Construction and Evaluation of an Efficient Live Attenuated Salmonella Choleraesuis Vaccine and Its Ability as a Vaccine Carrier to Deliver Heterologous Antigens

The gram-negative facultative intracellular pathogen Salmonella enterica serotype Choleraesuis, also known as S. Choleraesuis, is a major financial loss for the pig business. C500 is a vaccine strain that has been used for preventing S. Choleraesuis infection in pigs for many years in China. Although it possessed good immunogenicity and protection efficacy, it still showed severe side effects. The truncation of the key gene rpoS in C500 was believed to take the major responsibility for its attenuation. To achieve a good balance between attenuation and immunogenicity, rpoS was restored to an active state, and other essential virulent genes of crp, fur, phoP, and aroA were evaluated for their effects of deletion on safety and immunogenicity. Animal experiments demonstrated that C5001 (C500 rpoS+ Δcrp10) and C5002 (C500 rpoS+ Δfur9) showed an excellent ability to induce an immune response. To further decrease the endotoxic activity, the combination mutations of ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9 were introduced into the mutant strains to generate 1′-dephosphorylated lipid A. Animal experiments showed that SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9) induced higher levels of IgG and secreted IgA antibodies and provided a higher protection rate than SC1 (C500 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9) and SC2 (C500 rpoS+ Δcrp10 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9). We also evaluated the ability of SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9) as a vaccine carrier to deliver heterologous protein antigens and polysaccharide antigens. The results indicated that SC3 (C500 rpoS+ Δfur9 ΔpagL7 ΔpagP81:: Plpp lpxE ΔlpxR9) showed an excellent ability to deliver heterologous antigens and induce the host to produce high levels of antibodies. Together, these results indicate that we constructed a safe and efficient attenuated strain of the S. Choleraesuis vaccine, which demonstrated strong resistance to infection with wild-type S. Choleraesuis and can be employed as a universal vector for the delivery of recombinant antigens.


Introduction
More than 2000 serotypes of Salmonella enterica have been found, many of which are capable of infecting individuals as well as domestic animals with various kinds of diseases.Salmonella enterica serovar Typhi (S.Typhi) is the pathogen that causes typhoid fever, a systemic sickness that affects humans.Conversely, non-typhoidal Salmonella (NTS), such as Salmonella enterica serovar Choleraesuis (S.Choleraesuis), is one of the primary pathogens causing paratyphoid fever in piglets, often leading to many deaths and huge Vaccines 2024, 12, 249 2 of 19 economic losses for the pig industry due to simultaneous or secondary infection with other pathogens [1].It is also essential for public health that humans commonly contact S. Choleraesuis by eating contaminated poultry, eggs, pork, and beef [2].The most common clinical symptom of infection in humans is acute gastroenteritis.Young children and older people with low immunity are prone to the further development of bacteremia [3].Domestic animals have been routinely treated with antibiotics to prevent S. Choleraesuis infection.However, the multi-drug-resistant strains of S. Choleraesuis are increasing yearly, becoming a severe global problem [4].Therefore, developing vaccines for humans and animals is an attractive approach for controlling S. Choleraesuis infections.
Over the years, several vaccines have been developed specifically for pigs to prevent the infection of S. Choleraesuis [5][6][7][8][9].In China, the C500 vaccine was a live attenuated S. Choleraesuis vaccine derived from the C78-3 strain by a chemical approach and licensed for over 50 years to prevent piglet paratyphoid [10].However, C500 still exhibits a relatively high level of virulence because some pigs experienced severe side-effect reactions after the injected vaccination of C500, showing symptoms of vomiting, shivering, an elevated body temperature, and even death in some cases.Therefore, a safer and more effective vaccine against S. Choleraesuis is required.C500 can be continued to attenuate and reduce its virulence, but this process would affect the immunogenicity and alleviate protection efficacy [11].Hence, continued attenuation of C500 is not a good option for developing the vaccine.We must explore strategies to develop new vaccines that balance safety and immunogenicity.
There are several advantages of live oral attenuated vaccines over other forms of vaccination.Live oral attenuated vaccines induce local immune responses at the mucosal surface and systemic immune responses in the host body.They are easy to manufacture, suitable for large-scale vaccination in pig farms, and do not generate hazardous waste such as needles and syringes [12].An ideal oral attenuated Salmonella vaccine must overcome the unfavorable environment of low nutrients, high acidity, and bile in the stomach and intestine by oral immunization and colonizing the relevant tissues to induce local mucosal humoral immunity in the host [13].Virulence genes of Salmonella have been systematically studied, such as rpoS, crp, fur, phoPQ, and aroA.RpoS mediates the expression of multiple genes in response to environmental stress (including nutrient deficiency, heat, oxidation, and osmotic shock).It has been reported that RpoS enhances the ability of bacteria to overcome innate host defenses, including stomach acidity and the production of reactive oxygen radicals by immune cells [14,15].Furthermore, it has been shown that RpoS is essential for the persistence of bacteria in lymphoid organs such as Peyer's patch (gutassociated lymphoid tissue [GALT]), spleen, and liver, and thus in the initial stages of infection of the host [16][17][18].This led us to hypothesize that the presence of functional RpoS in attenuated S. Choleraesuis would enhance the viability of the strains in mice because of their ability to overcome the gastrointestinal environment in the host and enter the mucosaassociated lymphoid tissue.The crp gene encodes the cAMP receptor protein, which plays a crucial role in regulating glucose and amino acid metabolism, as well as controlling the production of pili and flagella [19].It has been reported that a Salmonella mutant missing the crp gene had reduced virulence in mice and demonstrated effective protection against wild-type Salmonella infection following immunization [20].Fur is a global regulator, which plays an essential role in iron uptake and acid resistance [21][22][23], and S. Typhimurium, with the deletion of the fur gene, can confer cross-protection against multiple Salmonella in mice [24].PhoP is a transcriptional activator that controls the expression of genes responsible for virulence and intracellular survival of Salmonella within macrophages [25].The phoP mutant is capable of eliciting a protective immune response in mice [26,27].The aroA gene is involved in the production of aromatic amino acids, which are rare in the host.Consequently, the removal of the aroA gene leads to a Salmonella phenotype characterized by a lack of essential nutrients [28].Attenuated Salmonella carrying the aroA mutant also protected mice against a fatal dose of infection of wild-type Salmonella [29].
Lipopolysaccharides (LPS) are present on the outer membranes of nearly all gramnegative bacteria and consist of lipid A, core oligosaccharide, and O-antigen polysaccharide [30,31].Lipid A is essential for activating TLR4-related pathways and Caspase 4/5/11-dependent pathways [32].With long-term evolution, Salmonella improves its ability to survive in different environments by modifying the lipid acyl chains and decorating phosphate groups.Previous studies indicated that 3-O-deacylated 4 -monophosphoryl lipid A (3D-MPL) can still induce dendritic cell maturation and Th1-biased immune responses while showing low endotoxicity as a vaccine adjuvant [33].Our previous study showed that LpxE selectively removes the 1-phosphate group of lipid A in Salmonella, resulting in a product that closely resembles monophosphoryl lipid A (MPL) [31].In addition, we demonstrated that the presence of the lpxE gene on the chromosome did not lead to a reduction in the ability of attenuated Salmonella with additional gene deletions to deliver heterologous antigens [31].Therefore, to improve the safety and maintain immunogenicity, we introduced the lpxE gene into the genome of attenuated S. Choleraesuis.
In the present study, we developed a novel low endotoxin activity recombinant attenuated vaccine SC3 (C500 rpoS + ∆fur9 ∆pagL7 ∆pagP81:: P lpp lpxE ∆lpxR9) based on the vaccine strain C500 of S. Choleraesuis.We evaluated its attributes in vitro and in vivo by quantifiably comparing them to C500 to show attenuation and safety.We also assessed the ability of SC3 (C500 rpoS + ∆fur9 ∆pagL7 ∆pagP81:: P lpp lpxE ∆lpxR9) as a vector to deliver heterologous antigens (GDH from S. suis and O-antigen polysaccharide of E. coli O9) for inducing immune responses in mice.

Construction of Plasmids and Mutant Strain
The allelic exchange method was utilized to introduce gene mutations in S. Choleraesuis, employing pYA4278 as described in the previous study [36].The transformation of S. Choleraesuis or E. coli was carried out using a technique known as electroporation.To select the transformants, LB agar plates supplemented with suitable antibiotics were employed.Specifically, the selection for Asd+ plasmids was performed on LB agar plates.A comprehensive list of the primers employed in this investigation can be found in Supplementary Table S1.To restore the rpoS gene, the C3545 genome was used as the template for cloning.We employed two sets of primers, namely, rpoS-F/rpoS-R, to amplify a DNA fragment of approximately 1656 bp containing the entire rpoS gene open-reading frame (ORF), the region upstream and the region downstream of the rpoS gene from the C3545 genome.The PCR products were purified by agarose gel (A620014, Sangon Biotech, China) and ligated to pYA4278 using Gibson Assembly Master Mix (E55510, New England Biolabs Biological Reagent Co., Ltd., MA, USA) to generate the suicide plasmid pSW001.Then the rpoS gene was introduced into C500 via allelic exchange by conjugation with the E. coli strain χ7213 [40] harboring the suicide plasmid of pSW001, to generate the strain C5000 (C500 rpoS + ).Subsequently, the genes of crp, fur, phop, and aroA were introduced into C5000 (C500 rpoS + ), respectively.In short, to delete the crp gene from the C5000 (C500 rpoS + ) genome, a 253 bp upstream DNA region and a 253 bp downstream DNA region were precisely amplified using PrimeSTAR Max DNA Polymerase (R045A, Takara Biotech Biological Reagent Co., Ltd., Kyoto, Japan) from the C500 genome by applying primers crp-1F/crp-1R and crp-2F/crp-2R, respectively.These two DNA segments were then fused via primer crp-1F and crp-2R with overlap PCR.The fused PCR products were purified by agarose gel (A620014, Sangon Biotech Biological Reagent Co., Ltd., Shanghai, China) and cloned into pYA4278 utilizing Gibson Assembly Master Mix to construct a new suicide plasmid (pSW002).The crp gene was introduced into C5000 (C500 rpoS + ) via allelic exchange by conjugation with the E. coli strain χ7213 [40] containing the suicide plasmid of pSW002, resulting in the crp-deficient strain C5001 (C500 rpoS + ∆crp10).The same approach was used to delete the fur, phop, and aroA genes to generate the plasmids pSW003, pSW004, and pW005 and then generate the corresponding deficient strains C5002 (C500 rpoS + ∆fur9), C5003 (C500 rpoS + ∆phoP11), and C5004 (C500 rpoS + ∆aroA12), respectively.

Phenotypic Determination of Bacteria
Bacterial strain phenotypes were identified in vitro, and each experiment was conducted not less than twice.The growth of Salmonella mutants was assessed in LB broth or LB broth containing 6% NaCl.Briefly, a single bacterial strain clone was selected, placed into LB broth, and cultured for the entire night at 37 • C and 180 rpm.The following day, the overnight culture was incubated under the same conditions after being diluted 100 times into the corresponding fresh medium.The bacterial solution's optical density value at 600 nm (OD 600 ) was determined every hour.Finally, the recorded results were summarized, and the growth curves of all mutant strains were plotted via the Graph-Pad Prism 8.0 software.As previously described, outer membrane proteins (OMPs) [38] and LPS [42,43] were isolated from S. Choleraesuis.The OMPs underwent electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and were subsequently treated with Coomassie brilliant blue.The bacterial strains of S. Choleraesuis went through silver staining to establish their LPS profile following the method outlined by Hitchcock and Brown [44].The swimming motility of the bacteria was evaluated by employing LB plates that were solidified using 0.3% agar (wt/vol), as previously delineated [45].In short, 0.3% agar LB plates were inoculated with 5 µL of bacterial suspension (roughly 8 × 10 6 CFU/mL) and then incubated for 8 h at 37 • C in an incubator.A ruler was used to determine the swimming diameter.

Animals
Female BALB/c mice (18-19 g) and New Zealand white rabbits were obtained from Chengdu Dashuo Laboratory Animal Technology Co., Ltd., Chengdu, China.To protect the welfare of the animals, care was given to them, and the tests were carried out in compliance with the recommendations made in the "Guide for the Care and Use of Laboratory Animals".The animal house's temperature, humidity, and ventilation were regularly checked to guarantee ideal climatic conditions.The mice received appropriate care and were managed by competent and trained personnel.Every attempt was carried out to mitigate animal suffering while the course of the experiments was being conducted.Following their arrival, a seven-day acclimation period was provided for all animals prior to the commencement of experiments.

Daily Observation of Mice
Daily observations were made on mice that received vaccinations.Monitoring was conducted for local reactions and any adverse effects, including abnormalities, a decline in overall health, and a decrease in food consumption.Additionally, evidence of unkempt fur, restlessness, diarrhea, morbidity, and mortality was recorded.

Immunity of Mice
Mice were immunized by oral administration.Briefly, 5 mL of appropriate fresh media was used to stationary culture a solitary bacterial strain clone overnight in a 37 • C incubator.On the following day, the cultures were diluted 1:100 within a 100 mL identical medium and then incubated at 37 • C until the OD 600 reached 0.85 (approximately 1 × 10 9 CFU/mL).The 100 mL suspension of bacterial strain was gathered through centrifugation at 5000 rpm under ambient temperature conditions and resuspended in 2 mL of buffered saline with gelatin (BSG), at which point the bacterial concentration was ap-proximately 1 × 10 9 CFU/20 µL.The mouse cage was removed from food and water for a duration of 6 h, and the animals were orally given 20 µL of BSG, including 10 9 CFU of the corresponding mutants.At 5 weeks, mice received a subsequent dose of the identical strain, matching the previous dosage.

Localization of S. Choleraesuis in Organs
To assess the bacterial load in organs after the mice were orally given 20 µL of BSG containing 1 × 10 9 CFU mutant strains, at specific time intervals, three mice from each group were humanely euthanized.The researchers gathered Peyer's patches (PP) from various locations on the surface of the small intestine.The bacterial load within the PP indicated the average bacterial load in the combined PP from every mouse.Samples from the spleen and liver were obtained and weighed individually.To obtain a homogenized sample, each sample was added to 1 mL of phosphate buffer saline (PBS).Subsequently, various dilutions of the samples ranging from 10 −1 to 10 −3 (depending on the specific tissue) were applied onto MacConkey agar and LB agar to measure the total amount of live bacteria present.MacConkey agar indicator plates are designed to exclude interference from E. coli in tissue homogenates.The bacterial loading was calculated based on the bacteria on the indicator plates.If there were no colonies observed on the LB plate after culturing the homogenizing 0.1 mL of the organ, the selenite cysteine broth (100212, Sigma-Aldrich Biological Reagent Co., Ltd., WI, USA) was used to inoculate the remaining PBS solution from each tissue sample the following day.Samples that showed positive results after being enriched in selenite cysteine broth at 37 • C overnight were noted to have a CFU count of less than 10 per gram.

Determination of Virulence of Mutants in Mice
To measure the 50% lethal dose (LD 50 ), C500 or C5000 (C500 rpoS + ) strains were grown to an OD 600 of about 0.85 (~10 9 CFU/mL) in 100 mL of LB broth at 37 • C.After centrifuging the strains at 5000 rpm under room temperature, the strains were suspended in 2 mL of BSG (~10 9 CFU/20 µL) and then adjusted at densities suitable for immunization.Mice were randomly divided into eight groups and orally infected with C500 or C5000 (C500 rpoS + ) at doses of 7 × 10 9 CFU/20 µL, 3.5 × 10 9 CFU/20 µL, 1.75 × 10 9 CFU/20 µL, and 7 × 10 8 CFU/20 µL, respectively.Every eight were infected with the same dose.Before being inoculated, all mice were fasted for 6 h.Mice were monitored daily for evidence of unkempt fur, restlessness, diarrhea, and morbidity, and mortality was recorded for 25 days after the challenge.The lethal dose 50 was computed using probit analysis on the SPSS 26.0 software [46].

Tissue Damage Caused by S. Choleraesuis
To investigate the spleen tissue damage caused by the mutants after the mice were orally given 20 µL of BSG containing 1 × 10 9 CFU mutant strains, we employed Hematoxylin and eosin (H&E) staining for histopathological analysis.On the sixth day following oral inoculation, two mice in each group were humanely euthanized and the spleen was taken to determine the scope of the organ damage brought by the mutants.The standard procedure was followed for fixing and processing tissues for H&E staining.We observed the morphological aspects of tissues to identify signs of inflammation, including infiltration of macrophages, accumulation, distortion, and abnormal red pulp areas in the spleen.

Ileal Loop Experiment on Rabbits
After an overnight fast, New Zealand white rabbits were given Zoletil to anesthetize through an ear vein.Ligate the ileum at 1 cm intervals to create loops 3-5 cm long.Separate loops were injected with mutant strains at a titer of 1 × 10 9 CFU, with a 1 mL volume.As a control, one of the loops was injected with LB broth.Closure of the abdominal musculature involved the use of 3-0 chromic gut sutures, followed by closure of the skin using 3-0 Ethilon sutures.Throughout the experiment, the rabbits were maintained at 37 • C using a thermal blanket.After 8 h, the rabbits were killed by intravenous air injection.The intestinal segment was ligated by laparotomy, and the intestinal tissue was fixed with 10% formalin for pathological section analysis by H&E staining.

Survival Assay
The protection rates of the attenuated S. Choleraesuis were assessed at 9 weeks post-immunization by the oral challenge of mice with 10 9 (~10,000 × LD 50 ) wild-type S. Choleraesuis C3545 in 20 µL BSG.C3545 is a clinical isolate from a pig with an LD 50 of 10 5 CFU/20 µL [41,47].Mice were monitored daily for evidence of unkempt fur, restlessness, diarrhea, and morbidity, and mortality was recorded for 25 days after the challenge.

Quantitative Analysis of Specific Antibody Levels by ELISA
Serum was collected by puncturing the mandibular vein to obtain blood, followed by centrifugation at 3500 rpm.To analyze the secretory IgA (S-IgA), the vaginal tract of each mouse was washed with 60 µL PBS, and the wash fluids were pooled together.The enzyme-linked immunosorbent assay (ELISA) was utilized to quantify the concentration of serum and vaginal wash antibodies against Salmonella LPS and OMPs induced by attenuated Salmonella strains, following established protocols [44,45].Briefly, solutions containing 200 ng per well of S. Choleraesuis-derived OMP or LPS were suspended in 100 µL of coating buffer composed of sodium carbonate-bicarbonate (pH 9.6).These solutions were then utilized to coat 96-well plates for an extended duration of time at a temperature of 4 • C. To generate standard curves for each antibody isotype, we coated plates with duplicate samples of purified mouse Ig isotype standard (1010-01, Southern Biotech Biological Reagent Co., Ltd., AL, USA).Each well was coated with 200 ng of the standard in 100 µL of coating buffer.The plates were first washed three times with tris buffered saline with 0.1% Tween 20 (TBST) and then blocked with a solution of 3% BSA (A602449, Sangon Biotech Biological Reagent Co., Ltd., AL, USA) for 2 h at 37 • C. Next, a 100 µL volume of the sample, diluted 100-fold or 10-fold, was added to each well in duplicate.The plates were then incubated for 1 h at 37 • C. To the standard curve, 100 µL of unconjugated IgG (0107-01, Southern Biotech Biological Reagent Co., Ltd., AL, USA), IgA (0106-01, Southern Biotech Biological Reagent Co., Ltd., AL, USA), IgG1 (0102-01, Southern Biotech Biological Reagent Co., Ltd., AL, USA), or IgG2a (0103-01, Southern Biotech Biological Reagent Co., Ltd., AL, USA) obtained from ordinary mice were diluted in a 2x serial in PBS.The unconjugated IgG was diluted from 500 ng/mL to 0.488 ng/mL, IgG1, and IgG2a from 1 µg/mL to 8 ng/mL, and the IgA was diluted from 500 ng/mL to 0.488 ng/mL.Following TBST washing, each well was added with a 1:5000 dilution of biotinylated goat anti-mouse IgA (1040-08, Southern Biotech Biological Reagent Co., Ltd., AL, USA), IgG1 (1070-08, Southern Biotech Biological Reagent Co., Ltd., AL, USA), IgG2a (1080-08, Southern Biotech Biological Reagent Co., Ltd., AL, USA), or IgG (1030-08, Southern Biotech Biological Reagent Co., Ltd., AL, USA).The plates were then incubated at 37 • C for 1 h.A 100 µL volume of p-nitrophenyl phosphate (N1891, Sigma-Aldrich Biological Reagent Co., Ltd., WI, USA) with a final concentration of 1 mg/mL was added to each of the wells for color development after they were previously incubated for an hour at 37 • C with a 1:3000 dilution of streptavidin-alkaline phosphatase conjugate (7100-04, Southern Biotech Biological Reagent Co., Ltd., AL, USA).Color development (absorbance) was read at 405 nm using an automated ELISA plate after appropriate incubation.Using linear regression in Excel (R 2 ≥ 0.95), the OD values at 405 nm were plotted against the representative concentrations of the diluted unconjugated antibody solutions to generate the standard curves.The corresponding standard curve was applied to calculate the total levels of antibodies in the samples.

Analysis of pSW-GDH and pSW-O9 Expression by Western Blot
To evaluate the heterologous antigen synthesis, the recombinant attenuated S. Choleraesuis strain SC3 ∆asd harboring the GDH protein and O-antigen of E. coli O9 were cultured overnight in LB medium with aeration at 37 • C. One milliliter of culture was collected from each passage and prepared for Western blot analysis.Western blot analysis was performed as previously described using anti-GDH and anti-E.coli O9 antisera [37,39].The following antibody was goat anti-rabbit immunoglobulin G (SAB4600068, Sigma-Aldrich Biological Reagent Co., Ltd., WI, USA), which was conjugated with horseradish peroxidase.

Statistical Analysis
The Graph-Pad Prism 8.0 software was utilized to conduct statistical calculations.Unless otherwise mentioned, numerical data were presented as means ± SEM.To determine the varying significance of bacterial motility, one-way or two-way ANOVA analysis was performed, followed by the application of Tukey's multiple comparisons test.The log-rank test was implemented to assess differences in mouse survival, with the Kaplan-Meier survival curve serving as the monitoring tool.To compare means, the least significant difference test was employed.A p-value of less than 0.05 was regarded as indicative of a significant difference.

Construction and Biological Characteristics of the C5000 Strain
To develop a new vaccine that maintains superior immunogenicity with moderate virulence based on the C500 strain, the complete rpoS gene was introduced into the corresponding position of the C500 genome by the homologous recombination method (Figure 1).The rpoS gene insertion in C500 was verified by PCR using the primers rpoS-F and rpoS-R with the expected amplicon size of 1656 bp from the wild-type C3545 and was confirmed by sequencing; the resultant mutant strain was named C5000 (C500 rpoS + ).cultured overnight in LB medium with aeration at 37 °C.One milliliter of culture was collected from each passage and prepared for Western blot analysis.Western blot analysis was performed as previously described using anti-GDH and anti-E.coli O9 antisera [37,39].The following antibody was goat anti-rabbit immunoglobulin G (SAB4600068, Sigma-Aldrich Biological Reagent Co., Ltd., WI, USA), which was conjugated with horseradish peroxidase.

Statistical Analysis
The Graph-Pad Prism 8.0 software was utilized to conduct statistical calculations.Unless otherwise mentioned, numerical data were presented as means ± SEM.To determine the varying significance of bacterial motility, one-way or two-way ANOVA analysis was performed, followed by the application of Tukey's multiple comparisons test.The log-rank test was implemented to assess differences in mouse survival, with the Kaplan-Meier survival curve serving as the monitoring tool.To compare means, the least significant difference test was employed.A p-value of less than 0.05 was regarded as indicative of a significant difference.

Construction and Biological Characteristics of the C5000 Strain
To develop a new vaccine that maintains superior immunogenicity with moderate virulence based on the C500 strain, the complete rpoS gene was introduced into the corresponding position of the C500 genome by the homologous recombination method (Figure 1).The rpoS gene insertion in C500 was verified by PCR using the primers rpoS-F and rpoS-R with the expected amplicon size of 1656 bp from the wild-type C3545 and was confirmed by sequencing; the resultant mutant strain was named C5000 (C500 rpoS + ).As a gastrointestinal pathogen, Salmonella has developed a variety of survival strategies to withstand harsh environments.After oral consumption, Salmonella upregulates many amino acid decarboxylase systems and synthesizes acid-activated proteins, including RpoS [18] and PhoPQ [27], to avoid an acidic pH and maintain pH homeostasis in the As a gastrointestinal pathogen, Salmonella has developed a variety of survival strategies to withstand harsh environments.After oral consumption, Salmonella upregulates many amino acid decarboxylase systems and synthesizes acid-activated proteins, including RpoS [18] and PhoPQ [27], to avoid an acidic pH and maintain pH homeostasis in the stomach.Therefore, we detected the growth curve of C5000 (C500 rpoS + ) in the hypertonic LB broth (LB containing 6% NaCl).The results showed that C5000 (C500 rpoS + ) grew faster than C500 (Figure 2A).In addition, we also monitored the growth curve of C5000 in N-minimal medium and N-minimal medium with different pH.The results indicated that the growth was consistent with that in the 6% NaCl LB broth (Supplementary Figure S1).
Vaccines 2024, 12, x FOR PEER REVIEW 9 of 19 stomach.Therefore, we detected the growth curve of C5000 (C500 rpoS + ) in the hypertonic LB broth (LB containing 6% NaCl).The results showed that C5000 (C500 rpoS + ) grew faster than C500 (Figure 2A).In addition, we also monitored the growth curve of C5000 in Nminimal medium and N-minimal medium with different pH.The results indicated that the growth was consistent with that in the 6% NaCl LB broth (Supplementary Figure S1).

Virulence Analysis of the C5000
To evaluate the effect of the rpoS gene in an activated state on the C500 genome in mice, this study compared the colonization and invasive capacity of C500 and C5000 (C500 rpoS + ) in the PP, spleen, and liver tissues of infected mice, as well as the LD50 of C5000 (C500 rpoS + ).The results showed that the colonization capacity of C5000 (C500 rpoS + ) in the PP tissues was significantly higher than C500 strains at 3 days after inoculation and exhibited a similar level at 7 and 18 days.Still, the colonization ability of C5000 (C500 rpoS + ) in the liver and spleen was significantly higher than C500 during the tested periods (Figure 2B).The histopathological analysis showed that a small number of neutrophils were present in the splenic tissue of the C500 group.In contrast, apparent neutrophil infiltration and forming infiltrative foci were observed in the C5000 (C500 rpoS + ) group (Figure 2C).The virulence of C5000 (C500 rpoS + ) in the BALB/c mice was only slightly higher than that of C500 (Supplementary Table S2).These results indicated that the rpoS gene was in an activated state in the C5000 (C500 rpoS + ), contributing to enhanced colonization in mice.

Virulence Analysis of the C5000
To evaluate the effect of the rpoS gene in an activated state on the C500 genome in mice, this study compared the colonization and invasive capacity of C500 and C5000 (C500 rpoS + ) in the PP, spleen, and liver tissues of infected mice, as well as the LD 50 of C5000 (C500 rpoS + ).The results showed that the colonization capacity of C5000 (C500 rpoS + ) in the PP tissues was significantly higher than C500 strains at 3 days after inoculation and exhibited a similar level at 7 and 18 days.Still, the colonization ability of C5000 (C500 rpoS + ) in the liver and spleen was significantly higher than C500 during the tested periods (Figure 2B).The histopathological analysis showed that a small number of neutrophils were present in the splenic tissue of the C500 group.In contrast, apparent neutrophil infiltration and forming infiltrative foci were observed in the C5000 (C500 rpoS + ) group (Figure 2C).The virulence of C5000 (C500 rpoS + ) in the BALB/c mice was only slightly higher than that of C500 (Supplementary Table S2).These results indicated that the rpoS gene was in an activated state in the C5000 (C500 rpoS + ), contributing to enhanced colonization in mice.

Phenotype Determination and Histopathological Analysis Induced by C5001, C5002, C5003, and C5004
The essential virulent genes of crp, fur, phoP, and aroA were deleted from C5000 (C500 rpoS + ) to yield C5001 (C500 rpoS + Δcrp10), C5002 (C500 rpoS + Δfur9), C5003 (C500 rpoS + ΔphoP11), and C5004 (C500 rpoS + ΔaroA12).We evaluated the phenotypes of each mutant strain growing in LB broth or agar plate at 37 °C (Figure 3A).All the strains showed similar growth rates except C5001 (C500 rpoS + Δcrp10), which grew slowest, and no differences were observed for each mutant strain derived from C500, but they were significantly slower than the wild-type strain C3545 in the motility test (Figure 3B).The SDS-PAGE gel indicated that the OMP profiles exhibited similar bands among all the strains, including C500 (Figure 3C).There were no apparent differences among the LPS profiles of these strains detected by silver staining (Figure 3D).To understand whether the crp, fur, phoP, and aroA mutations have any effect on inducing inflammation in the BALB/c mice, histopathological analysis was performed after the mutants infected mice 6 days later.The spleen of mice inoculated by C500, C5001 (C500 rpoS + Δcrp10), C5002 (C500 rpoS + Dfur9), and C5003 (C500 rpoS + DphoP11) showed some inflammatory foci but did not show severe pathological changes in the infected mice (Figure 4A).The results indicated that these S. Choleraesuis mutant strains exhibited low virulence during the experiment time.However, the spleen of mice infected by C5004 (C500 rpoS + ΔaroA12) had many neutrophils infiltrated.The infected mice showed ruffled fur, depression, weight loss, and even death, indicating that C5004 (C500 rpoS + ΔaroA12) is not attenuated sufficiently enough to be a vaccine candidate.Therefore, we excluded C5004 (C500 rpoS + ΔaroA12) in the subsequent study.As lipid A is its key stimulator to induce an inflammatory response in the host; thus, some mutations related to lipid A modification were introduced to reduce its endotoxic activity.To understand whether the crp, fur, phoP, and aroA mutations have any effect on inducing inflammation in the BALB/c mice, histopathological analysis was performed after the mutants infected mice 6 days later.The spleen of mice inoculated by C500, C5001 (C500 rpoS + ∆crp10), C5002 (C500 rpoS + ∆fur9), and C5003 (C500 rpoS + ∆phoP11) showed some inflammatory foci but did not show severe pathological changes in the infected mice (Figure 4A).The results indicated that these S. Choleraesuis mutant strains exhibited low virulence during the experiment time.However, the spleen of mice infected by C5004 (C500 rpoS + ∆aroA12) had many neutrophils infiltrated.The infected mice showed ruffled fur, depression, weight loss, and even death, indicating that C5004 (C500 rpoS + ∆aroA12) is not attenuated sufficiently enough to be a vaccine candidate.Therefore, we excluded C5004 (C500 rpoS + ∆aroA12) in the subsequent study.As lipid A is its key stimulator to induce an inflammatory response in the host; thus, some mutations related to lipid A modification were introduced to reduce its endotoxic activity.

Immunogenicity Assessment Induced by C5001, C5002, C5003, and C5004
To test the immunogenicity of the S. Choleraesuis mutant strains, the antibody responses against OMPs and LPS from S. Choleraesuis in mice were measured.Mice were immunized orally (10 9 CFU/20 µL) at 5 weeks after the initial immunization.Blood and vaginal secretions were collected at 8 weeks after the initial immunization to detect S-IgA and IgG antibody levels.The results showed that higher serum S-IgA and IgG antibody levels against OMPs from S. Choleraesuis were detected in the mice immunized with the S. Choleraesuis mutant strains compared to those with BSG (Figure 4B,C).The serum IgA and IgG levels against OMPs from S. Choleraesuis in mice immunized with C5003 (C500 rpoS + ΔphoP11) were similar to those in mice immunized with C500.Notably, the serum antibody levels of S-IgA and IgG against OMPs and LPS from S. Choleraesuis in mice immunized with C5001 (C500 rpoS + Δcrp10) and C5002 (C500 rpoS + Δfur9) were significantly higher than those in mice immunized with C500 and C5003 (C500 rpoS + DphoP11).These results indicated that all S. Choleraesuis mutant strains could induce high levels of S-IgA and IgG antibodies against OMPs and LPS from S. Choleraesuis in mice, especially C5001 (C500 rpoS + Δcrp10) and C5002 (C500 rpoS + Δfur9).
To determine whether the S. Choleraesuis mutant strains protect mice against S. Choleraesuis infection, mice were orally challenged with 20 µL of a lethal dose of wild-

Immunogenicity Assessment Induced by C5001, C5002, C5003, and C5004
To test the immunogenicity of the S. Choleraesuis mutant strains, the antibody responses against OMPs and LPS from S. Choleraesuis in mice were measured.Mice were immunized orally (10 9 CFU/20 µL) at 5 weeks after the initial immunization.Blood and vaginal secretions were collected at 8 weeks after the initial immunization to detect S-IgA and IgG antibody levels.The results showed that higher serum S-IgA and IgG antibody levels against OMPs from S. Choleraesuis were detected in the mice immunized with the S. Choleraesuis mutant strains compared to those with BSG (Figure 4B,C).The serum IgA and IgG levels against OMPs from S. Choleraesuis in mice immunized with C5003 (C500 rpoS + ∆phoP11) were similar to those in mice immunized with C500.Notably, the serum antibody levels of S-IgA and IgG against OMPs and LPS from S. Choleraesuis in mice immunized with C5001 (C500 rpoS + ∆crp10) and C5002 (C500 rpoS + ∆fur9) were significantly higher than those in mice immunized with C500 and C5003 (C500 rpoS + ∆phoP11).These results indicated that all S. Choleraesuis mutant strains could induce high levels of S-IgA and IgG antibodies against OMPs and LPS from S. Choleraesuis in mice, especially C5001 (C500 rpoS + ∆crp10) and C5002 (C500 rpoS + ∆fur9).
We also assessed the induced inflammation in mice after the mutants harboring ∆pagP81::P lpp lpxE ∆lpxR9 infected mice 7 days later; no noticeable histopathological changes were observed (Supplementary Figure S3).To further evaluate the endotoxic activity of these mutant strains, the rabbit ileal loops were used for this purpose.After being injected into ligated loops, mutant strains were cultured for eight hours.H&E-stained histological samples from loops treated with several mutant strains were viewed under a microscope.The C5002 (C500 rpoS + ∆fur9) strain caused severe damage to the mucosa of mice, resulting in a large necrotic infiltration of the epithelium and a large infiltration of polymorphonuclear leukocytes (PMN).In contrast, the mutant strain SC3 (C500 rpoS + ∆fur9 ∆pagL7 ∆pagP81::P lpp lpxE ∆lpxR9) induced some tissue destruction and no apparent PMN infiltration (Figure 5), indicating that the 1 -dephosphorylated lipid A in S. Choleraesuis aids in a reduction in the inflammatory response.

Construction of S. choleraesuis Mutant Strains with Low Endotoxic Activity and Evalu tions of Immunogenicity and Protection Rate
Our previous study showed that LpxE selectively removes the 1-phosphate gro lipid A in Salmonella, generating a product similar to monophosphoryl lipid A (MPL In this study, to further decrease the endotoxic activity, the combination mutation cluding ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9, which will generate 4′-monophosphory lipopolysaccharide, were introduced into the mutants of S. Choleraesuis, resulting i (C500 ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9), SC2 (C500 rpoS + Δcrp10 ΔpagL7 ΔpagP8 lpxE ΔlpxR9), and SC3 (C500 rpoS + Δfur9 ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9), respect These mutations did not impact their growth in LB media (Supplementary Figure S We also assessed the induced inflammation in mice after the mutants harb ΔpagP81::Plpp lpxE ΔlpxR9 infected mice 7 days later; no noticeable histopatholo changes were observed (Supplementary Figure S3).To further evaluate the endotox tivity of these mutant strains, the rabbit ileal loops were used for this purpose.After injected into ligated loops, mutant strains were cultured for eight hours.H&E-staine tological samples from loops treated with several mutant strains were viewed un microscope.The C5002 (C500 rpoS + Dfur9) strain caused severe damage to the muco mice, resulting in a large necrotic infiltration of the epithelium and a large infiltrati polymorphonuclear leukocytes (PMN).In contrast, the mutant strain SC3 (C500 Δfur9 ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9) induced some tissue destruction and no app PMN infiltration (Figure 5), indicating that the 1′-dephosphorylated lipid A in S. eraesuis aids in a reduction in the inflammatory response.To evaluate the immune response induced by S. Choleraesuis mutant strains in mice, serum and vaginal secretions were collected to detect S-IgA and IgG antibody levels 8 weeks after the primary immunization.As shown in the results (Figure 6A-D), antibody levels of mice immunized with the SC3 (C500 rpoS + ∆fur9 ∆pagL7 ∆pagP81::P lpp lpxE ∆lpxR9) were significantly higher than those in the control group and other groups.The results indicated that S. Choleraesuis mutant strains with 1 -dephosphorylated lipid A could reduce bacterial endotoxin while maintaining the induced immune response in mice.
weeks after the primary immunization.As shown in the results (Figure 6A-D), antibody levels of mice immunized with the SC3 (C500 rpoS + Δfur9 ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9) were significantly higher than those in the control group and other groups.The results indicated that S. Choleraesuis mutant strains with 1′-dephosphorylated lipid A could reduce bacterial endotoxin while maintaining the induced immune response in mice.

Evaluations of SC3 as a Vaccine Carrier to Deliver Heterologous Antigens
To evaluate the ability of SC3 (C500 rpoS + Δfur9 ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9) as a vector to deliver heterologous antigens, we selected the GDH protein antigen of S. suis and the O-polysaccharide antigen of E. coli O9.GDH is a glutamate dehydrogenase protein on the surface of S. suis, an important virulence factor and effective antigen of S. suis.The O-polysaccharide is a potent protective antigen of gram-negative bacteria.In this study, we evaluated the ability of SC3 (C500 rpoS + Δfur9 ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9) To determine whether the S. Choleraesuis mutant strains with low toxicity protect mice against S. Choleraesuis infection, mice were orally challenged with 20 µL of a lethal dose of wild-type S. Choleraesuis C3545 containing 10 9 (10,000 × LD 50 ) at 9 weeks after initial immunization.The results showed that SC3 (C500 rpoS + ∆fur9 ∆pagL7 ∆pagP81::P lpp lpxE ∆lpxR9) conferred 80% protection, and C5002 (C500 rpoS + ∆fur9) provided 60% protection (Figure 6E).

Evaluations of SC3 as a Vaccine Carrier to Deliver Heterologous Antigens
To evaluate the ability of SC3 (C500 rpoS + ∆fur9 ∆pagL7 ∆pagP81::P lpp lpxE ∆lpxR9) as a vector to deliver heterologous antigens, we selected the GDH protein antigen of S. suis and the O-polysaccharide antigen of E. coli O9.GDH is a glutamate dehydrogenase protein on the surface of S. suis, an important virulence factor and effective antigen of S. suis.The O-polysaccharide is a potent protective antigen of gram-negative bacteria.In this study, we evaluated the ability of SC3 (C500 rpoS + ∆fur9 ∆pagL7 ∆pagP81::P lpp lpxE ∆lpxR9) as a vector to deliver the GDH protein antigen and O-polysaccharide antigen.The results showed that the 48 kDa GDH protein and O-antigen of E. coli O9 were detected in SC3 ∆asd (C500 rpoS + ∆fur9 ∆pagL7 ∆pagP81::P lpp lpxE ∆lpxR9 ∆asd) (Figure 7A-C), indicating that the plasmids in SC3 ∆asd (C500 rpoS + ∆fur9 ∆pagL7 ∆pagP81::P lpp lpxE ∆lpxR9 ∆asd) are stable and able to synthesize both protein antigens and O-polysaccharide antigens.

Discussion
Salmonellosis caused by S. Choleraesuis in pigs leads to substantial economic losses in a large-scale pig farm.Clinical symptoms of this disease often show fever, depression, septicemia, arthritis, diarrhea, and even death in some cases [1].As these bacteria often spread quickly in industrialized pig farms by contaminated food and water, the vaccine is an efficient approach to prevent epidemics and control salmonellosis.C500 is a vaccine strain that has been widely used for preventing S. Choleraesuis infection in pigs for many years in China [48,49].Although C500 effectively prevents salmonellosis caused by S. Choleraesuis, it still shows a high level of side effects.The previous results indicated that further attenuation of C500 is not a good option for developing a live attenuated vaccine because this process would reduce its immunogenicity and protection efficacy [11].Therefore, we need to explore strategies to develop new vaccines with a good balance between safety and immunogenicity.In this work, we re-attenuated C500 to construct a safer and more immunogenic attenuated vector of S. Choleraesuis.
The previous genome sequence results showed that many genes were lost or inactivated in C500, including asr, ydgF, ydgD, ydgE, rpoS, and ptsG [50].The animal experiments indicated that the inactivation of the rpoS gene, a vital transcriptional regulator playing an essential role in Salmonella infection, was the main reason for its attenuation of C500 [10].Meanwhile, studies with S. Typhimurium and the mouse model of typhoid fever also demonstrated that RpoS played an essential role in mice colonization and survival [16][17][18].Considering these factors, the strategies to generate an effective vector of S. Choleraesuis were (1) to restore functional rpoS genes for enhancing the viability of the strains in mice because of their ability to overcome the gastrointestinal environment in the host and enter the mucosa-associated lymphoid tissue and (2) to remove essential virulence genes and to construct S. Choleraesuis vectors capable of delivering heterologous antigens.As we expected, C5000 (C500 rpoS + ) was more adapted to the gastrointestinal environment, thus contributing to colonization in mice (Figure 2B), although C5000 (C500 rpoS + ) did not exhibit significant virulence enhancement compared with C500 in mice (Supplementary Figure S1).This result indicates that functional RpoS in C5000 (C500 rpoS + ) can increase the persistence of bacteria in lymphoid tissues, which is consistent with previous studies [16][17][18].Despite earlier reports showing that the loss of functional RpoS was the main reason for the attenuation of C500 [10], we found that C5000 (C500 rpoS + ) did not restore to a wild-type virulence state in our detecting virulence in mice.This may be due to the inactivation of multiple other genes in addition to RpoS in C500, which collectively affect the virulence of C500.
We aimed to construct an attenuated S. Choleraesuis vaccine strain that has a persistent ability to stimulate intense primary and maintain lasting memory immune responses while retaining fewer side effects.In this work, we analyzed the general characteristics of S. Choleraesuis mutation strains both in vitro and in vivo.In the in vitro studies, the phenotypes of the S. Choleraesuis mutant strains were confirmed and were precisely the same as those in S. Typhimurium strains as we expected (Figure 3).In terms of safety based on the pathological analysis of the spleen, severe hyperemia and a massive inflammatory exudation in mice immunized with C5004 (C500 rpoS + ∆aroA12) were observed; moderate hyperemia and a small amount of inflammatory exudation in mice immunized with C500 and C5002 (C500 rpoS + Dfur9) and mild histopathological lesions in mice immunized with C5001 (C500 rpoS + Dcrp10) and C5003 (C500 rpoS + DphoP11) (Figure 4A) were also observed.Notably, some mice died after C5004 (C500 rpoS + ∆aroA12) was administered to them, indicating that C5004 (C500 rpoS + ∆aroA12) is not attenuated sufficiently enough to be a vaccine candidate.Therefore, we excluded C5004 (C500 rpoS + ∆aroA12) in the subsequent study.Although colonization can reflect the interaction between live attenuated strains and lymphoid tissues, which is closely related to the immunogenicity of the live attenuated strain.Considering the bacterial colonization of C5000 (C500 rpoS + ) in mice and the spleen pathological analysis of mice inoculated with mutant strains, it is unnecessary to reevaluate the colonization of mutant strains in mice.In in vivo experiments, all strains could induce a local mucosal immune response and systemic humoral immunity in mice at 9 weeks after the initial immunization.Mice were challenged with a lethal dose (10,000 × LD 50 ) of wild-type S. Choleraesuis C3545 at 9 weeks after the initial immunization.The results showed that although C5001 (C500 rpoS + ∆crp10) and C5002 (C500 rpoS + ∆fur9) can protect against wild-type S. Choleraesuis in mice (Figure 4D), further enhancing the immune response is required.
Institutional Review Board Statement: All animal experiments were designed and performed according to the principles of the "Guide for the Care and Use of Laboratory Animals" and the "Guidance for Experimental Animal Welfare and Ethical Treatment".We usually make an appointment in the online system with our registered personal information and carry out animal experiments after approval, and there is no license number for each animal experiment.The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review Board.
Informed Consent Statement: Consent for publication is not applicable.

Figure 1 .
Figure 1.A schematic diagram of the restoring function of the rpoS gene of C500.The rpoS gene is partially missing in the C500 genome.The position of the rpoS gene in the C500 genome, and the position of the rpoS gene in the C500 genome after restoration.

Figure 1 .
Figure 1.A schematic diagram of the restoring function of the rpoS gene of C500.The rpoS gene is partially missing in the C500 genome.The position of the rpoS gene in the C500 genome, and the position of the rpoS gene in the C500 genome after restoration.

Figure 2 .
Figure 2. The biological characteristics and virulence analysis of the C5000 (C500 rpoS + ).(A) Growth curves of S. Choleraesuis wild-type C3545, vaccine strain C500, and mutant strain C5000 (C500 rpoS + ) in hypertonic LB broth (LB with 6% NaCl).(B) The bacterial load in mice liver, spleen, and PP at 3, 7, and 18 days after oral administration (n = 3).The total count of Salmonella colonies was calculated and presented as CFU per g of liver, spleen, or PP.(C) Histopathological assessment.S. Choleraesuis was orally administered to mice, and the effects on their organs were examined and contrasted with organ samples from animals given BSG as a placebo.Changes in the spleen's histopathology were examined six days after administration.Blue arrows represent red blood cells, black arrows represent extramedullary hematopoietic cells, and green arrows represent macrophages.The red circle represents neutrophil infiltration.Images were captured with a magnification of 10 × (scale bars 250 µm) and an inset magnification of 40 × (scale bars 50 µm).Data are presented as the means ± SEM, and the superscript letters a, b, and c indicate p < 0.05 for comparisons with the C500 group at 3, 7, and 18 days, respectively.

Figure 2 .
Figure 2. The biological characteristics and virulence analysis of the C5000 (C500 rpoS + ).(A) Growth curves of S. Choleraesuis wild-type C3545, vaccine strain C500, and mutant strain C5000 (C500 rpoS + ) in hypertonic LB broth (LB with 6% NaCl).(B) The bacterial load in mice liver, spleen, and PP at 3, 7, and 18 days after oral administration (n = 3).The total count of Salmonella colonies was calculated and presented as CFU per g of liver, spleen, or PP.(C) Histopathological assessment.S. Choleraesuis was orally administered to mice, and the effects on their organs were examined and contrasted with organ samples from animals given BSG as a placebo.Changes in the spleen's histopathology were examined six days after administration.Blue arrows represent red blood cells, black arrows represent extramedullary hematopoietic cells, and green arrows represent macrophages.The red circle represents neutrophil infiltration.Images were captured with a magnification of 10× (scale bars 250 µm) and an inset magnification of 40× (scale bars 50 µm).Data are presented as the means ± SEM, and the superscript letters a, b, and c indicate p < 0.05 for comparisons with the C500 group at 3, 7, and 18 days, respectively.

Figure 4 .
Figure 4.The histopathological analysis and immunogenicity assessment induced by C5001 (C500 rpoS + Δcrp10), C5002 (C500 rpoS + Dfur9), C5003 (C500 rpoS + DphoP11), and C5004 (C500 rpoS + ΔaroA12).(A) The effects of recombinant attenuated S. Choleraesuis on organs were assessed by histopathological examination.The red arrows represent macrophages, and the red circles represent neutrophil infiltration.Images were captured with a magnification of 10× (scale bars 250 µm) and an inset magnification of 40× (scale bars 50 µm).(B,C) Recombinant attenuated S. Choleraesuis based on the C500 strain was investigated for immunogenicity by quantitative ELISA.The results displayed the precise levels of IgA and IgG antibodies, as measured by a standard curve, in mice orally inoculated with attenuated S. Choleraesuis at the scheduled weeks.The standard differences between the mice in each group were shown by the error bars.(D) Following the challenge, mortality was recorded for 25 days.Data are presented as the means ± SEM (n = 12), and the superscript letters a and b indicate p < 0.05 for comparisons with the BSG and C500 groups, respectively.

Figure 4 .
Figure 4.The histopathological analysis and immunogenicity assessment induced by C5001 (C500 rpoS + ∆crp10), C5002 (C500 rpoS + ∆fur9), C5003 (C500 rpoS + ∆phoP11), and C5004 (C500 rpoS + ∆aroA12).(A) The effects of recombinant attenuated S. Choleraesuis on organs were assessed by histopathological examination.The red arrows represent macrophages, and the red circles represent neutrophil infiltration.Images were captured with a magnification of 10× (scale bars 250 µm) and an inset magnification of 40× (scale bars 50 µm).(B,C) Recombinant attenuated S. Choleraesuis based on the C500 strain was investigated for immunogenicity by quantitative ELISA.The results displayed the precise levels of IgA and IgG antibodies, as measured by a standard curve, in mice orally inoculated with attenuated S. Choleraesuis at the scheduled weeks.The standard differences between the mice in each group were shown by the error bars.(D) Following the challenge, mortality was recorded for 25 days.Data are presented as the means ± SEM (n = 12), and the superscript letters a and b indicate p < 0.05 for comparisons with the BSG and C500 groups, respectively.

Figure 5 .
Figure 5.An ileal loop experiment on rabbits.Insertion of the lpxE gene on the S. Choleraesu tant's chromosome reduces inflammation but stimulates fluid secretion in rabbit ileal loops injecting 10 9 CFU of S. Choleraesuis strains into rabbit ileal loops for 8 h, the fluid secretion fro ileum was recorded, and the rabbit ileum was taken for H&E staining to observe histopathol abnormalities.The black arrows indicate tissue edema, the blue arrows indicate bleeding, the y arrows indicate neutrophils, and the green arrows indicate lymphocytes.Images were cap with a magnification of 10× (scale bars 250 µm) and an inset magnification of 40× (scale bars 50

Figure 5 .
Figure 5.An ileal loop experiment on rabbits.Insertion of the lpxE gene on the S. Choleraesuis mutant's chromosome reduces inflammation but stimulates fluid secretion in rabbit ileal loops.After injecting 10 9 CFU of S. Choleraesuis strains into rabbit ileal loops for 8 h, the fluid secretion from the ileum was recorded, and the rabbit ileum was taken for H&E staining to observe histopathological abnormalities.The black arrows indicate tissue edema, the blue arrows indicate bleeding, the yellow arrows indicate neutrophils, and the green arrows indicate lymphocytes.Images were captured with a magnification of 10× (scale bars 250 µm) and an inset magnification of 40× (scale bars 50 µm).

Figure 6 .
Figure 6.An immune response assay induced by recombinant attenuated S. Choleraesuis.Serum and vaginal secretions of mice were gathered at 8 weeks post-initial vaccination.Quantitative ELISA was applied to analyze specific IgA (A,B) and IgG (C,D) against S. Choleraesuis OMP and LPS.The results displayed the precise levels of antibodies, as measured by a standard curve, in mice orally inoculated with attenuated S. Choleraesuis at the scheduled weeks.The standard differences between the mice in each group were shown by the error bars.(E) Following the challenge, mortality was recorded for 25 days.Data are presented as the means ± SEM (n = 5), and the superscript letters a, b, and c indicate p < 0.05 for comparisons with the BSG, C500, and SC1 groups, respectively.

Figure 6 .
Figure 6.An immune response assay induced by recombinant attenuated S. Choleraesuis.Serum and vaginal secretions of mice were gathered at 8 weeks post-initial vaccination.Quantitative ELISA was applied to analyze specific IgA (A,B) and IgG (C,D) against S. Choleraesuis OMP and LPS.The results displayed the precise levels of antibodies, as measured by a standard curve, in mice orally inoculated with attenuated S. Choleraesuis at the scheduled weeks.The standard differences between the mice in each group were shown by the error bars.(E) Following the challenge, mortality was recorded for 25 days.Data are presented as the means ± SEM (n = 5), and the superscript letters a, b, and c indicate p < 0.05 for comparisons with the BSG, C500, and SC1 groups, respectively.

Figure 7 .
Figure 7. Evaluations of SC3 (C500 rpoS + Δfur9 ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9) as a vaccine carrier to deliver heterologous antigens.(A) The synthesis of S. Choleraesuis LPS in SC3 Δasd (pSW-O9).(B) The synthesis of E. coli O9 O-antigen in SC3 Δasd (pSW-O9).(C) The synthesis of Streptococcus suis GDH in SC3 Δasd (pSW-GDH).After strains SC3 Δasd (pSW-O9), SC3 Δasd (pSW-GDH), or SC3 Δasd reached an OD600 of 0.85 in LB broth, they were collected.For Western Blot analysis, the protein samples or LPS samples were separated on the SDS-PAGE, subsequently transferred onto nitrocellulose membranes, and analyzed with rabbit anti-Salmonella O7 serum, and rabbit anti-E.coli O9 serum, or rabbit anti-GDH serum.(D) Serum and vaginal secretions of mice were gathered at 8 weeks post-initial vaccination, and antibodies anti-GDH or anti-E.coli O9 LPS were detected by quantitative ELISA.The results displayed the precise levels of IgA and IgG antibodies, as measured by a standard curve, in mice orally inoculated with attenuated S. Choleraesuis at the scheduled weeks.The standard differences between the mice in each group were shown by the error bars.Data are presented as the means ± SEM (n = 5), and the superscript letters a and b indicate p < 0.05 for comparisons with the corresponding BSG group, respectively.

Figure 7 .
Figure 7. Evaluations of SC3 (C500 rpoS + ∆fur9 ∆pagL7 ∆pagP81::P lpp lpxE ∆lpxR9) as a vaccine carrier to deliver heterologous antigens.(A) The synthesis of S. Choleraesuis LPS in SC3 ∆asd (pSW-O9).(B) The synthesis of E. coli O9 O-antigen in SC3 ∆asd (pSW-O9).(C) The synthesis of Streptococcus suis GDH in SC3 ∆asd (pSW-GDH).After strains SC3 ∆asd (pSW-O9), SC3 ∆asd (pSW-GDH), or SC3 ∆asd reached an OD 600 of 0.85 in LB broth, they were collected.For Western Blot analysis, the protein samples or LPS samples were separated on the SDS-PAGE, subsequently transferred onto nitrocellulose membranes, and analyzed with rabbit anti-Salmonella O7 serum, and rabbit anti-E.coli O9 serum, or rabbit anti-GDH serum.(D) Serum and vaginal secretions of mice were gathered at 8 weeks post-initial vaccination, and antibodies anti-GDH or anti-E.coli O9 LPS were detected by quantitative ELISA.The results displayed the precise levels of IgA and IgG antibodies, as measured by a standard curve, in mice orally inoculated with attenuated S. Choleraesuis at the scheduled weeks.The standard differences between the mice in each group were shown by the error bars.Data are presented as the means ± SEM (n = 5), and the superscript letters a and b indicate p < 0.05 for comparisons with the corresponding BSG group, respectively.

Table 1 .
The bacterial strains and plasmids that were used in this study.