S . Choleraesuis. In fact,the LD 50 of rSC0012(pS-SaoA) was 38-fold higher than rSC0011(pS-SaoA) for 3-weeks-old mice with intraperitoneal injection. This result suggest that the ΔP fur88 ::TT araC P BAD fur mutation does different attenuation with S . Choleraesuis or S . Typhimurium,which may due to its complex role as a transcriptional activator of virulence in different strains [31]. The ΔP fur88 ::TT araC P BAD fur mutation can modify the iron regulated outer membrane protein, then strain with ΔP fur88 ::TT araC P BAD fur induced lower inflammation than the isogenic strain with ΔP crp527 ::TT araC P BAD crp mutation or wild type strain in mice. Theses results suggest that the S . Choleraesuis with ΔP fur88 ::TT araC P BAD fur mutation exhibits a lower tendency to trigger excessive inflammation while attenuated sufficiently. Through the oral vaccination route, a vaccine strain will endure the challenges of acid, bile, and antimicrobial peptides existing in the gastrointestinal tract. Once inside Peyer’s patch, it will face macrophagocytes and T cells during the process transferring to deep lymphatic tissues[33]. Fur is essential to Salmonella for accessorial an adaptive acid tolerance response[28]. Although both rSC0011 and rSC0012 had similar levels of Peyer’s patch colonization by oral immunization,we observed that the rSC0012 was cleared more rapidly than the rSC0011 in deeper lymphatic tissues, spleen, and liver of mice. These results suggest that strain rSC0012 was more attenuated than rSC0011 in deep lymphoid tissue, which maybe due to an increase in acid sensitivity cause by loss of Fur makes the cell more susceptible to killing by macrophages. Both rSC0012 and rSC0011 induced higher levels of IgA, IgG, IFN-γ, and IL4 responses with great colonization compared to strain rSC0018 with less colonization in mice.In general, the live Salmonella vaccines with RDAS display superior colonization level in lymphoid tissues during the invasion stage, leading to enhanced protection by effectively colonizing lymphoid tissues[10,35]. However, there are do exist high levels of colonization but low immunogenicity. A strain with the regulated delayed rfc mutation exhibits superior colonization and yet does not stimulate higher heterologous protection than a Δrfc strain without RDAS[36]. Thus, in addition to colonization level, other determining factors may exist to induce the enhanced protection achieved by regulated delayed attenuation. From the above, Selecting the proper mutation is critical for vaccine development with RDAS. This study confirmed above statement. Although the colonization of rSC0012(pS-SaoA) in mice was less than that of rSC0011(pS-SaoA), rSC0012(pS-SaoA) stimulated stronger serum IgG and mucosal IgA responses than the rSC0011(pS-SaoA). This phenomenon suggests that strain with regulated delayed fur mutation may stimulate stronger antibody response with fewer bacteria than strain with regulated delayed crp mutation. Both rSC0012 and rSC0011 aroused a Th1 cell-mediated response, as ostensived by the significant up-regulation of imprint Th1 cytokines, IFN-γ, the stimulator of Th1-type T cell immune response. It could partially be that the intracellular characteristics of Salmonella enterica cause them to be detected on the surface of APC through MHC-I molecules. This result is consistent with previous studies with S . Typhi [6]. Strain rSC0012 induced higher Th2 cell-mediated response than strain rSC0011, as aroused by the significant up-regulation of imprint Th2 cytokines, IL-4 and greater humoral responses than rSC0011 in mice. This phenomenon may be related to the secreting of heterologous antigen. The more heterologous antigens were secreted during infection, the more likely to trigger an IL-4-dominant response[44]. We found a larger secretion of the SaoA from rSC0012, which could be presented by MHC-II molecules. An additional factor may be due to an increase in acid sensitivity cause by loss of Fur makes the rSC0012 more susceptible to killing by macrophages, thus weakening rSC0012's ability to survive in macrophages. The presence of S . suis -specific IgA serves to promptly deliver the antigen to Peyer’s patch dendritic cells or phagocytes and also promptly excite the adaptive immunity during secondary exposure [37]. Generally speaking, the more attenuated the vaccine strain is, the lower its immunogenicity is[21,27], while, the more attenuated rSC0012 strain induced significantly higher IgA and IgG antibody responses to SaoA than the rSC0011 at 3 weeks after the initial immunization in juvenile mice. This may be the result of a equilibrium between attenuation and the ability to participation immune components and activate a controlled over-inflammatory response by the finely crafted strain[23]. The results presented herein highlight that strain rSC0012(pS-SaoA) with regulated delayed fur mutation has retained vaccine efficacy and adequate immunogenicity whilst being safer than previous strain rSC0011(pS-SaoA). Furthermore, the inclusion of the ΔP fur88 ::TT araC P BAD fur mutation may be able to decrease inflammation caused by live-attenuated Salmonella enterica serotype Choleraesuis vaccine, which has been an imperfection for other live-attenuated Salmonella enterica serotype Choleraesuis vaccine which transformed from wild type virulent strain. Both S. Choleraesuis and S. suis are major swine pathogens. Currently, there exist license live vaccines against S. Choleraesuis for swine, including Entersol® Salmonella T/C and SC-54 manufactured by Boehringer Ingelheim and Arugs SC/ST, respectively [38]. However, there is no licensed vaccine against S. suis . Preventing the diseases caused by SS 2 in swine with a combined vaccine is a long-sought goal. Conclusions Our results have shown the strains rSC0012(pS-SaoA) with regulated delayed fur mutation could confer higher protection against challenges with lethal doses of SS 2 or S . Choleraesuis C78-3. Thus, the use of attenuated S. Choleraesuis to develop a vaccine against S . suis will have the great potential to ease the burden of both pathogens. The regulated delayed fur mutation in the novel vaccine rSC0012 resulted in a well-safety, highly immunogenic, and effective vaccine in mice, this study has paved the way for testing in piglets. Our findings will aid the optimal of a S . Choleraesuis vaccine vector capable of eliciting a suitable immune response against other pathogens. Methods Animals Three-week-old female BALB/c mice were purchased from Animal Center of Yangzhou University, and kept one week before inoculation. All animal experiments were authorized by the Jiangsu Administrative Committee for Laboratory Animals (permission number SYXK-SU-2007-0005) and accorded to the Jiangsu Laboratory Animal Welfare and Ethics guidelines of the Jiangsu Administrative Committee of Laboratory Animals. All surgery was performed under anesthesia intraperitoneally injected with sodium pentobarbital, 40 mg per kilogram mouse weight. All the animals were humanely euthanized after the study by inhalation of CO 2 ,while injection with sodium pentobarbital, 40 mg per kilogram mouse weight, and all efforts were made to minimize suffering. Strains, plasmids, and culture conditions The strains, plasmids, used in this study are described in Table 1. C500, an approved live S. Choleraesuis vaccine strain attenuated by thallium compound in China, was used as an attenuation control [39]. The genetic characterization of this strain has been reported [40]. S . suis serotype 2 (SS2, CVCC3928) and S . Choleraesuis C78-3 (CVCC79103) were purchased from China Institute of Veterinary Drug Control.Plasmid pYA3493 is an Asd + vector with a P trc promoter. The asd gene from Salmonella was used as a unique plasmid marker to be used in asd mutants to constitute a balanced-lethal system [18]. LB medium[5],Nutrient broth (NB) and MacConkey agar (Difco) were used for phenotype characterization.When required, media were supplemented with 2,6-diaminopimelic acid (DAP;50 µg/mL),chloramphenicol (Cm; 25µg/mL),L-arabinose (0.2% wt/vol), D-mannose (0.2% wt/vol)or sucrose (5% wt/vol). The empty plasmid vector pYA3493 and expression vector pS‑SaoA were described on previous studies[5]. The saoA gene is under the control of the P trc promoter (Table 1). S. Choleraesuis vaccine vector strain rSC0012 harboring plasmid pS-SaoA (expression vector) or pYA3493 (control vector) were grown in LB broth with both 0.2% arabinose and 0.2% mannose. Selenite broth was used for enrichment of S. Choleraesuis from mice tissues. Strains were prepared as previously described [5,9,10,30]. Bacterial growth was monitored with a spectrophotometer at OD 600 and by direct plating for colony counts. Construction of S. Choleraesuis mutant strains . Four mutations ΔP fur88 ::TT araC P BAD fur , Δ pmi , Δ relA :: araC P BAD lacI TT, and Δ asdA were introduced into S. Choleraesuis C78-3 by conjugation with E. coli χ7213 harboring aforementioned suicide vectors as previously described [41]. The suicide vectors used are listed in Table 1 and Fig. 1A. To construct mutation ΔP fur88 ::TT araC P BAD fur , a 1,335-bp TT araC P BAD cassette were used to replace the 239-bp promoter sequence of the fur gene to achieve arabinose-regulated Fur synthesis (Figure 1A). The araC P BAD cassette contains a transcription terminator (TT) sequence to prevent araC transcription reading through adjoining genes. Plasmid for ΔP fur88 ::TT araC P BAD fur were confirmed by DNA sequencing. All the primers used have been reported [26]. Characterization of S. Choleraesuis mutations in vitro All mutations were confirmed by colony PCR using homologous primers [30]. The Δ asdA mutation was verified by growth with or without DAP in LB broth [18,19].Lipopolysaccharide (LPS) profiles were examined by silver staining in 12% sodium dodecyl sulfate-polyacrylamide (SDS) gel for the Δ pmi mutation [41,42]. The ΔP fur88 ::TT araC P BAD fur deletion-insertion mutation was verified by reduced production of Fur protein as arabinose concentrations decreased with the increased bacterial growth by western blot using anti-Fur antiserum[30]. The production of SaoA was verified by western blot using anti-SaoA antiserum, respectively [9,18,19,20,]. Salmonella subcellular fractionation To evaluate the subcellular localization of synthesized SaoA in the live attenuated S. Choleraesuis vaccine , cultures were grown in NB to an OD 600 of 0.8 and centrifuged at 13,200 × g for 5 min to collect supernatant and pellet. The culture supernatant was saved for later analysis of the bacterial secreted proteins. Periplasmic fractions were prepared using the lysozyme-osmotic shock method as previously described [43,44]. Equal volumes of supernatant,periplasmic, cytoplasmic samples were separated by SDS-PAGE. Proteins were then transferred to polyvinylidene fluoride membrane for western blot analysis using anti-SaoA antiserum [5]. The gel band were analyzed with ImageJ software (NIH) [45]. Preparation of SaoA and Salmonella outer membrane proteins (SOMPs) His-tagged SaoA fusion protein and Salmonella outer membrane proteins (SOMPs) were prepared as previous studies[5]. Determination of virulence in mice. Three-week-old female BALB/c mice were obtained from Animal Center of Yangzhou University. Mice were fasting for 6 h before inoculation.Recombination S. Choleraesuis vector strains were cultured in LB broth with D-0.2% mannose and 0.2% L-arabinose for 12 hours at 37°C as standing cultures. The cultures were diluted 1:50 in the same media and cultured at 37°C to an OD 600 of 0.9. Bacteria were collected by centrifugation at 13,200 × g for 5 min at room temperature and resuspended in PBS to densities suitable for the inoculation. Serial dilutions of the S. Choleraesuis strains were plated onto LB agar supplemented with 0.2% D-mannose and 0.2% L-arabinose to measure the actual densities. Groups of five mice were inoculated with different doses in 20 μl (oral immunization) or 100 μl (intraperitoneal immunization). The mice were observed for 4 weeks for death. The LD50s were calculated according the method of Reed and Muench [11]. Distribution of Salmonella in BALB/c mice A colonization assay for recombination S. Choleraesuis vector strains was performed as described previously[9, 11]. Three-week-old female BALB/c mice were divided into 7 groups with 25 mice in each group.Each mouse was orally inoculated with 1±0.2×10 9 CFU of S. Choleraesuis strains.Peyer’s patches, spleen, and liver of the mice were collected on days 3, 7, 14, 21 and 28 post-inoculated. The densities of bacteria in the tissues were determined using the method reported in previous studies[5,9,11,47]. The assay was performed twice, and the data were similar and pooled for analysis. Immunization of mice Bacteria were grown and collected as above. Serial dilutions of the S . Choleraesuis vaccine strains were plated onto LB agar supplemented with 0.2% D-mannose and 0.2% L-arabinose to determine the actual dose. Three-week-old female BALB/c mice were orally inoculated with 1±0.2×10 9 CFU of S. Choleraesuis vaccine strains containing either pS-SaoA or pYA3493. Mice were boosted inoculated with the same dose of the same strain after three weeks. About 50 μL of whole blood was collected by tail vein three weeks after primary inoculation and two weeks after boosting. Serum was separated from the whole-blood samples and stored at −70°C. Vaginal-wash samples in mice were collected at the indicated time and stored at −70 °C[9,35,46,47]. This experiment was performed in triplicate with each group receiving a similar dose of the vaccine strains. Tissue collection after Salmonella infection of mice Three-week-old female BALB/c mice were divided into 5 groups with 10 mice in each group. Groups of mice were orally inoculated with 1±0.3×10 9 CFU of Salmonella strains. Spleen and intestinum tenue of the mice were collected at 6 h and 12 h postinfection. The tissues were frozen with liquid nitrogen and then transferred to -70°C. Enzyme-linked immunosorbent assay (ELISA) Serum IgG antibody production against S. Choleraesuis outer membrane proteins (OMPs) and SS2 SaoA,and vaginal-wash IgA antibody production against SaoA in mice were evaluated by Enzyme-linked immunosorbent assay (ELISA)[5]. Cytokines in tissues were analyzed by sandwich ELISA using commercial kits (BD Biosciences) according to manufacturer’s instructions. The results from the two experiments were pooled for statistical analysis. Quantitative real -time PCR (qPCR) for cytokines For RNA isolation, gut tissues were homogenized and suspended in TRIzol® (Thermo Fisher Scientific,USA). Tubes were vortexed for 3 min to disrupt the tissues. Chloroform was added to TRIzol® -treated samples and the samples centrifuged at 13200×g for 10 min. The aqueous phase was separated out, and the RNA precipitated using precooled isopropanol. For quantitative real-time PCR, 1 µg of RNA was then reverse transcribed to cDNA. The primers were designed using Primer Blast (NCBI net) and synthesized by TSINGKE Biological Technology Co., Ltd. The sequences of the primers are listed in Table 3.Each sample were amplified using 7500 Fast Real -Time PCR Instrument (ABI,US) using Fast SYBR Green Master Mix (Thermo -Fisher Scientific). The results were using internal reference GAPDH as control for normalization, and the 2 ^ -ΔΔCt method was used to estimate the relative expression level of the mRNAs of target genes. Challenge with S. suis serotype 2 ( SS 2)and S. Choleraesuis (C78-3) in mice Twenty mice in each group were challenged with SS 2 by intraperitoneal (i.p.) injection with 2.4 × 10 8 CFU of SS 2 in 100 μl PBS at five weeks after primary immunization . The 50% lethal dose (LD 50 ) of SS 2 in BALB/c mice was 1.2 × 10 7 CFU. Another twenty mice in each group were challenged orally with 4.8 × 10 4 CFU of C78-3 in 20 μl PBS. The LD 50 of C78-3 in 3-week-old BALB/c mice was 9.5 × 10 2 CFU. Challenged mice were monitored for death daily for 15 days [9,25,26,35]. Statistical analysis Statistical analyses on ELISA were presented as the geometric means and standard deviations for all assays. A Mann-Whitney U Test ( GraphPad Software, Inc.) was applied to contrasting the distribution of the S . Choleraesuis in tissues of mice. The Kaplan-Meier method (SPSS Software) was used for obtain the survival fractions following i.p. challenge of immunized mice. A P value of 0.05 was considered statistically significant. Declarations Ethics approval and consent to participate Procedures involving the care and use of animals were approved by the Jiangsu Administrative Committee for Laboratory Animals (permission number SYXK-SU-2007-0005) and complied with the Jiangsu Laboratory Animal Welfare and Ethics guidelines of the Jiangsu Administrative Committee of Laboratory Animals. Consent for publication Not applicable. Availability of data and material All data generated or analysed during this study will be available from the corresponding author on reasonable request Competing interests None of the authors of this manuscript have any competing or financial interests. Funding This work was supported by the National Natural Science Foundation of China (31672516, 31172300, 30670079), supported by the Grant No. BE2016343 from Jiangsu province and the Priority Academic Program Development of Jiangsu Higher Education Institutions, supported by State Key Laboratory of Genetically Engineered Veterinary Vaccines (No.AGVSKL-ZD/ZY-201807). and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The funding bodies have not been involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Authors’ contributions Design of the study: YA-L, HY-S and SF-W. Performed experiments: YA-L, YZ-D, WW-G, J-F, YY-C. Wrote the manuscript: YA-L, DF-C, XB-W, MB, SF-W. All authors have read and approved the manuscript. Acknowledgements The authors thank to Roy Curtiss, III for kindly providing χ7213, pRE112, pYA3493, and the antibody of Fur. Authors' Information 1 College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, Jiangsu, China 2 Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China 3 Yebio Bioengineering Co., Ltd of Qingdao, Qingdao, 266114, China 4 Key Laboratory of Animal Infectious Diseases, Ministry of Agriculture, Yangzhou University, China 5 Jiangsu Key Laboratory of Preventive Veterinary Medicine, Yangzhou University, China 6 Yangzhou Uni-Bio Pharmaceutical Co., Ltd, Yangzhou, 225000, Jiangsu, China. 7 Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611-0880, USA Abbreviations Salmonella enterica serotype Choleraesuis: S .Choleraesuis; Streptococcus suis: S. suis; Sao:Surface-anchored protein;SS2: S.suis serotype 2;RDAS:Regulated delayed attenuated strategies;LB: Luria broth;LD 50 : 50% lethal dose. 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Strains and plasmids Strain or plasmid Relevant characteristics or genotype Source or reference E. coli strains BL21 F - pT hsdSB (rB - mB - al dcm (DE3) Invitrogen χ 7213 thi-1 thr-1 leuB6 fhuA21 lacY1 glnV44 asdA4 recA1 RP4 2-Tc::Mu pir; Km r Gift from Dr. Curtiss, III Salmonella Choleraesuis C78-3 Wild type, virulent, CVCC79103 Lab stock C500 S. Choleraesuis vaccine strain attenuated by chemical mutation , CVCC79500 [40] rSC0005 Δ pmi-2426, C78-3 [9] rSC0008 Δ pmi-2426 Δ relA199::araC P BAD lacI TT This study rSC0009 Δ pmi-2426 Δ relA199::araC P BAD lacI TT Δ P fur88 ::TT araC P BAD fur This study rSC0011 Δ P crp527 ::TT araC P BAD crp Δ pmi-2426 Δ relA199::araC P BAD lacI TT Δ asdA33 [9] rSC0012 Δ pmi-2426 Δ relA199::araC P BAD lacI TT Δ P fur88 ::TT araC P BAD fur Δ asdA33 This study rSC0018 Δ asdA33 C500 C78-3 Δ P fur88 ::TT araC P BAD fur C78-3 C78-3 Δ P crp527 ::TT araC P BAD crp C78-3 Streptococcus suis serotype 2 Wild type, virulent, CVCC3928 Lab stock Plasmids pYA3493 Asd + ; pBR ori , P trc promoter , β -lactamase signal sequence-based periplasmic secretion plasmid [19] pS-SaoA pYA3493 with SaoA, P trc promoter [5] pET28a expression vector, T7 promoter; Km r Novagen Suicide vector pRE112 sacB mobRP4 R6K oriV oriT Cm r [50] pDMS197 tet r sacB mobRP4 R6K oriV oriT [50] pYA3832 Δ P crp527 ::TT araC P BAD crp , pRE112 [ 46 ] pYA3546 Δ pmi-2426 , pDMS197 [ 46 ] pS003 Δ relA199::araC P BAD lacI TT, pRE112 [9] pS005 Δ P fur88 ::TT araC P BAD fur This study pYA3736 Δ asdA33 , pRE112 [9] Table 2. Virulence of rSC0012(pS-SaoA) in 3-week-old BALB/c mice Strain Description LD 50 (CFU) Oral i.p C78-3 Wild type 9.5 × 10 2 2.8 × 10 9 6.6 × 10 6 **, ## rSC0012(pS-SaoA) Δ P fur88 ::TT araC P BAD fur Δ pmi-2426 Δ relA199 :: araC P BAD lacI TT Δ asdA33 in C78-3 >5.8 × 10 9** 2.1 × 10 7 **, ## rSC0011(pS-SaoA) Δ P crp527 ::TT araC P BAD crp Δ pmi-2426 Δ relA199 :: araC P BAD lacI TT Δ asdA33 in C78-3 >1.0 × 10 9** 5.4 × 10 5 ** ** , P <0.01, compared with C78-3; ## , P <0.01, compared with rSC0011(pS-SaoA) Table 3 Primer sequences Gene Sequence （ 5 ’ -3 ’） GAPDH Forward CTT AGC ACC CCT GGC CAA G Reverse GAT GTT CTG GAG AGC CCC G IL-1 β Forward GTG TCT TTC CCG TGG ACC TT Reverse AAT GGG AAC GTC ACA CAC CA IL-6 Forward GGC GGA TCG GAT GTT GTG AT Reverse GGA CCC CAG ACA ATC GGT TG TNF- α Forward ATG AGC ACA GAA AGC ATG A Reverse AAG AGG CTG AGA CAT AGG C IL-8 Forward CTG CAA GAG ACT TCC ATC CAG Reverse AGT GGT ATA GAC AGG TCT GTT GG Supplementary Files NC3RsARRIVEGuidelinesChecklist2020.01.21.doc Cite Share Download PDF Status: Published Journal Publication published 07 May, 2020 Read the published version in BMC Veterinary Research → Version 4 posted Editorial decision: Accept 21 Apr, 2020 Editor assigned by journal 05 Apr, 2020 Submission checks completed at journal 04 Apr, 2020 Editor invited by journal 04 Apr, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4396","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":470847,"identity":"bdfece8c-b805-45db-b388-21bcdf729322","order_by":1,"name":"Yu-an Li","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yu-an","middleName":"","lastName":"Li","suffix":""},{"id":470848,"identity":"5a0bc70d-a2f8-4c56-97d4-f63e69911e3b","order_by":2,"name":"Yunyun Chen New","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yunyun","middleName":"Chen","lastName":"New","suffix":""},{"id":470849,"identity":"ad098422-b8bf-48cf-bb59-b8c211adbaf0","order_by":3,"name":"Yuan zhao Du","email":"","orcid":"","institution":"State Key Laboratory of Genetically Engineered Veterinary Vaccines","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"zhao","lastName":"Du","suffix":""},{"id":470850,"identity":"2c470697-672c-4b82-b866-9f0ac26f7b46","order_by":4,"name":"Weiwei Guo","email":"","orcid":"","institution":"State Key Laboratory of Genetically Engieered Veterinary Vaccine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Guo","suffix":""},{"id":470851,"identity":"cfc4e263-e71a-454b-8881-81e7d8783926","order_by":5,"name":"Dianfeng Chu","email":"","orcid":"","institution":"State Key Laboratory of Genetically Engineered Veterinary Vaccine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Dianfeng","middleName":"","lastName":"Chu","suffix":""},{"id":470852,"identity":"0a568ccd-f1f7-4db1-b177-95e7ebc68f1e","order_by":6,"name":"Juan Fan","email":"","orcid":"","institution":"Yangzhou Uni-Bio Pharmaceutical Co.，Ltd","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Fan","suffix":""},{"id":470853,"identity":"404dc538-f97c-42cf-a931-08f79d9548f8","order_by":7,"name":"Xiaobo Wang","email":"","orcid":"","institution":"Yangzhou University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaobo","middleName":"","lastName":"Wang","suffix":""},{"id":470854,"identity":"9a38dbc6-7af6-4a22-8ebf-0327880d557c","order_by":8,"name":"Matthew Bellefleur","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Bellefleur","suffix":""},{"id":470855,"identity":"14273af9-7cc3-4999-a58e-a004129beb5b","order_by":9,"name":"Shifeng Wang","email":"","orcid":"","institution":"University of Florida","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shifeng","middleName":"","lastName":"Wang","suffix":""},{"id":470856,"identity":"7ed80300-1d49-4b97-b675-0221f8c54a76","order_by":10,"name":"Huoying Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACAwYGNoaECghHggQtZ0jWwthGihZzifRnDx7OO2xvcID54G0eBrs8glosZ+SYGyRuO8xscIAt2ZqHIbmYsMNu5LBJALWwGRzgMZPmYTiQ2EBYS/ozicQ5h3kMDvB/I1ZLgplEYsNhCaAtbERqOfPGTCLhWLqB5GE2Y8s5BslEaDme/kzyR421Pd/x5oc33lTYEdbCIJAAIpsZGJjBJhBUDwT8B0BkHTFKR8EoGAWjYKQCAIIBOfKqANMyAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-4128-329X","institution":"Yangzhou University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Huoying","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2019-08-25 13:52:29","currentVersionCode":4,"declarations":"","doi":"10.21203/rs.2.13690/v4","doiUrl":"https://doi.org/10.21203/rs.2.13690/v4","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12917-020-02340-4","type":"published","date":"2020-05-07T20:15:07+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":879218,"identity":"0ba93ebc-3451-496f-ba77-3a68b266ab76","added_by":"auto","created_at":"2020-04-10 14:16:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2234758,"visible":true,"origin":"","legend":"Diagram of chromosomal mutation and phenotypes of the S. Choleraesuis vaccine strain rSC0012. (A) Schematic map of ΔPfur::TT araC PBAD fur deletion - insertion mutation; (B) Regulated decreased synthesis of Fur (ΔPfur::TT araC PBAD fur) and regulated delayed synthesis of SaoA in rSC0012(pS-SaoA) with the ΔrelA::araC PBAD lacI TT mutation. The rSC0012(pS-SaoA) strain was grown in NB with arabinose (Lane 1) when OD600 reach 0.8 and then diluted at a 1:10 ratio into fresh NB without arabinose. The process continued for 4 times (Lane 2 - 5); each lane was loaded around 4.6 × 107CFU cells. Synthesis of Fur and SaoA were detected by western blot using corresponding antiserum. M: protein marker; (C) LPS profile of Δpmi mutation in rSC0012 in NB grown with or without 0.2% mannose. Lanes: 1, wild type C78-3; 2, C500; 3, rSC0012 with mannose; 4, rSC0012 without mannose; (D) Growth curves of rSC0012 with and without DAP, rSC0012(pYA3493) or rSC0012(pS-SaoA) in LB were measured with a spectrophotometer (OD600) at the indicated time intervals.","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4396/v4/Figure 1.png"},{"id":879219,"identity":"e0524a58-2965-4358-9f58-869b0c8d2b5f","added_by":"auto","created_at":"2020-04-10 14:16:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1108869,"visible":true,"origin":"","legend":"Synthesis of SaoA in Salmonella Choleraesuis vector rSC0011, rSC0012 and rSC0018. (A) Subcellular fractions of SaoA in rSC0011(pS-SaoA), rSC0012(pS-SaoA), and rSC0018(pS-SaoA) from cells grown in NB detected by western blot. The number showed relative densitometry in one of the three representative experiments. (B) Densitometry analysis of the SaoA protein in rSC0011(pS-SaoA), rSC0012(pS-SaoA), and rSC0018(pS-SaoA) by using Image J software (Image J2 PMID 26153368). The assay was repeated 3 times. *P \u003c 0.05, **P \u003c 0.01. #，P\u003c0.05, ##，P \u003c 0.01, for rSC0012(pS-SaoA) compared to rSC0011(pS-SaoA)","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4396/v4/Figure 2.png"},{"id":879220,"identity":"a4e4bfd5-1a33-45ed-bbc0-3fff11c50793","added_by":"auto","created_at":"2020-04-10 14:16:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12130197,"visible":true,"origin":"","legend":"Colonization of Salmonella Choleraesuis rSC0012 in BALB/c mice at diferent time points. Mice were orally inoculated with 1.0 ± 0.3 × 109 CFU of the indicated strains. The numbers of bacteria loads in the Peyer’s patches (A),spleen (B) and liver (C) of mice at 3 d, 7 d, 14 d, 21 d and 28 d after inoculation were plotted. Bars represent the arithmetic mean ± standard deviations from two separate experiments each with 5 mice per group. *, P\u003c0.05; **, P \u003c 0.01, for rSC0018 compared to rSC0012 or to rSC0011 with either pYA3493 or pS-SaoA; #, P\u003c0.05, ##, P \u003c 0.01, for rSC0011 compared to rSC0012 or to rSC0011 with either pYA3493 or pS-SaoA; $$, P \u003c 0.01, for C78-3 compared to rSC0011, rSC0012 and rSC0018 with either pYA3493 or pS-SaoA, as indicated. The data were collected from two independent experiments.","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4396/v4/Figure 3.png"},{"id":879221,"identity":"48ceb6c7-90f6-4760-9b03-2855c8c86577","added_by":"auto","created_at":"2020-04-10 14:16:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15549223,"visible":true,"origin":"","legend":"Antibody responses in ten mice. Serum IgG responses to SaoA (A), S. Choleraesuis OMPs (B) and vaginal wash IgA responses to SaoA (C) were measured by ELISA at weeks 3 and 5. Each triangle represents one mouse. Error bars represent variation between mice. Significant differences were indicated. *, P\u003c0.05; ** P\u003c 0.01, for Salmonella carrying pS-SaoA compared to each other; #, P\u003c0.05; ##, P\u003c 0.01, for the titers of antibody at 3 weeks after immunization were compared to those at 5 weeks after immunization. No immune responses were detected to antigen tested in mice immunized with PBS or in pre-immune sera from vaccinated mice (reciprocal titer \u003c1:50). The assay was performed in duplicate and repeated at least 3 times.","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4396/v4/Figure 4.png"},{"id":879222,"identity":"6b276eae-43aa-4b60-ba6c-3283cca9b72e","added_by":"auto","created_at":"2020-04-10 14:16:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":11535963,"visible":true,"origin":"","legend":"Cytokines levels in ten mice immunized with the S. Choleraesuis vaccines. IFN-γ (A) and IL-4 (B) in spleens 7 d after the booster dose were assayed with an EILSA kit. A PBS control were also included. (C) the ratio of IFN-γ to IL-4. The significant differences between groups of each strain was indicated. The assay was performed in duplicate and repeated at least 3 times. *, P\u003c0.05; ** P \u003c 0.01, for strains rSC0011, rSC0012, rSC0018 with either pYA3493 or pS-SaoA compared each other; #, P\u003c0.05; ##, P \u003c 0.01, for the strains rSC0011, rSC0012, rSC0018 with pS-SaoA compared to these strains with pYA3493 respectively at 7 d and 14 d post immunization.","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4396/v4/Figure 5.png"},{"id":879223,"identity":"c933e3e3-bd0f-4add-84db-ed7bd3cb6b1c","added_by":"auto","created_at":"2020-04-10 14:16:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":14427571,"visible":true,"origin":"","legend":"Induction of inflammatory cytokines at 6 h and 12 h post-infection in ten mice immunized with different Salmonella strains. Analysis of RNA transcript levels by qPCR showed that all mutant strains induced less inflammatory cytokine production than the wild-type strain C78-3 at both 6 h (A and C) and 12 h (B and D) post-infection. \u0026, P\u003c0.05, C78-3 ΔPfur88 TT araC PBAD fur compared to C78-3; #, P\u003c0.05, ##, P \u003c 0.01, rSC0012(pS-SaoA) compared to rSC0011(pS-SaoA); *, P\u003c0.05, **, P \u003c 0.01, strains with ΔPfur88 TT araC PBAD fur mutation compared to C78-3.","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4396/v4/Figure 6.png"},{"id":879224,"identity":"4dd7d713-42b6-452f-ac4a-dda69c9c0942","added_by":"auto","created_at":"2020-04-10 14:16:37","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":16019649,"visible":true,"origin":"","legend":"Protection in mice. Groups of 40 mice were orally immunized twice at 3 weeks intervals with indicated strains. Half of the mice were challenged orally with 50×LD50 of C78-3 and the other half were intraperitoneally injected with 20×LD50 of SS2 at 2 weeks after the 2nd immunization.","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4396/v4/Figure 7.png"},{"id":15666814,"identity":"d5316901-3141-4343-a4c1-4f7f524dae08","added_by":"auto","created_at":"2021-11-18 13:38:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1843500,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4396/v4/ad1c62c9-564d-4a7f-a2cf-a2a435a1420b.pdf"},{"id":879217,"identity":"1f759d14-795c-45ab-ae54-de5b2a989845","added_by":"auto","created_at":"2020-04-10 14:16:34","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":702464,"visible":true,"origin":"","legend":"","description":"","filename":"NC3RsARRIVEGuidelinesChecklist2020.01.21.doc","url":"https://assets-eu.researchsquare.com/files/rs-4396/v4/NC3Rs ARRIVE Guidelines Checklist 2020.01.21.doc"}],"financialInterests":"","formattedTitle":"\u003cp\u003eLive-attenuated \u003cem\u003eSalmonella enterica\u003c/em\u003e serotype Choleraesuis vaccine with regulated delayed \u003cem\u003efur\u003c/em\u003e mutation confer protection against \u003cem\u003eStreptococcus suis\u003c/em\u003e in mice \u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003e\u003cem\u003eStreptococcus\u003c/em\u003e\u003cem\u003esuis\u003c/em\u003e is a pandemic pathogen responsible for\u0026nbsp;a\u0026nbsp;wide range of invasive diseases such as pneumonia, meningitis and bacteraemia in both humans and pigs[\u003ca href=\"#_ENREF_1\"\u003e1\u003c/a\u003e,2]. \u003cem\u003eS. suis\u003c/em\u003e type 2 (SS2) is the most frequently and virulent isolated from both humans and pigs among all serotypes reported to date [1,3]. The surface-anchored protein (Sao) is a highly conserved membrane-anchored protein and proved to be a immunogenic vaccine candidate[4]. However, Sao formulated with Emulsigen-Plus\u0026reg; provides only partial protection to mice against SS2 infection [3]. In our previous study, a recombinant attenuated \u003cem\u003eSalmonella enterica\u003c/em\u003e serotype Choleraesuis vaccine strain rSC0016 carrying \u003cem\u003esaoA\u003c/em\u003e gene, provided full protection to mice against SS2 challenge[5].\u0026nbsp;From the above, an effective delivery system such as live \u003cem\u003eSalmonella enterica\u003c/em\u003e serotype Choleraesuis play a crucial role to the effectiveness of Sao.\u003c/p\u003e\n\u003cp\u003eThe use of intracellular\u003cem\u003e Salmonella enterica\u003c/em\u003e as a vehicle to deliver heterologous protective antigens against pathogens is an attractive strategy. Curtiss et al. developed the RDAS (Regulated Delayed Attenuated Strategies), which enable live \u003cem\u003esalmonella\u003c/em\u003e vaccine effectively colonize lymphoid tissues during the invasion stage because of its wild-type aggressiveness and then be full attenuated by silencing the virulence factor, while stimulate both strong cellular and humoral immunity in the immunized mice[6]. Several ways were used to implement this strategy (RDAS). One way is the reverse synthesis of lipopolysaccharide O-antigen by \u003cem\u003epmi\u003c/em\u003e mutation[7]. Another way is to replace the upstream regulatory and promoter sequences of virulence genes with a tightly regulated \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e activator\u0026ndash;promoter [8]. This strategy has been successfully used for \u003cem\u003eS\u003c/em\u003e. Typhimurium and \u003cem\u003eS\u003c/em\u003e. Typhi[6, 7, 8]. With this strategy, we construct a regulated delayed \u003cem\u003eS.\u003c/em\u003e Choleraesuis vaccine strain rSC0011 with \u0026Delta;P\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003ecrp\u003c/em\u003e and \u003cem\u003epmi\u003c/em\u003e mutations[9]. rSC0011 delivering \u003cem\u003eS. suis\u003c/em\u003e antigens were effective to induce protective immunity against SS2 in mice, but it occasionally caused enteritidis.\u003c/p\u003e\n\u003cp\u003eWe sought to improve our \u003cem\u003eS\u003c/em\u003e. Choleraesuis candidate vector vaccine by using alternative mutation or introducing new mutation to decreasing its potential to induce enteritidis and enhance immunogenicity. Fur is an important regulatory protein in \u003cem\u003eSalmonella\u003c/em\u003e. In the presence of iron, Fur acts as a repressor of iron-controlled genes and mounts an adaptive acid tolerance response [8]. Synthesis of Fur in a vaccine strain during growth confers acid tolerance and maintains iron homeostasis. A decrease of Fur synthesis in\u003cem\u003e Salmonella \u003c/em\u003eleads to acid sensitivity and iron acquisition [\u003ca href=\"#_ENREF_9\"\u003e9\u003c/a\u003e]. Curtiss et al. reported that a \u003cem\u003eS. \u003c/em\u003eTyphimurium strain with an arabinose regulated delayed\u003cem\u003e fur\u003c/em\u003e mutation is highly immunogenic [10]. In these consideration, an arabinose regulated delayed \u003cem\u003efur\u003c/em\u003e mutation (\u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e:: TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e) was introduced into a \u003cem\u003eS\u003c/em\u003e. Choleraesuis vaccine strain with multiple preexist mutations (\u0026Delta;\u003cem\u003epmi-2426 \u003c/em\u003e\u0026Delta;\u003cem\u003erelA199::araC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e\u0026nbsp;\u003cem\u003elacI\u003c/em\u003e TT \u0026Delta;\u003cem\u003easdA33\u003c/em\u003e) to generate strain rSC0012. A plasmid pS-SaoA[5], encoding \u003cem\u003esaoA \u003c/em\u003efrom \u003cem\u003eSS\u003c/em\u003e2, was transformed into this strain. We evaluated the virulence, immunogenicity and protection against challenge with virulent \u003cem\u003eSS\u003c/em\u003e2 or \u003cem\u003eS\u003c/em\u003e. Choleraesuis C78-3.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eConstruction and characterization of the \u003cem\u003eS.\u003c/em\u003e Choleraesuis vaccine strain rSC0012\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFur is a ferric uptake regulator that is involved not only in iron metabolism, uptake, and transport, but also invasion and survival of \u003cem\u003eS.\u003c/em\u003e Typhimurium in the hosts [11-13]. The absence of Fur attenuates \u003cem\u003eS.\u003c/em\u003e Typhimurium [6,14]. To improve the safety and increase the immunogenicity of \u003cem\u003eS.\u003c/em\u003e Choleraesuis vector, a new strain, rSC0012, was generated with an arabinose regulated \u003cem\u003efur\u003c/em\u003e, \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e:: TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e (Figure 1A).\u003c/p\u003e\n\u003cp\u003eThe phenotypes of the mutations \u0026Delta;P\u003csup\u003efur88\u003c/sup\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csup\u003eBAD\u003c/sup\u003e\u0026nbsp;\u003cem\u003efur\u003c/em\u003e and \u0026Delta;\u003cem\u003erelA\u003c/em\u003e::\u003cem\u003earaC\u003c/em\u003e P\u003csup\u003eBAD \u003c/sup\u003e\u003cem\u003elacI\u003c/em\u003e TT were confirmed by western blot analysis (Figure 1B). The level of Fur synthesis decreased with arabinose dilution (Figure 1B). The presence of mutation \u0026Delta;\u003cem\u003erelA\u003c/em\u003e::\u003cem\u003earaC\u003c/em\u003e P\u003csup\u003eBAD \u003c/sup\u003e\u003cem\u003elacI\u003c/em\u003e TT in rSC0012(pS-SaoA) were confirmed by the increased synthesis of SaoA (Figure 1B) due to the derepression of P\u003csub\u003etrc\u003c/sub\u003e promoter on plasmid in rSC0012, which resulted from reduced LacI production whose production was controlled by arabinose. The \u003cem\u003epmi\u003c/em\u003e gene encodes 6-phosphomannose isomerase that interconverts fructose-6-phosphate and mannose-6-phosphatein in\u003cem\u003e Salmonella\u003c/em\u003e [7]. Because mannose is required for O-antigen synthesis, the \u0026Delta;\u003cem\u003epmi\u003c/em\u003e mutation enables the strain rSC0012 to display a smooth LPS pattern in nutrient broth in the presence of mannose and a rough pattern in the absence of mannose (Figure 1C). The \u0026Delta;\u003cem\u003easdA\u003c/em\u003e mutation enables the strain rSC0012 to have an obligate requirement for DAP [18], which can be complemented with a vector harboring the \u003cem\u003easd\u003c/em\u003e gene then eliminates the need for antibiotic resistance genes for plasmid maintenance[19]. The growth rates of rSC0012 with DAP, rSC0012(pS-SaoA), and rSC0012(pYA3493) were similar (Figure 1D). rSC0012 could grow only with DAP (Figure 1D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntigen synthesis and plasmid stability in \u003cem\u003eS. Choleraesuis\u003c/em\u003e rSC0012\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStable maintenance of plasmids and the production of heterologous antigens are critical to ensure efficacy of recombinant live vaccines. The SaoA protein is a highly conserved surface protective antigen among \u003cem\u003eS.\u003c/em\u003e\u003cem\u003esuis\u003c/em\u003e serotypes [2, 5]. Using live attenuated \u003cem\u003eS. \u003c/em\u003eCholeraesuis vector delivering SaoA antigen from \u003cem\u003eS. suis\u003c/em\u003e will allow to develop a bivalent vaccine against both \u003cem\u003eS\u003c/em\u003e. Choleraesuis and \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esuis\u003c/em\u003e. The stabilities of pS-SaoA and pYA3493 in rSC0012 were evaluated by continuous culturing for 50 generations. The stabilities of both Asd\u003csup\u003e+\u003c/sup\u003e plasmids, pS-SaoA and pYA3493, were 100% in rSC0012 (data not shown). All rSC0012 colonies examined (100 clones/generation) by endonuclease digestion possessed the Asd+ plasmid pS-SaoA or pYA3493. The 34-kDa SaoA protein was detected in cells obtained from both the first and 50th generations of rSC0012(pS-SaoA) (data not shown), indicating the stability of plasmid and stable synthesis of SaoA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of secreted SaoA in \u003cem\u003eS. \u003c/em\u003eCholeraesuis rSC0012 \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe production levels of SaoA in various subcellular fractions from pS-SaoA-carrying strains, rSC0011, rSC0012, and rSC0018, were determined (Figure 2A-B). rSC0018 is a derivative of C500 [5, 20], a licensed live \u003cem\u003eS. \u003c/em\u003eCholeraesuis vaccine attenuated by chemical methods in China, with an \u003cem\u003easdA \u003c/em\u003emutation. The results showed that the level of the SaoA protein produced in the cytoplasm by strain rSC0012(pS-SaoA) was significantly lower than strain rSC0011(pS-SaoA) (Figure 2B; #, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05), but significantly higher in supernatant when compared with rSC0011(pS-SaoA) and rSC0018(pS-SaoA) (Figure 2B; **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). The SaoA protein produced in the periplasm fraction of rSC0012(pS-SaoA) was significantly higher than strain rSC0018(pS-SaoA)(Figure 2A-B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLower virulence of \u003cem\u003eS.\u003c/em\u003e Choleraesuis rSC0012 \u003cem\u003ein vivo\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the virulence of strains with mutations \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e:: TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e or \u0026Delta;P\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003ecrp\u003c/em\u003e, the LD\u003csub\u003e50\u003c/sub\u003e values of rSC0012 and rSC0011 were tested in 3-week-old BALB/c mice, which represents young mice. rSC0018 was used as an attenuation control. All strains carried the expression plasmid pS-SaoA. The results revealed that the LD\u003csub\u003e50\u003c/sub\u003es of rSC0011(pS-SaoA), rSC0012(pS-SaoA), and rSC0018(pS-SaoA) were at least 10\u003csup\u003e9\u003c/sup\u003e CFU by oral inoculation; whereas, the LD\u003csub\u003e50 \u003c/sub\u003eof wild-type C78-3 was 9.5\u0026times;10\u003csup\u003e2\u003c/sup\u003e CFU (Table 2). Following intraperitoneal infection, the LD\u003csub\u003e50\u003c/sub\u003e of rSC0012(pS-SaoA) was 38.89-fold higher than that of rSC0011(pS-SaoA) and 3.2-fold higher than that of rSC0018(pS-SaoA) in mice. These results indicated that the virulence of rSC0012(pS-SaoA) harboring \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e:: TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e was significantly lower than that of rSC0011(pS-SaoA) harboring\u0026Delta;P\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003ecrp\u003c/em\u003e by intraperitoneal route in young mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue d\u003c/strong\u003e\u003cstrong\u003eistribution of \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eS. \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eCholeraesuis \u003c/strong\u003e\u003cstrong\u003estrains in \u003c/strong\u003e\u003cstrong\u003eBALB/c \u003c/strong\u003e\u003cstrong\u003emice \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFur and Crp are important regulatory proteins in \u003cem\u003eSalmonella\u003c/em\u003e. Inactivation of the \u003cem\u003efur \u003c/em\u003eand \u003cem\u003ecrp\u003c/em\u003e genes, attenuates the organism [6,21]. To quantitatively the colonization of the \u003cem\u003eS. \u003c/em\u003eCholeraesuis strains containing regulated delayed \u003cem\u003efur\u003c/em\u003e or \u003cem\u003ecrp\u003c/em\u003e mutations, 3-week-old BALB/c mice were orally inoculated with rSC0011(pYA3493), rSC0012(pYA3493), rSC0018(pYA3493), rSC0011(pS-SaoA), rSC0012(pS-SaoA), rSC0018(pS-SaoA), or wild-type C78-3 (Figure 3A\u0026ndash;F). The mice inoculated with wild-type \u003cem\u003eS. \u003c/em\u003eCholeraesuis C78-3 died 3\u0026ndash;5 days after inoculation, whereas the mice infected orally with vaccine strains rSC0011, rSC0012, rSC0018 containing either plasmid pS-SaoA or pYA3493 survived. The bacteria titers of wild-type strain C78-3 in Peyer\u0026rsquo;s patches, spleen, and liver were significantly higher than those of vaccine strains rSC0011, rSC0012, rSC0018 containing either plasmid pS-SaoA or pYA3493 at 3 days after inoculation (Figure 3A\u0026ndash;C). The titers of bacteria in Peyer\u0026rsquo;s patches were similar for strains rSC0012(pYA3493), rSC0012(pS-SaoA), rSC0011(pYA3493), and rSC0011(pS-SaoA) at 3-21 days post-inoculation(Figure 3A). At 3d, 7 d, 14 d, 21 d and 28 d, the numbers of rSC0012(pS-SaoA) were significantly higher than those of attenuated vaccine strains rSC0018(pS-SaoA). Same for two control vector, the numbers of rSC0012(pYA3493) were significantly higher than those of attenuated vaccine strains rSC0018(p YA3493) and there was no significant difference with same strain with expression or control vector. respectively (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; Fig. 3A). These results indicating that the colonization abilities of strains rSC0012(pS-SaoA) and rSC0012(pYA3493) in Peyer\u0026rsquo;s patches were higher than that of rSC0018 with either pYA3493 or pS-SaoA.\u003c/p\u003e\n\u003cp\u003eIn spleen, the titers of rSC0012(pYA3493) and rSC0012(pS-SaoA) were similar to those of vaccine strains rSC0018(pYA3493) and rSC0018(pS-SaoA) at 7, 14, 21, 28 days after inoculation (Figure 3B). The titers of strains rSC0011(pYA3493) and rSC0011(pS-SaoA) were significantly higher than those of rSC0012(pYA3493), rSC0012(pS-SaoA), rSC0018(pYA3493), and rSC0018(pS-SaoA), at 3, 7, 14, and 21 days, respectively (Figure 3B).\u003c/p\u003e\n\u003cp\u003eIn liver, the titers of rSC0012(pYA3493) and rSC0012(pS-SaoA) were similar to those of vaccine strains rSC0018(pYA3493) and rSC0018(pS-SaoA) at 3, 14, 21, 28 days after inoculation (Figure 3C). At 3d, 7 d, 14 d, 21 d and 28 d post-inoculation, the titers of rSC0012(pS-SaoA) were significantly lower than those of rSC0011(pS-SaoA), same for two control vector, the numbers of rSC0012(pYA3493) were significantly lower than those of strains rSC0011(pYA3493).respectively(\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; Fig. 3C), whereas the strain rSC0012(pYA3493) was the fastest to be cleared in liver (Figure 3C). These results indicated that the \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e:: TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e mutation impaired the colonization in the liver of \u003cem\u003eS\u003c/em\u003e. Choleraesuis vaccine strains. In a summary, the \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e:: TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e mutation reduced the colonization ability of \u003cem\u003eS.\u003c/em\u003e Choleraesuis vaccine strains in mice spleen and liver but not in the Peyer\u0026rsquo;s patches.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e\u003cstrong\u003entibody responses in mice immunized with \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eS. \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eCholeraesuis \u003c/strong\u003e\u003cstrong\u003estrains \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll of the mice immunized with strains containing pS-SaoA developed anti-SaoA antibodies (Figure 4A). rSC0012(pS-SaoA) induced significantly higher anti-SaoA IgG titer than did rSC0011(pS-SaoA) 3 weeks after the immunization in 3-week-old mice (Figure 4A; *, P\u0026lt;0.05). Although the anti-SaoA IgG titer of rSC0012(pS-SaoA) were slightly higher than that of rSC0011(pS-SaoA) 5 weeks after immunization, they were not significantly different (Figures 4A). Compared to mice immunized with rSC0018(pS-SaoA), higher serum IgG titers against SaoA were detected in mice immunized with both rSC0011(pS-SaoA) and rSC0012(pS-SaoA) at 3 weeks and 5 weeks postimmunization (Figure 4A, *, P\u0026lt;0.05, **, P\u0026lt;0.01). After boosting, higher titers of anti-SaoA IgG were observed in mice immunized with all three strains containing pS-SaoA (Figure 4A, #, P\u0026lt;0.05). All three attenuated recombinant Salmonella strains induced significant OMP titers after the first immunization in mice (Figure 4B). A significant boosting of serum antibody responses to OMPs was observed after the second immunization, (Figure 4B; #, P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003eMucosal IgA anti-SaoA responses were detected at week 3 in mice immunized with all three attenuated strains containing pS-SaoA. rSC0018(pS-SaoA) induced lower titers of anti-SaoA IgA than rSC0012(pS-SaoA) or rSC0011(pS-SaoA) did in mice (Figure 4C, *, P\u0026lt;0.05, **, P\u0026lt;0.01). Anti-SaoA IgA levels detected in the rSC0012(pS-SaoA) immunized group were significantly higher than those induced in the rSC0011(pS-SaoA) immunized group at 3 and 5 weeks after the immunization (Figure 4C, *, P\u0026lt;0.05, **, P\u0026lt;0.01). These results indicated that strain rSC0012(pS-SaoA) harboring \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e:: TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e elicited a stronger immune response than strain rSC0011(pS-SaoA) harboring \u0026Delta;P\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003ecrp\u003c/em\u003e did, especially elicit a stronger mucosal immune response.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIFN-\u0026gamma; or IL-4 production induced by \u003cem\u003eS. \u003c/em\u003eCholeraesuis \u003c/strong\u003e\u003cstrong\u003estrains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further evaluate the effect of the \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e:: TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e and \u0026Delta;P\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003ecrp\u003c/em\u003e mutations in a strain with multiple preexist mutations on Th1/Th2 immune responses, the levels of IFN-\u0026gamma; and IL-4 in the spleen tissues of mice 7 days and 14 days after booster immunization were measured. The results showed that the titers of IFN-\u0026gamma; and IL-4 induced in mice immunized with rSC0012, and rSC0011 with either pYA3493 or pS-SaoA were significantly higher than those induced in mice inoculated by strain rSC0018 with either pYA3493 or pS-SaoA at both 7 and 14 days after booster (Figure 5A, B; *, \u003cem\u003eP\u0026lt;\u003c/em\u003e0.01,**, \u003cem\u003eP\u0026lt;\u003c/em\u003e0.05). Although rSC0011(pS-SaoA) induced a slightly higher level of IFN-\u0026gamma; in mice than rSC0012(pS-SaoA) did, no significant difference was observed in 3-week-old mice at both 7 and 14 days after booster (Figure 5A-C). However, rSC0011(pYA3493) elicited a higher level of IFN-\u0026gamma; than rSC0012(pYA3493) did at 7 days after booster. The titers of IL-4 induced by rSC0012 with either pS-SaoA or pYA3493 in spleen tissues were significantly higher than those induced by rSC0011 with either pS-SaoA or pYA3493 in 3-week-old mice (Figure 5B; *, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01) at 7 and 14 days after booster. Of note, rSC0012(pS-SaoA) induced a dominant Th2 immune response (IFN-\u0026gamma; \u0026lt; IL-4, IFN/IL-4\u0026lt;1) in 3-week-old mice (Figure 5C); whereas, rSC0011(pS-SaoA) elicited a moderately dominant Th1 immune response (IFN-\u0026gamma; \u0026gt; IL-4, IFN/IL-4\u0026gt;1) in 3-week-old mice (Figure 5C, *, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **, \u003cem\u003eP\u0026lt;\u003c/em\u003e0.01). These results indicated the isogenic strain with two different mutations, \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e or \u0026Delta;P\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003ecrp\u003c/em\u003e, affected different branches of immune responses. In additional, the titers of IFN-\u0026gamma; and IL-4 induced by the three attenuated strains harboring pS-SaoA in immunized mice were higher than those induced by the same strains harboring the emptor vector pYA3493 (Figure 5A\u0026ndash;C; #, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ##, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01), suggesting that the protein, SaoA, might augment the immune responses in mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInduction of inflammation in mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe inflammatory properties of intestinal tissue were investigated in mice after immuned vaccine strains and wild type strain,C78-3.The expression of cytokine genes, TNF\u0026alpha;, IL-1\u0026beta;, IL-6 and IL-8, were assessed by quantitative real -time PCR in gut tissue samples at 6 hours and 12 hours postinfection. All strains with mutation \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e showed significantly lower transcription levels of cytokine genes IL-1\u0026beta;, IL-6, IL-8 and TNF\u0026alpha; than the wild -type strain, C78-3 at both 6 h and 12 h postinfection (Fig 6A-D,*, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, **, \u003cem\u003eP\u0026lt;\u003c/em\u003e0.01). The C78-3 with mutation \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e induced significantly lower transcription levels of cytokine genes IL-1\u0026beta;, IL-6,IL-8 and TNF\u0026alpha; than the same strain with \u0026Delta;P\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003ecrp\u003c/em\u003e mutation at both 6 hours and 12 hours postinfection (Fig 6 C and D, \u0026amp;, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, \u0026amp;\u0026amp;, \u003cem\u003eP\u0026lt;\u003c/em\u003e0.01). At 6 hours postinfection, strain rSC0012( pS-SaoA) showed significantly lower transcription levels of cytokine genes IL-1\u0026beta; , IL-6, IL-8 and TNF\u0026alpha; than strain rSC0011( pS-SaoA) (Fig 6A) , a similar trend was seen with IL-1\u0026beta; , IL-6, IL-8 and TNF\u0026alpha; at 12 h, although the differences were not significant for IL-6 and TNF\u0026alpha; (Fig 6B,#, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, ##, \u003cem\u003eP\u0026lt;\u003c/em\u003e0.01). These results suggest that the addition of the \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e mutations could decreased the inflammatory potential of strains rSC0012( pS-SaoA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComparison of the protective immunity induced by \u003cem\u003eS. \u003c/em\u003eCholeraesuis \u003c/strong\u003e\u003cstrong\u003estrains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the protective immunity conferred by rSC0012(pS-SaoA), the mice in each immunized group were challenged orally with 50\u0026times;LD\u003csub\u003e50\u003c/sub\u003e of the virulent \u003cem\u003eS\u003c/em\u003e. Choleraesuis C78-3 strain or 20\u0026times;LD\u003csub\u003e50\u003c/sub\u003e of the virulent SS2 strain at 14 days post-boost immunization. After challenge with C78-3, the results revealed 100% protection in mice immunized with either strain rSC0011(pS-SaoA) or strain rSC0012(pS-SaoA), suggesting full protection. Mice immunized with the rSC0018(pS-SaoA) strains resulted in 20% survival. In contrast, all the mice in the PBS group succumbed to the challenge after 4 days. There were no significant difference between the groups immunized with rSC0012(pS-SaoA) and rSC0011(pS-SaoA), though both displayed significantly higher levels of protection than the group immunized with rSC0018(pS-SaoA) (Figure 7A). After the SS2 challenge, immunization with the rSC0011(pS-SaoA), rSC0012(pS-SaoA) and rSC0018(pS-SaoA) strains resulted in 80% survival, 95% survival and 16.7% survival with the lethal SS2 challenge, respectively. In contrast, all the mice in the PBS group succumbed to the challenge after 2 days.\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe ultimate goal of an engineered live vaccine strain relies on achieving the proper balance between immunogenicity and attenuation [22,23]. Achieving that goal will restrict unacceptable reactogenicity to avoid over-excitation of inflammatory responses, but sufficient metabolic activity should be maintained to enable the live vaccine to reach deep lymphatic tissues and induce protective immunity[24]. The development of bacterial vaccine relies on a combination of defined mutations[23,25,26]. In order to enhancing the immunogenicity of vaccine strains or to disarm them, multiple independent defined mutations were introduced into \u003cem\u003eSalmonella\u003c/em\u003e to generate new recombinant attenuated vaccine strains[6,25,23,27]. By coalescent proper mutations, vaccine strains can be befittingly designed to avoid unacceptable reactogenicity and enhanced immunogenicity [21]. Our previous live attenuated\u003cem\u003e S\u003c/em\u003e. Choleraesuis vaccine vector rSC0011 occasionally caused enteritidis in mice[9]. One of the ways to address this problem is by incorporating a \u003cem\u003esopB\u003c/em\u003e mutation[5,25]. In this paper, another way to improve the \u003cem\u003eS\u003c/em\u003e. Choleraesuis vector was reported.\u003c/p\u003e\n\u003cp\u003eFur is an important regulatory proteins of \u003cem\u003eSalmonella\u003c/em\u003e, which has been implicated in the acid tolerance response since \u003cem\u003efur\u003c/em\u003e mutants are acid sensitive and cause altered expression of several acid shock proteins [28,29]. A \u003cem\u003eS. \u003c/em\u003eTyphimurium strain with an arabinose regulated \u003cem\u003efur\u003c/em\u003e mutation is adequately attenuated and highly immunogenic [6,30]. However, \u003cem\u003eS\u003c/em\u003e. Typhimurium belong to group B, while \u003cem\u003eS\u003c/em\u003e. Choleraesuis group C. Whether the \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur \u003c/em\u003emutation that results in the proper balance between attenuation and immunogenicity of \u003cem\u003eS\u003c/em\u003e. Typhimurium is also appropriate for attenuated \u003cem\u003eS\u003c/em\u003e. Choleraesuis vaccine has not been reported previously.\u003c/p\u003e\n\u003cp\u003eWe corroborated the \u003cem\u003eS\u003c/em\u003e. Choleraesuis strain rSC0012 (\u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur \u003c/em\u003e\u0026Delta;\u003cem\u003epmi \u003c/em\u003e\u0026Delta;\u003cem\u003erelA\u003c/em\u003e::\u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003elacI \u003c/em\u003eTT \u0026Delta;\u003cem\u003easdA\u003c/em\u003e) displayed a regulated decrease of Fur production in the absence of arabinose. In a previous publication, \u003cem\u003eS\u003c/em\u003e. Typhimurium strain with a single \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e mutation has shown higher virulent than \u003cem\u003eS\u003c/em\u003e. Typhimurium with a single \u0026Delta;P\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003ecrp \u003c/em\u003emutation by an oral immunization. Unlike report from Curtiss et al in \u003cem\u003eS\u003c/em\u003e. Typhimurium [6], our studies did not showed that the \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e mutation has higher virulent than \u0026Delta;P\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003ecrp \u003c/em\u003emutation in\u003cem\u003e S\u003c/em\u003e. Choleraesuis. In fact,the LD\u003csub\u003e50 \u003c/sub\u003eof rSC0012(pS-SaoA) was 38-fold higher than rSC0011(pS-SaoA) for 3-weeks-old mice with intraperitoneal injection. This result suggest that the \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur \u003c/em\u003emutation does different attenuation with \u003cem\u003eS\u003c/em\u003e. Choleraesuis or \u003cem\u003eS\u003c/em\u003e. Typhimurium,which may due to its complex role as a transcriptional activator of virulence in different strains [31].\u003c/p\u003e\n\u003cp\u003eThe \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e mutation can modify the iron regulated outer membrane protein, then strain with \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e induced lower inflammation than the isogenic strain with \u0026Delta;P\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003ecrp \u003c/em\u003emutation or wild type strain in mice. Theses results suggest that the \u003cem\u003eS\u003c/em\u003e. Choleraesuis with \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT\u003cem\u003e araC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003efur \u003c/em\u003emutation exhibits a lower tendency to trigger excessive inflammation while attenuated sufficiently.\u003c/p\u003e\n\u003cp\u003eThrough the oral vaccination route, a vaccine strain will endure the challenges of acid, bile, and antimicrobial peptides existing in the gastrointestinal tract. Once inside Peyer\u0026rsquo;s patch, it will face macrophagocytes and T cells during the process transferring to deep lymphatic tissues[33]. Fur is essential to \u003cem\u003eSalmonella\u003c/em\u003e for accessorial an adaptive acid tolerance response[28]. Although both rSC0011 and rSC0012 had similar levels of Peyer\u0026rsquo;s patch colonization by oral immunization,we observed that the rSC0012 was cleared more rapidly than the rSC0011 in deeper lymphatic tissues, spleen, and liver of mice. These results suggest that strain rSC0012 was more attenuated than rSC0011 in deep lymphoid tissue, which maybe due to an increase in acid sensitivity cause by loss of Fur makes the cell more susceptible to killing by macrophages.\u003c/p\u003e\n\u003cp\u003eBoth rSC0012 and rSC0011 induced higher levels of IgA, IgG, IFN-\u0026gamma;, and IL4 responses with great colonization compared to strain rSC0018 with less colonization in mice.In general, the live \u003cem\u003eSalmonella\u003c/em\u003e vaccines with RDAS display superior colonization level in lymphoid tissues during the invasion stage, leading to enhanced protection by effectively colonizing lymphoid tissues[10,35]. However, there are do exist high levels of colonization but low immunogenicity. A strain with the regulated delayed \u003cem\u003erfc\u003c/em\u003e mutation exhibits superior colonization and yet does not stimulate higher heterologous protection than a \u003cem\u003e\u0026Delta;rfc\u003c/em\u003e strain without RDAS[36]. Thus, in addition to colonization level, other determining factors may exist to induce the enhanced protection achieved by regulated delayed attenuation. From the above, Selecting the proper mutation is critical for vaccine development with RDAS. This study confirmed above statement. Although the colonization of rSC0012(pS-SaoA) in mice was less than that of rSC0011(pS-SaoA), rSC0012(pS-SaoA) stimulated stronger serum IgG and mucosal IgA responses than the rSC0011(pS-SaoA). This phenomenon suggests that strain with regulated delayed \u003cem\u003efur\u003c/em\u003e mutation may stimulate stronger antibody response with fewer bacteria than strain with regulated delayed \u003cem\u003ecrp\u003c/em\u003e mutation.\u003c/p\u003e\n\u003cp\u003eBoth rSC0012 and rSC0011 aroused a Th1 cell-mediated response, as ostensived by the significant up-regulation of imprint Th1 cytokines, IFN-\u0026gamma;, the stimulator of Th1-type T cell immune response. It could partially be that the intracellular characteristics of \u003cem\u003eSalmonella enterica\u003c/em\u003e cause them to be detected on the surface of APC through MHC-I molecules. This result is consistent with previous studies with \u003cem\u003eS\u003c/em\u003e. Typhi [6]. Strain rSC0012 induced higher Th2 cell-mediated response than strain rSC0011, as aroused by the significant up-regulation of imprint Th2 cytokines, IL-4 and greater humoral responses than rSC0011 in mice. This phenomenon may be related to the secreting of heterologous antigen. The more heterologous antigens were secreted during infection, the more likely\u0026nbsp;to trigger an IL-4-dominant response[44]. We found a larger secretion of the SaoA from rSC0012, which could be presented by MHC-II molecules. An additional factor may be due to an increase in acid sensitivity cause by loss of Fur makes the rSC0012 more susceptible to killing by macrophages, thus weakening rSC0012's ability to survive in macrophages. The presence of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esuis\u003c/em\u003e-specific IgA serves to promptly deliver the antigen to Peyer\u0026rsquo;s patch dendritic cells or phagocytes and also promptly excite the adaptive immunity during secondary exposure [37]. Generally speaking, the more attenuated the vaccine strain is, the lower its immunogenicity is[21,27], while, the more attenuated rSC0012 strain induced significantly higher IgA and IgG antibody responses to SaoA than the rSC0011 at 3 weeks after the initial immunization in juvenile mice. This may be the result of a equilibrium between attenuation and the ability to participation immune components and activate a controlled over-inflammatory response by the finely crafted strain[23].\u003c/p\u003e\n\u003cp\u003eThe results presented herein highlight that strain rSC0012(pS-SaoA) with regulated delayed \u003cem\u003efur \u003c/em\u003emutation has retained vaccine efficacy and adequate immunogenicity whilst being safer than previous strain rSC0011(pS-SaoA). Furthermore, the inclusion of the \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT\u003cem\u003earaC\u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003efur \u003c/em\u003emutation may be able to decrease inflammation caused by live-attenuated \u003cem\u003eSalmonella\u003c/em\u003e\u003cem\u003eenterica\u003c/em\u003e serotype Choleraesuis vaccine, which has been an imperfection for other live-attenuated \u003cem\u003eSalmonella\u003c/em\u003e\u003cem\u003eenterica\u003c/em\u003e serotype Choleraesuis vaccine which transformed from wild type virulent strain. Both \u003cem\u003eS.\u003c/em\u003e Choleraesuis and \u003cem\u003eS. suis\u003c/em\u003e are major swine pathogens. Currently, there exist license live vaccines against \u003cem\u003eS. Choleraesuis \u003c/em\u003efor swine, including Entersol\u0026reg; \u003cem\u003eSalmonella\u003c/em\u003e T/C and SC-54 manufactured by Boehringer Ingelheim and Arugs SC/ST, respectively [38]. However, there is no licensed vaccine against \u003cem\u003eS. suis\u003c/em\u003e. Preventing the diseases caused by\u003cem\u003e SS\u003c/em\u003e2 in swine with a combined vaccine is a long-sought goal.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results have shown the strains rSC0012(pS-SaoA) with regulated delayed \u003cem\u003efur\u003c/em\u003e mutation could confer higher protection against challenges with lethal doses of\u003cem\u003e SS\u003c/em\u003e2 or \u003cem\u003eS\u003c/em\u003e. Choleraesuis C78-3. Thus, the use of attenuated \u003cem\u003eS.\u003c/em\u003e Choleraesuis to develop a vaccine against \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esuis\u003c/em\u003e will have the great potential to ease the burden of both pathogens. The regulated delayed \u003cem\u003efur\u003c/em\u003e mutation in the novel vaccine rSC0012 resulted in a well-safety, highly immunogenic, and effective vaccine in mice, this study has paved the way for testing in piglets. Our findings will aid the optimal of a \u003cem\u003eS\u003c/em\u003e. Choleraesuis vaccine vector capable of eliciting a suitable immune response against other pathogens.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree-week-old female BALB/c mice were purchased from Animal Center of Yangzhou University, and kept one week before inoculation. All animal experiments were authorized by the Jiangsu Administrative Committee for Laboratory Animals (permission number SYXK-SU-2007-0005) and accorded to the Jiangsu Laboratory Animal Welfare and Ethics guidelines of the Jiangsu Administrative Committee of Laboratory Animals. All surgery was performed under anesthesia intraperitoneally injected with sodium pentobarbital, 40 mg per kilogram mouse weight. All the animals were humanely euthanized after the study by inhalation of CO\u003csub\u003e2\u003c/sub\u003e,while injection with sodium pentobarbital, 40 mg per kilogram mouse weight, and all efforts were made to minimize suffering.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStrains, plasmids, and culture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe strains, plasmids, used in this study are described in Table 1. C500, an approved live \u003cem\u003eS. \u003c/em\u003eCholeraesuis vaccine strain attenuated by thallium compound in China, was used as an attenuation control [39]. The genetic characterization of this strain has been reported [40]. \u003cem\u003eS\u003c/em\u003e. suis serotype 2 (SS2, CVCC3928) and \u003cem\u003eS\u003c/em\u003e. Choleraesuis C78-3 (CVCC79103) were purchased from China Institute of Veterinary Drug Control.Plasmid pYA3493 is an Asd\u003csup\u003e+\u003c/sup\u003e vector with a P\u003csub\u003etrc\u003c/sub\u003e promoter. The \u003cem\u003easd\u003c/em\u003e gene from \u003cem\u003eSalmonella\u003c/em\u003e was used as a unique plasmid marker to be used in \u003cem\u003easd\u003c/em\u003e mutants to constitute a balanced-lethal system [18]. LB medium[5],Nutrient broth (NB) and MacConkey agar (Difco) were used for phenotype characterization.When required, media were supplemented with 2,6-diaminopimelic acid (DAP;50\u0026nbsp;\u0026micro;g/mL),chloramphenicol (Cm; 25\u0026micro;g/mL),L-arabinose (0.2% wt/vol), D-mannose (0.2% wt/vol)or sucrose (5% wt/vol). The empty plasmid vector pYA3493 and expression vector pS‑SaoA were described on previous studies[5]. The \u003cem\u003esaoA\u003c/em\u003e gene is under the control of the P\u003csub\u003etrc\u003c/sub\u003e promoter (Table 1). \u003cem\u003eS. \u003c/em\u003eCholeraesuis vaccine vector strain rSC0012 harboring plasmid pS-SaoA (expression vector) or pYA3493 (control vector) were grown in LB broth with both 0.2% arabinose and 0.2% mannose. Selenite broth was used for enrichment of \u003cem\u003eS. \u003c/em\u003eCholeraesuis from mice tissues. Strains were prepared as previously described [5,9,10,30]. Bacterial growth was monitored with a spectrophotometer at OD\u003csub\u003e600\u003c/sub\u003e and by direct plating for colony counts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of \u003cem\u003eS. \u003c/em\u003eCholeraesuis\u003c/strong\u003e\u003cstrong\u003emutant\u003c/strong\u003e\u003cstrong\u003e strains\u003c/strong\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFour mutations \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e, \u0026Delta;\u003cem\u003epmi\u003c/em\u003e, \u0026Delta;\u003cem\u003erelA\u003c/em\u003e::\u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u003cem\u003elacI \u003c/em\u003eTT, and \u0026Delta;\u003cem\u003easdA\u003c/em\u003e were introduced into \u003cem\u003eS. \u003c/em\u003eCholeraesuis C78-3 by conjugation with \u003cem\u003eE. coli\u003c/em\u003e \u0026chi;7213 harboring aforementioned suicide vectors as previously described [41]. The suicide vectors used are listed in Table 1 and Fig. 1A. To construct mutation \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e\u003cem\u003e, \u003c/em\u003ea 1,335-bp TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e cassette were used to replace the 239-bp promoter sequence of the \u003cem\u003efur\u003c/em\u003e gene to achieve arabinose-regulated Fur synthesis (Figure 1A). The \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003ecassette contains a transcription terminator (TT) sequence to prevent \u003cem\u003earaC\u003c/em\u003e transcription reading through adjoining genes. Plasmid for \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur\u003c/em\u003e were confirmed by DNA sequencing. All the primers used have been reported [26].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of \u003cem\u003eS. \u003c/em\u003eCholeraesuis mutations \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll mutations were confirmed by colony PCR using homologous primers [30]. The \u0026Delta;\u003cem\u003easdA\u003c/em\u003e mutation was verified by growth with or without DAP in LB broth [18,19].Lipopolysaccharide (LPS) profiles were examined by silver staining in 12% sodium dodecyl sulfate-polyacrylamide (SDS) gel for the \u0026Delta;\u003cem\u003epmi\u003c/em\u003e mutation [41,42]. The \u0026Delta;P\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur \u003c/em\u003edeletion-insertion mutation was verified by reduced production of Fur protein as arabinose concentrations decreased with the increased bacterial growth by western blot using anti-Fur antiserum[30]. The production of SaoA was verified by western blot using anti-SaoA antiserum, respectively [9,18,19,20,].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSalmonella\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e subcellular fractionation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the subcellular localization of synthesized SaoA in the live attenuated \u003cem\u003eS. \u003c/em\u003eCholeraesuis vaccine\u003cem\u003e, \u003c/em\u003ecultures were grown in NB to an OD\u003csub\u003e600\u003c/sub\u003e of 0.8 and centrifuged at 13,200 \u0026times; g for 5 min to collect supernatant and pellet. The culture supernatant was saved for later analysis of the bacterial secreted proteins. Periplasmic fractions were prepared using the lysozyme-osmotic shock method as previously described [43,44]. Equal volumes of supernatant,periplasmic, cytoplasmic samples were separated by SDS-PAGE. Proteins were then transferred to polyvinylidene fluoride membrane for western blot analysis using anti-SaoA antiserum [5]. The gel band were analyzed with ImageJ software (NIH) [45].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of SaoA and \u003cem\u003eSalmonella \u003c/em\u003eouter membrane proteins (SOMPs) \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHis-tagged SaoA fusion protein and \u003cem\u003eSalmonella\u003c/em\u003e outer membrane proteins (SOMPs) were prepared as previous studies[5].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of virulence in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree-week-old female BALB/c mice were obtained from Animal Center of Yangzhou University. Mice were fasting for 6 h before inoculation.Recombination \u003cem\u003eS. \u003c/em\u003eCholeraesuis vector strains were cultured in LB broth with D-0.2% mannose and 0.2% L-arabinose for 12 hours at 37\u0026deg;C as standing cultures. The cultures were diluted 1:50 in the same media and cultured at 37\u0026deg;C to an OD\u003csub\u003e600\u003c/sub\u003e of 0.9. Bacteria were collected by centrifugation at 13,200 \u0026times; g for 5 min at room temperature and resuspended in PBS to densities suitable for the inoculation. Serial dilutions of the \u003cem\u003eS. \u003c/em\u003eCholeraesuis strains were plated onto LB agar supplemented with 0.2% D-mannose and 0.2% L-arabinose to measure the actual densities. Groups of five mice were inoculated with different doses in 20 \u0026mu;l (oral immunization) or 100 \u0026mu;l (intraperitoneal immunization). The mice were observed for 4 weeks for death. The LD50s were calculated according the method of Reed and Muench [11].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDistribution of \u003cem\u003eSalmonella\u003c/em\u003e in BALB/c mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA colonization assay for recombination \u003cem\u003eS. \u003c/em\u003eCholeraesuis vector strains was performed as described previously[9, 11]. Three-week-old female BALB/c mice were divided into 7 groups with 25 mice in each group.Each mouse was orally inoculated with 1\u0026plusmn;0.2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU of \u003cem\u003eS. \u003c/em\u003eCholeraesuis strains.Peyer\u0026rsquo;s patches, spleen, and liver of the mice were collected on days 3, 7, 14, 21 and 28 post-inoculated. The densities of bacteria in the tissues were determined using the method reported in previous studies[5,9,11,47]. The assay was performed twice, and the data were similar and pooled for analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunization of mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBacteria were grown and collected as above. Serial dilutions of the \u003cem\u003eS\u003c/em\u003e. Choleraesuis vaccine strains were plated onto LB agar supplemented with 0.2% D-mannose and 0.2% L-arabinose to determine the actual dose. Three-week-old female BALB/c mice were orally inoculated with 1\u0026plusmn;0.2\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU of \u003cem\u003eS. \u003c/em\u003eCholeraesuis vaccine strains containing either pS-SaoA or pYA3493. Mice were boosted inoculated with the same dose of the same strain after three weeks. About 50 \u0026mu;L of whole blood was collected by tail vein three weeks after primary inoculation and two weeks after boosting. Serum was separated from the whole-blood samples and stored at \u0026minus;70\u0026deg;C. Vaginal-wash samples in mice were collected at the indicated time and stored at \u0026minus;70 \u0026deg;C[9,35,46,47]. This experiment was performed in triplicate with each group receiving a similar dose of the vaccine strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue collection after \u003cem\u003eSalmonella\u003c/em\u003e infection of mice \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree-week-old female BALB/c mice were divided into 5 groups with 10 mice in each group. Groups of mice were orally inoculated with 1\u0026plusmn;0.3\u0026times;10\u003csup\u003e9\u003c/sup\u003e CFU of \u003cem\u003eSalmonella\u003c/em\u003e strains. Spleen and intestinum tenue of the mice were collected at 6 h and 12 h postinfection. The tissues were frozen with liquid nitrogen and then transferred to -70\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-linked immunosorbent assay (ELISA) \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum IgG antibody production against \u003cem\u003eS. \u003c/em\u003eCholeraesuis outer membrane proteins (OMPs) and SS2 SaoA,and vaginal-wash IgA antibody production against SaoA in mice were evaluated by Enzyme-linked immunosorbent assay (ELISA)[5]. Cytokines in tissues were analyzed by sandwich ELISA using commercial kits (BD Biosciences) according to manufacturer\u0026rsquo;s instructions. The results from the two experiments were pooled for statistical analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real -time PCR (qPCR) for cytokines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor RNA isolation, gut tissues were homogenized and suspended in TRIzol\u0026reg; (Thermo Fisher Scientific,USA). Tubes were vortexed for 3 min to disrupt the tissues. Chloroform was added to TRIzol\u0026reg; -treated samples and the samples centrifuged at 13200\u0026times;g for 10 min. The aqueous phase was separated out, and the RNA precipitated using precooled isopropanol. For quantitative real-time PCR, 1 \u0026micro;g of RNA was then reverse transcribed to cDNA. The primers were designed using Primer Blast (NCBI net) and synthesized by TSINGKE Biological Technology Co., Ltd. The sequences of the primers are listed in Table 3.Each sample were amplified using 7500 Fast Real -Time PCR Instrument (ABI,US) using Fast SYBR Green Master Mix (Thermo -Fisher Scientific). The results were using internal reference GAPDH as\u0026nbsp;control\u0026nbsp;for\u0026nbsp;normalization, and the 2 ^\u003csup\u003e-\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method was used to estimate the relative expression level of the mRNAs of target genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChallenge with\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e S. suis \u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eserotype 2 (\u003cem\u003eSS\u003c/em\u003e2)and \u003cem\u003eS.\u003c/em\u003e Choleraesuis (C78-3) in mice \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty mice in each group were challenged with \u003cem\u003eSS\u003c/em\u003e2 by intraperitoneal (i.p.) injection with 2.4 \u0026times; 10\u003csup\u003e8 \u003c/sup\u003eCFU of \u003cem\u003eSS\u003c/em\u003e2 in 100 \u0026mu;l PBS at five weeks after primary immunization . The 50% lethal dose (LD\u003csub\u003e50\u003c/sub\u003e) of \u003cem\u003eSS\u003c/em\u003e2 in BALB/c mice was 1.2 \u0026times; 10\u003csup\u003e7 \u003c/sup\u003eCFU. Another twenty mice in each group were challenged orally with 4.8 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e CFU of C78-3 in 20 \u0026mu;l PBS. The LD\u003csub\u003e50\u003c/sub\u003e of C78-3 in 3-week-old BALB/c mice was 9.5 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e CFU. Challenged mice were monitored for death daily for 15 days [9,25,26,35].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analyses on ELISA were presented as the geometric means and standard deviations for all assays. A Mann-Whitney U Test ( GraphPad Software, Inc.) was applied to contrasting the distribution of the \u003cem\u003eS\u003c/em\u003e. Choleraesuis in tissues of mice. The Kaplan-Meier method (SPSS Software) was used for obtain the survival fractions following i.p. challenge of immunized mice. A \u003cem\u003eP \u003c/em\u003evalue of 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProcedures involving the care and use of animals were approved by the Jiangsu Administrative Committee for Laboratory Animals (permission number SYXK-SU-2007-0005) and complied with the Jiangsu Laboratory Animal Welfare and Ethics guidelines of the Jiangsu Administrative Committee of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study will be available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone of the authors of this manuscript have any competing or financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (31672516, 31172300, 30670079), supported by the Grant No. BE2016343 from Jiangsu province and the Priority Academic Program Development of Jiangsu Higher Education Institutions, supported by State Key Laboratory of Genetically Engineered Veterinary Vaccines (No.AGVSKL-ZD/ZY-201807). and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). The funding bodies have not been involved in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDesign of the study: YA-L, HY-S and SF-W. Performed experiments: YA-L, YZ-D, WW-G, J-F, YY-C. Wrote the manuscript: YA-L, DF-C, XB-W, MB, SF-W. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank to Roy Curtiss, III for kindly providing \u0026chi;7213, pRE112, pYA3493, and the antibody of Fur.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003e College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, Jiangsu, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2 \u003c/sup\u003eJiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e3\u003c/sup\u003e Yebio Bioengineering Co., Ltd of Qingdao, Qingdao, 266114, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e4\u003c/sup\u003e Key Laboratory of Animal Infectious Diseases, Ministry of Agriculture, Yangzhou University, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e5\u003c/sup\u003e Jiangsu Key Laboratory of Preventive Veterinary Medicine, Yangzhou University, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e6\u003c/sup\u003e Yangzhou Uni-Bio Pharmaceutical Co., Ltd, Yangzhou, 225000, Jiangsu, China.\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e7\u003c/sup\u003e Department of Infectious Diseases and Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611-0880, USA\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eSalmonella enterica\u003c/em\u003e serotype Choleraesuis:\u003cem\u003eS\u003c/em\u003e.Choleraesuis;\u003cem\u003eStreptococcus suis: \u003c/em\u003e\u003cem\u003eS. suis;\u003c/em\u003eSao:Surface-anchored protein;SS2:\u003cem\u003eS.suis\u003c/em\u003e serotype 2;RDAS:Regulated delayed attenuated strategies;LB: Luria broth;LD\u003csub\u003e50\u003c/sub\u003e: 50% lethal dose. Antigen-Presenting Cells: APC; Major Histocompatibility ComplexⅠ: MHCⅠ; T helper type 1: Th1; Phosphate buffer saline:PBS; Lipopolysaccharide:LPS;Sodium dodecyl sulfate-polyacrylamide: SDS.\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003e[1] Seele J, Hillermann L M, Beineke A, et al. The immunoglobulin M-degrading enzyme of \u003cem\u003eStreptococcus suis\u003c/em\u003e, IdeS suis, is a highly protective antigen against serotype 2[J]. Vaccine, 2015, 33(19):2207-2212.\u003c/p\u003e\n\u003cp\u003e[2] The protective protein Sao (surface antigen one) is not a critical virulence factor for \u003cem\u003eStreptococcus suis\u003c/em\u003e serotype 2[J]. Microbial Pathogenesis, 2014, 67-68:31-35.\u003c/p\u003e\n\u003cp\u003e[3] Li Y, Martinez G, Gottschalk M, et al. Identification of a Surface Protein of \u003cem\u003eStreptococcus suis\u003c/em\u003e and Evaluation of Its Immunogenic and Protective Capacity in Pigs[J]. 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J Bacteriol, 1983, 154.\u003c/p\u003e\n\u003cp\u003e[40] Witholt B, Boekhout M, Brock M, et al. An efficient and reproducible procedure for the formation of spheroplasts from variously grown \u003cem\u003eEscherichia\u003c/em\u003e coli[J]. Analytical Biochemistry, 1976, 74(1):160-170.\u003c/p\u003e\n\u003cp\u003e[41] Kang, H. Y . Immune Responses to Recombinant Pneumococcal PspA Antigen Delivered by Live Attenuated \u003cem\u003eSalmonella\u003c/em\u003e\u003cem\u003eenterica\u003c/em\u003e Serovar Typhimurium Vaccine[J]. Infection and Immunity, 2002, 70(4):1739-1749.\u003c/p\u003e\n\u003cp\u003e[42] Schindelin J, Rueden C T, Hiner M C, et al. The ImageJ ecosystem: An open platform for biomedical image analysis[J]. Molecular Reproduction and Development, 2015, 82(7-8):518-529.\u003c/p\u003e\n\u003cp\u003e[43] Rd C S . CHROMOSOMAL ABERRATIONS ASSOCIATED WITH MUTATIONS TO BACTERIOPHAGE RESISTANCE IN ESCHERICHIA COLI.[J]. Journal of Bacteriology, 1965, 89(1):28.\u003c/p\u003e\n\u003cp\u003e[44] Xinxin Z, Sheng L, Qinlong D, et al. Regulated delayed attenuation enhances the immunogenicity and protection provided by recombinant, \u003cem\u003eSalmonella\u003c/em\u003e, \u003cem\u003eenterica\u003c/em\u003e, serovar Typhimurium vaccines expressing serovar Choleraesuis O-polysaccharides[J]. Vaccine, 2018, 36(33):5010-5019.\u003c/p\u003e\n\u003cp\u003e[45] Wang S, Li Y, Scarpellini G, et al. \u003cem\u003eSalmonella\u003c/em\u003e Vaccine Vectors Displaying Delayed Antigen Synthesis In Vivo To Enhance Immunogenicity[J]. Infection and Immunity, 2010, 78(9):3969-3980.\u003c/p\u003e\n\u003cp\u003e[46] Kang H Y, Dozois C M, Tinge S A, et al. Transduction-Mediated Transfer of Unmarked Deletion and Point Mutations through Use of Counterselectable Suicide Vectors[J]. Journal of Bacteriology, 2002, 184(1):307-312.\u003c/p\u003e\n\u003cp\u003e[47] Pashine A, John B, Rath S, George A, Bal V. Th1 dominance in the immune response to live \u003cem\u003eSalmonella\u003c/em\u003e typhimurium requires bacterial invasiveness but not persistence[J]. Int Immunol. 1999 Apr;11(4):481-9.\u003c/p\u003e"},{"header":"Tables","content":"\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eTable 1. Strains and plasmids\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"margin-left: 5.4pt; border-collapse: collapse; border: none;\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eStrain or plasmid\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eRelevant characteristics or genotype\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eSource or reference\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 27.6pt;\"\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 27.6pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eE. coli \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003estrains\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 27.6pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 27.6pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eBL21\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eF\u003cspan style=\"position: relative; top: -5.5pt;\"\u003e- \u003c/span\u003e\u003cem\u003epT hsdSB\u003c/em\u003e(rB\u003cspan style=\"position: relative; top: -5.5pt;\"\u003e- \u003c/span\u003emB\u003cspan style=\"position: relative; top: -5.5pt;\"\u003e-\u003c/span\u003e\u003cem\u003eal dcm \u003c/em\u003e(DE3)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eInvitrogen\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026chi;\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e7213\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003ethi-1 thr-1 leuB6 fhuA21 lacY1 glnV44 asdA4 recA1 RP4 2-Tc::Mu pir; Km\u003csup\u003er\u003c/sup\u003e\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eGift from Dr. Curtiss, III\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eSalmonella\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e Choleraesuis\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eC78-3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eWild type, virulent, \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eCVCC79103\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eLab stock\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eC500\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; background: white;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eS. \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eCholeraesuis vaccine strain attenuated by chemical mutation , CVCC79500\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[40]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: top;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003erSC0005\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epmi-2426, \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eC78-3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[9]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003erSC0008\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epmi-2426 \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003erelA199::araC\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e P\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003elacI\u003c/em\u003e TT\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eThis study\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003erSC0009\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"margin: 0in; margin-bottom: .0001pt; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003epmi-2426 \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003erelA199::araC\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e P\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003elacI\u003c/em\u003e TT\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eP\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003efur88\u003c/span\u003e\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003efur \u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eThis study\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003erSC0011\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"margin: 0in; margin-bottom: .0001pt; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eP\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003ecrp527\u003c/span\u003e\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003ecrp\u003c/em\u003e\u0026nbsp;\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003epmi-2426\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003erelA199::araC\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e P\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003elacI\u003c/em\u003e TT \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003easdA33\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[9]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003erSC0012\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"margin: 0in; margin-bottom: .0001pt; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003epmi-2426 \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003erelA199::araC\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e P\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003elacI\u003c/em\u003e TT\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eP\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003efur88\u003c/span\u003e\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003efur \u003c/em\u003e\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003easdA33\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eThis study\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003erSC0018\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003easdA33\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eC500\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eC78-3 \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eP\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003efur88\u003c/span\u003e\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003efur\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eC78-3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eC78-3 \u003c/span\u003e\u003cspan style=\"font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eP\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003ecrp527\u003c/span\u003e\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003ecrp\u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eC78-3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eStreptococcus suis \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eserotype 2\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eWild type, virulent, CVCC3928\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eLab stock\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003ePlasmids\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epYA3493\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; text-autospace: none;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eAsd\u003csup\u003e+\u003c/sup\u003e; pBR \u003cem\u003eori\u003c/em\u003e , \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eP\u003csub\u003etrc\u003c/sub\u003e promoter\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e, \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026beta;\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e-lactamase signal sequence-based periplasmic secretion plasmid\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[19]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epS-SaoA\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epYA3493 with SaoA, P\u003csub\u003etrc\u003c/sub\u003e promoter\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[5]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epET28a\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eexpression vector, T7 promoter; Km\u003csup\u003e\u003cspan style=\"position: relative; top: -5.5pt;\"\u003er\u003c/span\u003e\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eNovagen\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eSuicide vector\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epRE112\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003esacB mobRP4 R6K oriV oriT Cm\u003csup\u003e\u003cspan style=\"position: relative; top: -5.5pt;\"\u003er\u003c/span\u003e\u003c/sup\u003e\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[50]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epDMS197\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003etet\u003csup\u003er\u003c/sup\u003e sacB mobRP4 R6K oriV oriT\u003c/span\u003e\u003c/em\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[50]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epYA3832\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eP\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003ecrp527\u003c/span\u003e\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003ecrp\u003c/em\u003e, pRE112\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[\u003ca href=\"#_ENREF_25\"\u003e\u003cspan style=\"color: black; text-decoration: none; text-underline: none;\"\u003e46\u003c/span\u003e\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epYA3546\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epmi-2426\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e,\u003cem\u003e\u0026nbsp;\u003c/em\u003epDMS197\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[\u003ca href=\"#_ENREF_25\"\u003e\u003cspan style=\"color: black; text-decoration: none; text-underline: none;\"\u003e46\u003c/span\u003e\u003c/a\u003e]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epS003\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003erelA199::araC \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eP\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003elacI\u003c/em\u003e TT, pRE112\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[9]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epS005\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eP\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003efur88\u003c/span\u003e\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003e\u003cspan style=\"position: relative; top: 4.5pt;\"\u003eBAD\u003c/span\u003e\u003c/sub\u003e\u0026nbsp;\u003cem\u003efur \u003c/em\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eThis study\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 83.4pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"111\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003epYA3736\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 238.8pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"318\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%; vertical-align: middle;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: SimSun; color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003easdA33\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e, pRE112\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 87.2pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt;\" width=\"116\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e[9]\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eTable 2. Virulence of rSC0012(pS-SaoA) in 3-week-old BALB/c mice\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"margin-left: 34.8pt; border-collapse: collapse; border: none;\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 30.6pt;\"\u003e\n\u003ctd style=\"width: 84.95pt; border: none; border-top: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 30.6pt;\" width=\"113\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eStrain\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 288.95pt; border: none; border-top: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 30.6pt;\" width=\"385\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eDescription\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 255.05pt; border: none; border-top: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 30.6pt;\" colspan=\"2\" width=\"340\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eLD\u003csub\u003e50\u003c/sub\u003e (CFU)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 30.6pt;\"\u003e\n\u003ctd style=\"width: 84.95pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 30.6pt;\" width=\"113\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 288.95pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 30.6pt;\" width=\"385\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 127.45pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 30.6pt;\" width=\"170\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eOral\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 127.6pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 30.6pt;\" width=\"170\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003ei.p\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 30.6pt;\"\u003e\n\u003ctd style=\"width: 84.95pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 30.6pt;\" width=\"113\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eC78-3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 288.95pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 30.6pt;\" width=\"385\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eWild type\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 127.45pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 30.6pt;\" width=\"170\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e9.5\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026times;\u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e10\u003csup\u003e2\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 127.6pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 30.6pt;\" width=\"170\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026lt;10\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 60.3pt;\"\u003e\n\u003ctd style=\"width: 84.95pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 60.3pt;\" width=\"113\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003erSC0018(pS-SaoA)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 288.95pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 60.3pt;\" width=\"385\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003easdA33 \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003ein\u003cem\u003e\u0026nbsp;\u003c/em\u003ea live attenuated \u003cem\u003eS. \u003c/em\u003eCholeraesuis vaccine strain C500 \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 127.45pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 60.3pt;\" width=\"170\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026gt;2.8\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026times;\u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e10\u003csup\u003e9\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 127.6pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 60.3pt;\" width=\"170\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"color: black;\"\u003e6.6\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026times;\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e10\u003c/span\u003e\u003csup\u003e\u003cspan style=\"color: black; position: relative; top: -3.5pt;\"\u003e6\u003c/span\u003e\u003c/sup\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;**, ##\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 68.55pt;\"\u003e\n\u003ctd style=\"width: 84.95pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 68.55pt;\" width=\"113\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003erSC0012(pS-SaoA)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 288.95pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 68.55pt;\" width=\"385\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eP\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u0026nbsp;\u003cem\u003efur \u003c/em\u003e\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003epmi-2426 \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003erelA199\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e::\u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u0026nbsp;\u003cem\u003elacI \u003c/em\u003eTT\u0026nbsp; \u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003easdA33\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e in C78-3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 127.45pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 68.55pt;\" width=\"170\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026gt;5.8\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026times;\u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e10\u003csup\u003e9**\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 127.6pt; border: none; padding: 0in 5.4pt 0in 5.4pt; height: 68.55pt;\" width=\"170\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"color: black;\"\u003e2.1\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026times;\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e10\u003c/span\u003e\u003csup\u003e\u003cspan style=\"color: black; position: relative; top: -3.5pt;\"\u003e7\u003c/span\u003e\u003c/sup\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;**, ##\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 60.3pt;\"\u003e\n\u003ctd style=\"width: 84.95pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 60.3pt;\" width=\"113\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003erSC0011(pS-SaoA)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 288.95pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 60.3pt;\" width=\"385\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003eP\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u0026nbsp;\u003cem\u003ecrp \u003c/em\u003e\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003epmi-2426 \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003erelA199\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e::\u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e\u0026nbsp;\u003cem\u003elacI \u003c/em\u003eTT \u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026Delta;\u003c/span\u003e\u003cem\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003easdA33 \u003c/span\u003e\u003c/em\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003ein C78-3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 127.45pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 60.3pt;\" width=\"170\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026gt;1.0\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026times;\u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003e10\u003csup\u003e9**\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 127.6pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 5.4pt 0in 5.4pt; height: 60.3pt;\" width=\"170\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%;\"\u003e\u003cspan style=\"color: black;\"\u003e5.4\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e\u0026times;\u003c/span\u003e\u003cspan style=\"color: black;\"\u003e10\u003c/span\u003e\u003csup\u003e\u003cspan style=\"color: black; position: relative; top: -3.5pt;\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003cspan style=\"color: black;\"\u003e\u0026nbsp;**\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black; position: relative; top: -3.5pt;\"\u003e**\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e, \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, \u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003ecompared with \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eC78-3; \u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black; position: relative; top: -3.5pt;\"\u003e##\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e,\u003cem\u003e P\u003c/em\u003e\u0026lt;0.01, \u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black;\"\u003ecompared with \u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003erSC0011(pS-SaoA)\u003c/span\u003e\u003cspan style=\"font-family: 'Times New Roman',serif; color: black; position: relative; top: -3.5pt;\"\u003e \u003csup\u003e\u0026nbsp;\u0026nbsp;\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eTable 3 Primer sequences \u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"margin-left: 5.4pt; border-collapse: collapse;\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 31.55pt;\"\u003e\n\u003ctd style=\"width: 101.2pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: .15pt .15pt 0in .15pt; height: 31.55pt;\" width=\"135\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eGene\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: .7pt .65pt 0in .65pt; height: 31.55pt;\" width=\"123\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003cem\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003e\u003c/span\u003e\u003c/strong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp; \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 359.65pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: .7pt .65pt 0in .65pt; height: 31.55pt;\" width=\"480\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cstrong\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eSequence\u003c/span\u003e\u003c/strong\u003e \u003cspan style=\"font-size: 12.0pt; line-height: 200%; color: black;\"\u003e（\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e5\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; color: black;\"\u003e\u0026rsquo;\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e-3\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; color: black;\"\u003e\u0026rsquo;）\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 29.75pt;\"\u003e\n\u003ctd style=\"width: 101.2pt; border: none; padding: .15pt .15pt 0in .15pt; height: 29.75pt;\" width=\"135\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eGAPDH\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; border: none; padding: .7pt .65pt 0in .65pt; height: 29.75pt;\" width=\"123\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eForward \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 359.65pt; border: none; padding: .7pt .65pt 0in .65pt; height: 29.75pt;\" width=\"480\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eCTT AGC ACC CCT GGC CAA G\u0026nbsp; \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 31.55pt;\"\u003e\n\u003ctd style=\"width: 101.2pt; padding: .15pt .15pt 0in .15pt; height: 31.55pt;\" width=\"135\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; padding: .7pt .65pt 0in .65pt; height: 31.55pt;\" width=\"123\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eReverse \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 359.65pt; padding: .7pt .65pt 0in .65pt; height: 31.55pt;\" width=\"480\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eGAT GTT CTG GAG AGC CCC G\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 31.55pt;\"\u003e\n\u003ctd style=\"width: 101.2pt; padding: .15pt .15pt 0in .15pt; height: 31.55pt;\" width=\"135\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eIL-1\u003c/span\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; color: black;\"\u003e\u0026beta;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; padding: .7pt .65pt 0in .65pt; height: 31.55pt;\" width=\"123\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eForward \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 359.65pt; padding: .7pt .65pt 0in .65pt; height: 31.55pt;\" width=\"480\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eGTG TCT TTC CCG TGG ACC TT\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 31.55pt;\"\u003e\n\u003ctd style=\"width: 101.2pt; padding: .15pt .15pt 0in .15pt; height: 31.55pt;\" width=\"135\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; padding: .7pt .65pt 0in .65pt; height: 31.55pt;\" width=\"123\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eReverse \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 359.65pt; padding: .7pt .65pt 0in .65pt; height: 31.55pt;\" width=\"480\"\u003e\n\u003cp style=\"text-align: left; line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; line-height: 200%; font-family: 'Times New Roman',serif; color: black;\"\u003eAAT GGG AAC GTC ACA CAC CA\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 31.55pt;\"\u003e\n\u003ctd style=\"width: 101.2pt; padding: .15pt .15pt 0in .15pt; height: 31.55pt;\" width=\"135\"\u003e\n\u003cp style=\"line-height: 200%;\"\u003e\u003cspan style=\"font-size: 12.0pt; 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Choleraesuis) vaccine vector could be used to deliver heterologous antigens to prevent and control pig diseases. We have previously shown that a live-attenuated \u003cem\u003eS\u003c/em\u003e. Choleraesuis vaccine candidate strain rSC0011 (ΔP\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ecrp \u003c/em\u003eΔ\u003cem\u003epmi-2426 \u003c/em\u003eΔ\u003cem\u003erelA199\u003c/em\u003e::\u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003elacI \u003c/em\u003eTT Δ\u003cem\u003easdA33\u003c/em\u003e, Δ, deletion, TT, terminator) delivering SaoA, a conserved surface protein in most of \u003cem\u003eS\u003c/em\u003e. \u003cem\u003esuis\u003c/em\u003e serotypes, provided excellent protection against \u003cem\u003eS. suis\u003c/em\u003e challenge, but occasionally lead to morbidity (enteritidis) in vaccinated mice (approximately 1 in every 10 mice). Thus, alternated attenuation method was sought to reduce the reactogenicity of strain rSC0011. Herein\u003cu\u003e, \u003c/u\u003ewe described another recombinant attenuated \u003cem\u003eS. \u003c/em\u003eCholeraesuis vector, rSC0012 (ΔP\u003csub\u003efur88\u003c/sub\u003e:: TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD \u003c/sub\u003e\u003cem\u003efur \u003c/em\u003eΔ\u003cem\u003epmi-2426 \u003c/em\u003eΔ\u003cem\u003erelA199\u003c/em\u003e:: \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003elacI \u003c/em\u003eTT Δ\u003cem\u003easdA33\u003c/em\u003e) with regulated delayed \u003cem\u003efur\u003c/em\u003e mutation to avoid inducing disease symptoms while exhibiting a high degree of immunogenicity. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe strain rSC0012 strain with the ΔP\u003csub\u003efur88\u003c/sub\u003e::TT \u003cem\u003earaC \u003c/em\u003eP\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003efur\u003c/em\u003e mutation induced less production of inflammatory cytokines than strain rSC0011 with the ΔP\u003csub\u003ecrp527\u003c/sub\u003e::TT \u003cem\u003earaC\u003c/em\u003e P\u003csub\u003eBAD\u003c/sub\u003e \u003cem\u003ecrp\u003c/em\u003e mutation in mice. When delivering the same pS-SaoA plasmid, the intraperitoneal LD\u003csub\u003e50\u003c/sub\u003e of rSC0012 was 18.2 times higher than that of rSC0011 in 3-week-old BALB/C mice. rSC0012 with either pS-SaoA or pYA3493 was cleared from spleen and liver tissues 7 days earlier than rSC0011 with same vectors after oral inoculation. The strain rSC0012 synthesizing SaoA induced high titers of anti-SaoA antibodies in both systemic (IgG in serum) and mucosal (IgA in vaginal washes) sites, as well as increased level of IL-4, the facilitator of Th2-type T cell immune response in mice. The recombinant vaccine rSC0012(pS-SaoA) conferred high percentage of protection against \u003cem\u003eS. suis\u003c/em\u003e or \u003cem\u003eS\u003c/em\u003e. Choleraesuis challenge in BALB/C mice.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e The live-attenuated \u003cem\u003eSalmonella enterica\u003c/em\u003e serotype Choleraesuis vaccine rSC0012(pS-SaoA) with regulated delayed \u003cem\u003efur\u003c/em\u003e mutation provides a foundation for the development of a safe and effective vaccine against \u003cem\u003eS\u003c/em\u003e. Choleraesuis and \u003cem\u003eS. suis\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Live-attenuated Salmonella enterica serotype Choleraesuis vaccine with regulated delayed fur mutation confer protection against Streptococcus suis in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":4,"date":"2020-04-10 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