Immunogenic potential and protective efficacy of a sptP deletion mutant of Salmonella Enteritidis as a live vaccine for chickens against a lethal challenge.

Salmonella Enteritidis (SE) is a highly adapted pathogen causing severe economic losses in the poultry industry worldwide. Chickens infected by SE are a major source of human food poisoning. Vaccination is an effective approach to control SE infections. This study evaluated the immunogenicity and protective efficacy of a SE sptP deletion mutant (C50336ΔsptP) as a live attenuated vaccine (LAV) candidate in chickens. 14 day-old specific pathogen-free (SPF) chickens were intramuscularly immunized with various doses of C50336ΔsptP. Several groups of chickens were challenged with the virulent wild-type SE strain Z-11 via the same route at 14 days post vaccination. Compared to the control group, the groups vaccinated with 1 × 106, 1 × 107 and 1 × 108 colony-forming units (CFU) of C50336ΔsptP exhibited no clinical symptoms after immunization. Only slight pathological changes occurred in the organs of the 1 × 109 CFU vaccinated group. C50336ΔsptP bacteria were cleared from the organs of immunized chickens within 14 days after vaccination. Lymphocyte proliferation and serum cytokine analyses indicated that significant cellular immune responses were induced after the vaccination of C50336ΔsptP. Compared to the control group, specific IgG antibody levels increased significantly in vaccinated chickens, and the levels increased markedly after the challenge. The 1 × 107, 1 × 108, and 1 × 109 CFU vaccinated chickens groups showed no clinical symptoms or pathological changes, and no death after the lethal challenge. Whereas severe clinical signs of disease and pathological changes were observed in the control group chickens after the challenge. These results suggest that a single dose of C50336ΔsptP could be an effective LAV candidate to against SE infection in chickens.

The SseL protein inhibits the intracellular NF-κB pathway to enhance the virulence of Salmonella Pullorum in a chicken model.

To persist in the host, Salmonella is known to facultatively parasitize cells to escape the immune response. Intracellular Salmonella enterica can replicate using effector proteins translocated across the Salmonella-containing vacuolar membrane via a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). One of these factors, Salmonella secreted factor L (SseL), is a deubiquitinase that contributes to the virulence of Salmonella Typhimurium in mice by inhibiting the cellular NF-κB inflammatory pathway. However, the nature of its effect on the NF-κB pathway is controversial, and little research has been performed in other animal models. In this study, the SseL of Salmonella Pullorum was studied, and chickens were used as an infection model. An sseL gene deletion strain, a complementation strain and a eukaryotic expression plasmid were used to clarify the means by which SseL regulates Salmonella virulence and the cellular inflammatory response. SseL significantly enhanced the virulence of Salmonella Pullorum in chickens and suppressed activation of the cellular NF-κB pathway, thus inhibiting cellular inflammatory cytokine expression.

Recombinant Salmonella expressing SspH2-EscI fusion protein limits its colonization in mice.

BACKGROUND: Activation of inflammasome contributes to the clearance of intracellular bacteria. C-terminus of E. coli EscI protein can activate NLRC4 (NLR family, CARD domain containing-4) inflammasome in macrophages. The purpose of this study was to determine if activation of NLRC4 inflammasome by EscI can reduce the colonization of Salmonella in mice. RESULTS: A recombinant S. typhimurium strain expressing fusion protein of the N-terminal SspH2 (a Salmonella type III secretion system 2 effector) and C-terminal EscI was constructed and designated as X4550(pYA3334-SspH2-EscI). In vitro assay showed that X4550(pYA3334-SspH2-EscI) significantly enhanced IL-1ß and IL-18 secretion (P < 0.05) and pyroptotic cell death of mouse peritoneal macrophages, compared with those infected with control strain, X4550(pYA3334-SspH2). In vivo studies showed that colonization of X4550(pYA3334-SspH2-EscI) in both spleen and liver were significantly lower than that of X4550(pYA3334-SspH2) (P < 0.05). The bacterial counts of X4550(pYA3334-SspH2-EscI) in mice decreased, while those of X4550(pYA3334-SspH2) increased over the time after infection. Additionally, X4550(pYA3334-SspH2-EscI) induced a less pathological alteration in spleen and liver than X4550(pYA3334-SspH2). CONCLUSION: Fusion protein SspH2-EscI may be translocated into macrophages and activate NLRC4 inflammasome, which limits Salmonella colonization in spleen and liver of mice.

Signature-tagged mutagenesis screening revealed a novel smooth-to-rough transition determinant of Salmonella enterica serovar Enteritidis.

BACKGROUND: Salmonella enterica serovar Enteritidis (S. Enteritidis) has emerged as one of the most important food-borne pathogens for humans. Lipopolysaccharide (LPS), as a component of the outer membrane, is responsible for the virulence and smooth-to-rough transition in S. Enteritidis. In this study, we screened S. Enteritidis signature-tagged transposon mutant library using monoclonal antibody against somatic O9 antigen (O9 MAb) and O9 factor rabbit antiserum to identify novel genes that are involved in smooth-to-rough transition. RESULTS: A total of 480 mutants were screened and one mutant with transposon insertion in rfbG gene had smooth-to-rough transition phenotype. In order to verify the role of rfbG gene, an rfbG insertion or deletion mutant was constructed using λ-Red recombination system. Phenotypic and biological analysis revealed that rfbG insertion or deletion mutants were similar to the wild-type strain in growth rate and biochemical properties, but the swimming motility was reduced. SE Slide Agglutination test and ELISA test showed that rfbG mutants do not stimulate animals to produce agglutinating antibody. In addition, the half-lethal dose (LD50) of the rfbG deletion mutant strain was 106.6 -fold higher than that of the parent strain in a mouse model when injected intraperitoneally. CONCLUSIONS: These data indicate that the rfbG gene is involved in smooth-to-rough transition, swimming motility and virulence of S. Enteritidis. Furthermore, somatic O-antigen antibody-based approach to screen signature-tagged transposon mutants is feasible to clarify LPS biosynthesis and to find suitable markers in DIVA-vaccine research.

Identification by PCR signature-tagged mutagenesis of attenuated Salmonella Pullorum mutants and corresponding genes in a chicken embryo model.

A key feature of the fowl-specific pathogen Salmonella Pullorum is its vertical transmission to progeny via the egg. In this study, PCR signature-tagged mutagenesis identified nine genes of a strain of S. Pullorum that contributed to survival in the chicken embryo during incubation. The genes were involved in invasion, cell division, metabolism and bacterial defence. The competition index in vivo and in vitro together with a virulence evaluation for chicken embryos of all nine mutant strains confirmed their attenuation.

Influence of Salmonella enterica serovar Pullorum pathogenicity island 2 on type III secretion system effector gene expression in chicken macrophage HD11 cells.

Salmonella pathogenicity island 2 (SPI2) can encode type III secretion system 2 (T3SS2) which plays an important role in systemic disease development through delivering different effector proteins into host cells. Here, the influence of Salmonella Pullorum pathogenicity island 2 on T3SS2 effector gene expression was studied using qRT-PCR in chicken macrophage HD11 cells. Our results showed that all the detected genes (including pseudogenes sifB, sspH2 and steC) can express in HD11 cells of S. Pullorum infection; deletion of SPI2 of S. Pullorum did not significantly affect the expression of genes cigR, gtgA, slrP, sopD, sseK1, steB and steC, but had a significant effect on the expression of genes pipB2, sifB, sopD2, sseJ, sseL, sspH2, steD, sifA, pipB and steA at different degrees. These results suggest that SPI2 can significantly affect the expression of some T3SS2 effector genes. Some effectors may have secretion pathways other than T3SS2 and pseudogenes may play roles in the process of S. Pullorum infection.

O-polysaccharide is important for Salmonella Pullorum survival in egg albumen, and virulence and colonization in chicken embryos.

The pathogen Salmonella Pullorum is the causative agent of persistent systemic infection of poultry, leading to economic losses in developing countries due to morbidity, mortality and reduction in egg production. These infections may result in vertical transmission to eggs or progeny. Limited information is available regarding the mechanisms involved in the survival of Salmonella Pullorum in egg albumen and developing chicken embryos. Hence, we investigated the role of O-polysaccharide in the contamination of eggs and the colonization of chicken embryos. Compared with the wild-type strain, the isogenic waaL mutant exhibited an O-antigen-deficient rough phenotype, and increased sensitivity to egg albumen and chicken serum, as well as reduced adherence to DF-1 cells. Infection with Salmonella Pullorum lacking O-polysaccharide resulted in significantly reduced embryo lethality and bacterial colonization. These results suggest that O-polysaccharide is essential for Salmonella Pullorum colonization in eggs, both post-lay and developing embryos. The chicken embryo infection model could be used to characterize the interaction between Salmonella Pullorum and developing embryos, and it will also contribute to the development of more rational vaccines to protect laying hens and embryos.

Construction and characterization of a cigR deletion mutant of Salmonella enterica serovar Pullorum.

Salmonella enterica serovar Pullorum (S. Pullorum) is the causative agent of pullorum disease (PD) and results in severe economic losses to the poultry industry. As a Salmonella type III secretion system 2 (T3SS2) effector and predicted membrane protein, CigR is encoded by the cigR gene within Salmonella pathogenicity island 3 (SPI3). In order to research the influence of the cigR gene on S. Pullorum, a cigR mutant of S. Pullorum S06004 was constructed by the lambda Red recombination system, and then its characterization was analysed. Lack of cigR did not affect the growth and biochemical properties, but resulted in decreased biofilm formation. The mutant strain was stable with the deletion of the cigR gene. Macrophage infection assay and in vivo competition assay showed that the mutant strain increased the replication and/or survival ability in the HD11 cell line and in chickens compared to that of the parent strain, the median lethal dose (LD50) of the mutant strain was one-fifth of the parent strain for 2-day-old chickens when injected intramuscularly. These results demonstrate CigR plays roles in biofilm formation and pathogenicity of S. Pullorum, deletion of cigR can significantly decrease biofilm formation and significantly increase virulence.

High-Efficiency, Two-Step Scarless-Markerless Genome Genetic Modification in Salmonella enterica.

We present a two-step method for scarless-markerless genome genetic modification in Salmonella enterica based on the improved suicide plasmid pGMB152. The whole LacZYA gene can provide a lacZ-based blue/white screening strategy for fast selection of double-crossover mutants by allelic exchange. The high efficiency of this genetic engineering strategy permits the study of gene function by gene knockin, site-directed mutagenesis, and gene knockout to construct live attenuated vaccines.

[Deletion-mutant construction, prokaryotic expression and characterization of peptidal-prolyl cis-trans isomerase SlyD from Salmonella enteritidis].

Objective: Salmonella enterica serovar enteritidis is an important food-borne pathogen of human and animal. To further study the function of SlyD associated with virulence and regulation in stress responses of Salmonella Enteritidis, we constructed slyD gene-deletion mutant,, expressed it in E. coli, and characterized the PPIase enzyme obtained. Methods: The slyD gene-deletion mutant of Salmonella enteritidis C50041 was constructed by suicide plasmid mediated homologous recombination. Salmonella enteritidis slyD prokaryotic expression vector was carried out in E. coli, and PPIase activity of recombination SlyD was measured in protease-coupling assay with chymotrypsin. For amino acids conservation studies, functional domain searches and secondary structure predictions, the BLAST, SMART, TMHMM, SignalP, PHD and SWISS MODEL were used. Results: Salmonella enteritidis C50041 ΔslyD mutant strain was successfully constructed. The growth rate of slyD-deleted strain was identified consistent with its parent strain C50041. A soluble recombinant SlyD protein was expressed in Escherichia coli BL21(DE3) cells and confirmed by SDS-PAGE. Catalytic activity confirmed that the SlyD protein was biologically active. Bioinformatic analysis showed that Salmonella Enteritidis SlyD as a multifaceted protein including three separated domains, the FKBP type peptidal-prolyl cis-trans isomerase domain, the IF chaperone domain and the metal-binding domain. Conclusion: Salmonella enteritidis C50041 ΔslyD mutant strain and soluble SlyD protein was obtained, and the present study may provide a basis for further study of the role of SlyD in Salmonella enteritidis.

A porcine reproductive and respiratory syndrome virus (PRRSV) vaccine candidate based on the fusion protein of PRRSV glycoprotein 5 and the Toll-like Receptor-5 agonist Salmonella Typhimurium FljB.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is characterized by severe reproductive failure and severe pneumonia in neonatal pigs and is caused by PRRS virus (PRRSV). Glycoprotein 5 (GP5) from PRRSV is a key inducer of neutralizing antibodies. Flagellin, a toll-like receptor 5 (TLR-5) agonist, is an effective inducer of innate immune responses. This study presents a novel PRRSV vaccine candidate based on the adjuvant effect of Salmonella Typhimurium FljB fused with PRRSV GP5. RESULTS: A truncated rGP5 gene lacking the signal peptide and transmembrane sequences was amplified and inserted into prokaryotic expression vectors, pColdI or pGEX-6p-1. Salmonella Typhimurium flagellin fljB was amplified and inserted into the plasmid pCold-rGP5, generating recombinant plasmid pCold-rGP5-fljB. Histidine (His)-tagged rGP5 and fusion protein rGP5-FljB were induced with isopropyl-ß-d-thiogalactoside, verified by SDS-PAGE and western blotting, and purified via Ni-NTA affinity columns. The TLR-5-specific bioactivity of fusion protein rGP5-FljB was determined by detecting the expression levels of the cytokine IL-8 in HEK293-mTLR5 cells by sandwich ELISA. The purified endotoxin-free proteins were administered intraperitoneally in a C3H/HeJ mouse model. The results show that immunization with the fusion protein rGP5-FljB induced a significantly enhanced GP5-specific and PRRSV-specific IgG response that persisted for almost 5 weeks. Co-administration of the rGP5 with R848 or Alum also yielded a higher IgG response than administration of rGP5 alone. The IgG1/IgG2a ratio in the rGP5-FljB immunization group was significantly higher (9-fold) than that in the rGP5 alone group and was equivalent to the response in the rGP5 + Alum immunization group, suggesting a strong Th2 immune response was induced by the fusion protein. CONCLUSIONS: Purified fusion protein rGP5-FljB is capable of activating the innate immune response, as demonstrated by the results of our TLR-5-specific bioactivity assay, and FljB has adjuvant activity, as shown by the results from our administration of rGP5-FljB in a mouse model. Our findings confirm that FljB could serve as an excellent adjuvant for the production of GP5-specific and PRRSV-specific IgG antibodies as part of an induction of a robust humoral immune response.

Immunogenicity and protective efficacy of Salmonella enterica serovar Pullorum pathogenicity island 2 mutant as a live attenuated vaccine candidate.

BACKGROUND: Salmonella enterica serovar Pullorum (S. Pullorum) causes Pullorum disease (PD), a severe systemic disease of poultry and results in considerable economic losses in developing countries. In order to develop a safe and immunogenic vaccine, the immunogenicity and protective efficacy of S06004ΔSPI2, a Salmonella pathogenicity island 2 (SPI2) deleted mutant of S. Pullorum was evaluated in 2-day old chickens. RESULTS: Single intramuscular vaccination with S06004ΔSPI2 (2 × 10(7) CFU) of chickens revealed no differences in body weight or clinical symptoms compared to control group. S06004ΔSPI2 bacteria can colonize and persistent in liver and spleen of vaccinated chickens approximately 14 days, and specific humoral and cellular immune responses were significantly induced. Vaccination of chickens offered efficient protection against S. Pullorum strain S06004 and S. Gallinarum strain SG9 challenge, respectively, at 10 days post vaccination (dpv) based on mortality and clinical symptoms compared to control group. CONCLUSIONS: These findings suggest that S06004ΔSPI2 appears to be a highly immunogenic and efficient live attenuated vaccine candidate.

Identification and immune functional characterization of pigeon TLR7.

Toll-like receptor 7 (TLR7) is activated by single-stranded RNA and synthetic imidazoquinoline components, and induces interferon production. In this study, we cloned the TLR7 gene from King pigeon (Columba livia). The TLR7 open reading frame is 3144 bp and encodes a 1047-amino acid protein, consisting of a canonical TLR composition with 15 leucine-rich repeats (LRRs). Amino acid-inserting modifications were found at position 15 of LRR2, LRR11, LRR13, and LRR14 and position 10 of LRR10. The tissue distribution of pigeon TLR7 suggests that immune-associated tissues, especially the spleen and liver, have high TLR7 expression. HEK293T cells transfected with pigeon TLR7 plasmid responded to the agonist R848, indicating a functional TLR7 homolog. Following R848 stimulation of pigeon peripheral blood mononuclear cells, the levels of IFN-γ, IL-6, IL-8, CCL5, and IL-10 mRNA, assessed using quantitative real-time PCR, were significantly up-regulated. After Newcastle disease virus vaccine strain LaSota inoculation and agonist R848 injection, the level of TLR7 mRNA in the spleen of pigeons increased significantly in the R848-injected group, but decreased in the LaSota-inoculated group at three day post-infection (d.p.i.). The mRNA levels of inflammatory cytokines and chemokines were significantly upregulated in both LaSota-inoculated and R848-injected groups. Triggering pigeon TLR7 leads to robust up-regulation of inflammatory cytokines and chemokines, suggesting an important role in the innate immune response.

[Inflammasome responses in macrophages induced by C-terminal peptides of Escherichia coli EscI protein].

OBJECTIVE: To analyze NLRC4 inflammasome responses in macrophages induced by C-terminal of Escherichia coli EscI protein. METHODS: NLRC4 inflammasome responses in mouse peritoneal macrophages were analyzed after delivery of the peptides containing C-terminal amino acid sequences of E. coli EscI protein in vitro. RESULTS: The peptides containing C-terminal 15 amino acids of EscI protein could significantly activate NLRC4 inflammasome responses in macrophages pre-stimulated with lipopolysaccharide. Intracellular caspase-1 was activated and pyroptotic dead cells were found after peptides delivery. The contents of cytokines, IL-1ß and IL-18, in supernatants were elevated significantly compared with that of the control (P < 0.05). Besides, through comparison of IL-1ß contents under different stimulation conditions, 4 h incubation after peptides delivery (peptides: lipofectamine 2000 = 70 µg/µL) could obviously promote the secretion of IL-1ß. CONCLUSION: Peptides containing C-terminal 15 amino acids of E. coli EscI protein can significantly induce NLRC4 inflammasome activation in macrophages.

[Construction and characterization of Salmonella Pathogenicity Island 2 (SPI-2) deletion mutant of Salmonella Pullorum S06004].

OBJECTIVE: To research the pathogenicity of Salmonella Pathogenicity Island 2 (SPI-2) deletion mutant of Salmonella Pullorum and preliminary explore the feasibility of developing safe attenuated Salmonella Pullorum candidate vaccine strain. METHODS: The SPI-2 (-40 kb) deletion mutant of Salmonella Pullorum S06004 was constructed using the λ-red recombinant system. Then the biological characteristics such as growth rate, biochemical properties, genetic stability and virulence were evaluated between the deletion mutant strain S06004ΔSPI2 and its parent strain S06004. RESULTS: S06004ΔSPI2 was successfully constructed. The growth rate and biochemical properties of S06004ΔSPI2 were consistent with those of its parent strain S06004. The mutant was stable with the deletion of SPI-2. Chicken lethal test showed that the LD50 of S06004ΔSPI2 was 252 times higher than the parent strain S06004. CONCLUSION: The virulence of S06004ΔSPI2 was obviously attenuated. This study provided basic data for further study of the functions of SPI-2, and implied its potential to develop attenuated Salmonella vaccine.

Nifuratel reduces Salmonella survival in macrophages by extracellular and intracellular antibacterial activity.

Salmonella are intracellular bacterial pathogens for which, as with many of the other Enterobacteriaceae, antibiotic resistance is becoming an increasing problem. New antibiotics are being sought as recommended by the World Health Organization and other international institutions. These must be able to penetrate macrophages, and infect the major host cells and the Salmonella-containing vacuole. This study reports screening a small library of Food and Drug Administration (FDA)-approved drugs for their antibacterial effect in macrophages infected with a rapid-multiplying mutant of Salmonella Enteritidis. The most effective drug that was least toxic for macrophages was Nifuratel, a nitrofuran antibiotic already in use for parasitic infections. In mice, it provided 60% protection after oral infection with a lethal S. Enteritidis dose with reduced bacterial numbers in the tissues. It was effective against different serovars, including multidrug-resistant strains of Salmonella Typhimurium, and in macrophages from different host species and against Listeria monocytogenes and Shigella flexneri. It reduced IL-10 and STAT3 production in infected macrophages which should increase the inflammatory response against Salmonella. IMPORTANCE Salmonella can keep long-term persistence in host's macrophages to evade cellular immune defense and antibiotic attack and exit in some condition and reinfect to cause salmonellosis again. In addition to multidrug resistance, this infection circle causes Salmonella clearance difficult in the host, and so there is a great need for new antibacterial agents that reduce intramacrophage Salmonella survival to block endogenous Salmonella reinfection.

Safety and protective efficacy of Salmonella Pullorum spiC and rfaH deletion rough mutant as a live attenuated DIVA vaccine candidate.

Salmonella enterica serovar Pullorum (S. Pullorum) causes pullorum disease (PD), which is an acute systemic disease, in chickens, and leads to serious economic losses in many developing countries because of its high morbidity and mortality rate in young chicks. The live-attenuated vaccine is considered to be an effective measure to control the Salmonella infection. In addition, the DIVA (differentiation of infected and vaccinated animals) feature without the interference of serological monitoring of Salmonella infection is an important consideration in the development of the Salmonella vaccine. In this study, we evaluated the immunogenicity and protective efficacy of a S. Pullorum rough mutant S06004ΔspiCΔrfaH as a live attenuated DIVA vaccine candidate in chickens. The S06004ΔspiCΔrfaH exhibited a significant rough lipopolysaccharides (LPS) phenotype which was agglutinated with the acriflavine, not with the O9 mono antibody. Compared to the wild-type, 50% lethal dose (LD50) of the rough mutant increased 100-fold confirmed its attenuation. The mutant strain also showed a decreased bacterial colonization in the spleen and liver. The immunization with the mutant strain had no effect on the body weight and no tissue lesions were observed in the liver and spleen. The high level of the S. Pullorum-specific IgG titers in the serum indicated that significant humoral immune responses were induced in the immunization group. The cellular immune responses were also elicited from the analysis of lymphocyte proliferation and expression of cytokines in the spleen. In addition, the S06004ΔspiCΔrfaH immunized group exhibited a negative response for the serological test, while the wild-type S06004 infection group was strongly positive for the serological test showing a DIVA capability. The survival rates in the vaccinated chickens were 87% after intramuscular challenge with wild-type S. Pullorum, while the survival rates were 20% in the control groups. Overall, these results have demonstrated that the rough mutant S06004ΔspiCΔrfaH strain can be developed as an efficient live attenuated DIVA vaccine candidate to control the systemic S. Pullorum infection without the interference of salmonellosis monitoring program in poultry.

A phage for the controlling of Salmonella in poultry and reducing biofilms.

As a natural alternative to traditional antimicrobials, phages are being recognised as highly effective control agents for Salmonella and other foodborne bacteria. Due to the high diversity of Salmonella serotypes and the emergence of phage-resistant strains, attempting to isolate more widespread, strictly lytic Salmonella phages is highly warranted. In this study, a lytic phage, LP31, was isolated from poultry faecal samples. Transmission electron microscopy revealed that the phage had a polyhedral head and a retraction-free tail, indicative of the Siphoviridae family. Adsorption rate experiments showed that LP31 required the participation of lipopolysaccharides, but not flagella, during phage adsorption. Host profile identification showed that LP31 could lyse most Salmonella Enteritidis (S. Enteritidis) (96.15%, N = 104) and Salmonella Pullorum (S. Pullorum) (96.67%, N = 60). Initial applications found that LP31 reduced the concentration of static S. Enteritidis on metal surfaces (0.951 log10 cfu/ml) and in the faeces of chicks (2.14 log10 cfu/g). Notably, LP31 could almost completely remove biofilms formed by S. Enteritidis and S. Pullorum in 1 h. These findings suggest that LP31 has a good prevention and control effect against biofilms and planktonic antibiotic-resistant Salmonella, and is therefore a potentially promising biocontrol agent for controlling the spread of Salmonella in the poultry and food processing industries.

Safety of the Salmonella enterica serotype Dublin strain Sdu189-derived live attenuated vaccine-A pilot study.

Salmonella enterica serovar Dublin (S. Dublin) is an important zoonotic pathogen with high invasiveness. In the prevention and control of the Salmonella epidemic, the live attenuated vaccine plays a very important role. To prevent and control the epidemic of S. Dublin in cattle farms, the development of more effective vaccines is necessary. In this study, we constructed two gene deletion mutants, Sdu189ΔspiC and Sdu189ΔspiCΔaroA, with the parental strain S. Dublin Sdu189. The immunogenicity and protective efficacy were evaluated in the mice model. First, both mutant strains were much less virulent than the parental strain, as determined by the 50% lethal dose (LD50) for specific pathogen-free (SPF) 6-week-old female BALB/c mice. Second, the specific IgG antibody level and the expression level of cytokine TNF-α, IFN-γ, IL-4, and IL-18 were increased significantly in the vaccinated mice compared to the control group. In addition, the deletion strains were cleared rapidly from organs of immunized mice within 14 d after immunization, while the parental strain could still be detected in the spleen and liver after 21 d of infection. Compared with the parental strain infected group, no obvious lesions were detected in the liver, spleen, and cecum of the deletion strain vaccinated groups of mice. Immunization with Sdu189ΔspiC and Sdu189ΔspiCΔaroA both provided 100% protection against subsequent challenges with the wild-type Sdu189 strain. These results demonstrated that these two deletion strains showed the potential as live attenuated vaccines against S. Dublin infection. The present study established a foundation for screening a suitable live attenuated Salmonella vaccine.

The Loss of focA Gene Increases the Ability of Salmonella Enteritidis to Exit from Macrophages and Boosts Early Extraintestinal Spread for Systemic Infection in a Mouse Model.

Salmonella Enteritidis (SE) can spread from the intestines to cause systemic infection, mainly involving macrophages. Intramacrophage Salmonella exits and reinfects neighboring cells, leading to severe disease. Salmonella genes involved in exiting from macrophages are not well understood or fully identified. A focA::Tn5 mutant was identified by an in vitro assay, with increased ability to exit from macrophages. A defined SEΔfocA mutant and its complemented derivative strain, SEΔfocA::focA, were constructed to confirm this phenotype. Although the lethal ability of focA mutants was similar to that of the parental SE in mice, it was isolated earlier from the liver and spleen than the parental SE. focA mutants induced higher levels of proinflammatory IL-12 and TNF-α compared with the parental SE and SEΔfocA::focA. focA mutants showed higher cytotoxicity and lower formate concentrations than SE and SEΔfocA::focA, whereas there was no change in pyroptosis, apoptosis and flagella formation ability. These current data suggest that the focA gene plays an important role in regulating intramacrophage Salmonella exiting and extraintestinal spread in mice, although the specific mechanism requires further in-depth studies.