Bacterial strains, plasmids, and growth conditions
All bacterial strains were routinely grown in Luria Bertani (BD, Sparks, MD, USA) medium with agitation at 37°C using appropriate antibiotics as selection markers wherever applicable. All the bacterial strains and plasmids used in the present study are listed in Table 1. In this study, an attenuated SG strain was developed with the deletion of lon, rfaL, and arnT or pagL genes as a therapeutic strain for Salmonellosis. For this, the SG 914 strain (Δlon) [14] was engineered to develop the SG JOL3015 strain (Δlon ΔrfaL ΔarnT) and SG JOL3016 strain (Δlon ΔrfaL ΔpagL) applying the λ red recombination technique described elsewhere with modifications [15]. This recombineering approach inserts a chloramphenicol resistance (catR) gene into the chromosome by replacing the target gene. Briefly, the parent SG strain was transformed with a helper plasmid, pKD46, and induced to express recombinase with L-arabinose for homologous recombination. The linear DNA cassette of the catR gene flanked by a rfaL gene homologous sequence was amplified from pKD3 and electroporated (Harvard Apparatus, USA) in pKD46-transformed Salmonella. The rfaL-deleted mutant colonies were screened by plating on LB media containing chloramphenicol. Colonies were confirmed by inner primers and transformed with pCP20 plasmid to eliminate the FRT-flanked catR through flippase production. The catR deletion was confirmed by flanking primers, as listed in Table 2. The procedure was reprised to include arnT and pagL deletion in the respective strains. A commercially available vaccine SG9R was procured (9R VAC®, Komipharm International Co. Ltd., Siheung, Korea) for the comparative study.
Bacterial growth kinetics of the engineered Salmonella Gallinarum strains
Bacterial growth of the engineered attenuated SG strain was evaluated alongside wild type and commercial strain, SG9R. Bacterial cultures grown overnight were inoculated (1%, v/v) to 50 mL LB broth, incubated at 37oC in a shaking incubator at 200 rpm. Optical density at 600 nm (OD600) was measured using an Infinite M200 spectrophotometer (Tecan, Seestrasse, Switzerland) every 4 hours in a 96-well plate (200 μL). Growth kinetics were further validated by enumerating colony-forming units (CFUs) from serially diluted broth cultures. Samples were taken every 4 h, serially diluted, spread (100 μL) on LB agar plates, and incubated at 37°C. Plates containing 30-300 colonies were counted to determine CFUs.
Auto-aggregation assay
The clustering ability of bacteria under culture conditions was assessed using an auto-aggregation assay. Overnight bacterial cultures were prepared and inoculated at a 1:100 dilution in LB broth. The cultures were then incubated at 37°C for 24 h. Subsequently, the optical density (OD) of the upper layer of the culture (collected without disturbing the culture, OD600 pre-resuspension) and the re-suspended culture (OD600 post-resuspension) after vortexing were measured at 600 nm. The level of auto-aggregation was determined as a percentage using the formula: [(OD600 post-resuspension – OD600 pre-resuspension) / OD600 post-resuspension] × 100.
Hemolysis assay
Overnight cultures of the wild-type and mutant strains were grown, followed by centrifugation at 8000 rpm for 10 minutes to collect the culture supernatants. To eliminate bacterial contaminants, the supernatants were filtered using 0.2 μm membrane filters (BioFACT, Parit Jamil, Malaysia). The resulting sterile solutions were then mixed into a 10% chicken red blood cell (RBC) suspension at a 4:1 ratio and incubated in a shaking incubator at 37°C for 12 hours. A control was prepared by adding LB broth to the RBC suspension at the same ratio. After incubation, the suspensions were centrifuged at 2000 rpm for 5 minutes [16]. Hemolysis rates were determined by absorbance measurements at 570 nm using a multi-well plate reader (Tecan, Männedorf, Switzerland).
Acriflavine agglutination test
Lack of O-antigen components and confirmation of rough phenotype was conducted by acriflavine agglutination test [17]. Bacteria cultures were grown on LB agar plates for 24 h, and selected bacterial colonies were collected and mixed into 30 μL of 0.2% acriflavine solution on glass slides. Cells were gently mixed, interacted for 2 min., and observed under a microscope at 40 × magnification or by the naked eye.
Western blot of lipopolysaccharides
Bacterial lipopolysaccharides were extracted using a phenolic-based extraction method using an LPS extraction kit (iNtRON Biotechnology, Seoul, South Korea) following the manufacturer’s recommendations. The LPS samples were separated on 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Western blotting was performed using a mouse monoclonal antibody against Salmonella O antigen at 1:1000 (cat. no. 10R-S103b, Fitzgerald, MA, USA) and goat anti-mouse IgG-HRP conjugate at 1:5000 dilution (cat. no. 1030-05, SouthernBiotech, Birmingham, AL 35209 USA). All steps were conducted according to a previously described procedure [18].
Adhesion and Invasion
The adhesion and invasion capability of SG strains were evaluated in Hela and chicken peripheral blood mononuclear cells (PBMCs) in vitro. Overnight cultures of bacterial cells were re-inoculated to LB medium as 1% inoculum and incubated for 3 h to reach 0.4 – 0.6 absorbance at OD600. Cells were collected by centrifugation at 12000 × g for 5 min and washed with phosphate-buffered saline. Blood was collected from the wing vein of the bird, following the standard procedure, and PBMCs were isolated using Ficoll-Paque PLUS density gradient media (Cytiva, Uppsala, Sweden) [19]. Collected macrophages and Hela cells were seeded in 12 well plates at 2 × 106 and 2 × 105 cells/ well. The PBMCs were incubated for 5 h for attachment and then the media was changed. When the Hela cells reached 60 – 70% confluence, bacterial adhesion and invasion were conducted. Cells were infected with SG WT, SG9R, and the attenuated strains at 40 multiplicity of infection (MOI), 30 min for adhesion and 2.5 h for invasion. In the invasion assay, bacteria were incubated with cells for 2.5 h to invade the cells. The noninfected bacteria were eliminated by 2 h gentamycin treatment (100 µg/mL). Adhered or invaded cells were retrieved by lysis of monolayers using 0.25% Triton X-100 and the bacterial enumeration was done by counting on Brilliant Green Agar (BGA) plates (BD Difco).
Cell survival and cytotoxicity assay
Salmonella-induced cell cytotoxicity was assessed using an IncuCyte live imaging system (Essen Bioscience, MI, USA). HeLa cells were seeded at a density of 5×10⁵ cells/mL in 12-well plates. Cells were infected with Salmonella at a multiplicity of infection (MOI) of 40 for 2 h. Post-infection, cells were washed twice with phosphate-buffered saline (PBS) to remove dead cells, non-adherent bacteria, and debris. Subsequently, non-infected cells were eliminated by treatment with Gentamycin (100 μg/mL) for 2 h. Following this, cells were treated with propidium iodide (5 µL/mL, cat. no. 556463, BD Biosciences, California, USA) and monitored via imaging at 6 h intervals over 24 h.
Safety evaluation of detoxified SG strains in chicken
The safety of detoxified Salmonella Gallinarum (SG) strains was assessed following intramuscular (IM) inoculation at varying concentrations in female brown-layer chickens. Four-week-old chickens (n = 12) were inoculated either with the SG wild-type (WT JOL422), attenuated strains JOL3015 and JOL3016, or a commercial vaccine strain, SG9R. Post-inoculation, the birds were monitored for morbidity and mortality associated with fowl typhoid (FT). Clinical parameters such as body temperature, abnormal behavior, anorexia, and feed intake were observed to detect any adverse effects caused by SG infection.
To evaluate bacterial persistence in vital organs, the attenuated strains JOL3015 and JOL3016 were administered at concentrations of 1×10⁷ CFU/bird (Low) and 1×10⁸ CFU/bird (High), SG9R and SG WT JOL422 were administered at a concentration of 1×10⁷. Three chickens from each group were sacrificed at 3, 7, and 14 days post-inoculation (dpi) for sample collection. Chickens were euthanized, and the spleen and liver were aseptically collected. The collected organs were homogenized in PBS using a mechanical homogenizer (IKA T 10 basic ULTRA-TURRAX, Germany) and plated on BGA at 10-fold serial dilutions to quantify bacterial load. Additionally, cloacal swabs were collected using sterilized cotton swabs in 1 mL PBS to evaluate bacterial shedding for environmental safety. The swab samples were thoroughly mixed, serially diluted in PBS, and plated on BGA. Body weight changes were monitored at three-day intervals up to 15 days post-inoculation to assess the impact on body weight gain.
Histopathological evaluation of organ damage
The evaluation of harm inflicted by the attenuated SG strains on specific organs namely, the liver, spleen, and cecum was conducted through a detailed histopathological examination using hematoxylin and eosin (H&E) staining. Three birds per group were sacrificed on the seventh-day post-inoculation, and the organs were collected and fixed in 10% formalin. The tissues were then sectioned into 3 µm slices, fixed, and processed according to a standard protocol for H&E staining. This process involved dehydration, clearing, embedding, and staining to allow for clear visualization of tissue architecture. A comprehensive investigation of potential tissue damage was performed using a Zeiss Axio Imager.M2 microscope (Carl Zeiss AB, Stockholm, Sweden). Microscopic examination allowed for the assessment of cellular and structural integrity and images were documented for further analysis.
Quantification of cytokines
Chickens inoculated with engineered SG strains underwent endotoxicity assessment through the quantification of inflammatory cytokines. Serum samples were collected on Day 3 post-inoculation. To evaluate in vitro endotoxicity induced by the structural modification of bacterial LPS, LPS was extracted from SG mutants JOL3015, JOL3016, SG9R, and the SG WT strain using an LPS extraction kit (iNtRON, Korea). The PBMCs were isolated from 8-week-old chickens and cultured in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) in 12-well plates at a density of 5×10⁵ cells per well. PBMCs were treated with isolated LPS at 100 ng/mL concentrations. The levels of inflammatory cytokines, including TNF-α, IL-1β, and IFN-γ, were measured using commercial sandwich-ELISA kits following the manufacturer's instructions.
Briefly, for the TNF-α assay, micro-ELISA plates pre-coated with an antibody specific to chicken TNF-α (Cat. No. MBS2509660, MyBioSource, San Diego, USA) were incubated with serum samples and standards for 90 minutes at 37°C. A biotinylated detection antibody specific to chicken TNF-α and an Avidin-Horseradish Peroxidase (HRP) conjugate were successively added to the microplate wells and incubated. After washing away free components, a substrate solution was added, and the enzyme-substrate reaction was stopped with a stop solution. Optical density at 450 nm was measured using an Infinite M200 spectrophotometer (Tecan). The concentration of TNF-α in the samples was estimated using a reference standard. Similarly, IL-1β (Cat. No. MBS2702032, MyBioSource) and IFN-γ (Cat. No. MBS2700893, MyBioSource) levels were quantified using comparable procedures, adapted from the manufacturer's instructions.
Immunization and challenge against fowl typhoid using attenuated SG strain.
The immune response elicited by inoculation with attenuated SG strains was evaluated in 4-week-old female brown chickens. Birds (n = 8) were intramuscularly immunized with JOL3015 and JOL3016 strains at a concentration of 1×10⁷ CFU/200 μL per bird. A commercial vaccine strain, SG9R, was used as a comparative control and administered intramuscularly at the same concentration. Additional groups served as PBS and naïve controls. After two weeks, birds received a booster inoculation with the attenuated SG strains. Serum and cloacal swab samples were collected at intervals up to five weeks from the initial inoculation. These samples were used to measure levels of IgY and IgA antibodies.
Two weeks after the booster inoculation, blood samples were collected, and PBMCs were isolated. Flow cytometry was performed to quantify the cell-mediated immune response by assessing T-cell counts. Three weeks after booster application, chickens were challenged with wild-type SG strain (SG WT JOL422) via the oral route using 1×106 CFU/200 µL per bird. Post-challenge survival rate was evaluated by monitoring for up to 15 days. Animals were sacrificed at the end of the experiment to examine gross morphological distortion in the vital organs. Bacterial persistence in the spleen and liver of immunized chickens was investigated to elucidate the bacterial load. In addition, the spleen and liver tissues were collected for H&E staining as described elsewhere [20].
ELISA Humoral and mucosal immune responses
Salmonella-specific systemic IgY and mucosal IgA responses in immunized birds were quantified using an indirect ELISA. For this assay, 96-well plates were coated with 400 ng/well of crude soluble protein extracted from the SG wild-type strain JOL422, dissolved in a carbonate-bicarbonate buffer. The plates were incubated overnight at 4°C to allow for proper antigen coating. The following day, plates were blocked with 5% skim milk for 1 h at room temperature (RT) to prevent non-specific binding. Serum samples were diluted 1:50 for IgY detection, while undiluted cloacal swab samples were used for IgA detection. Samples were added to the wells and incubated for 2 h at RT. After the incubation period, the plates were washed three times with PBS-T (PBS containing 0.05% Tween 20) to remove any unbound antibodies. Subsequently, the plates were incubated with secondary antibodies: goat anti-chicken IgY-HRP (Bethyl Laboratories, Texas, USA) for IgY detection and goat anti-chicken IgA-HR-P (Bethyl Laboratories, Texas, USA) for IgA detection following the manufacturer's instructions at 1:3000 dilution. The plates were incubated for 1 h at RT and washed with PBS-T to remove excess secondary antibodies. The colorimetric detection was carried out by adding an O-phenylenediamine dihydrochloride substrate (Sigma, Missouri, USA). The reaction was allowed to proceed for 15-30 minutes in the dark at RT until sufficient color development was achieved. The enzyme-substrate reaction was stopped by adding 50 μL of 2N sulfuric acid. The optical density (OD) was measured at 492 nm using an Infinite M200 microplate reader (Tecan). The absorbance values obtained were used to quantify the levels of IgY and IgA antibodies in the serum and cloacal swab samples, respectively.
Flow cytometry
The cell-mediated immune responses were investigated by evaluating T-lymphocyte subsets via flow cytometry analysis. Two weeks after the booster immunization, blood was collected from all groups (n = 5) to isolate PBMCs. Mononuclear cells were separated from whole blood using density gradient centrifugation according to the manufacturer’s instructions. Briefly, blood was diluted 1:1 with phosphate-buffered saline (PBS; pH 7.4) to a final volume of 2 mL and carefully layered over 2 mL of Ficoll-Paque PLUS density gradient media (Cytiva, Uppsala, Sweden) in a centrifuge tube. The samples were centrifuged at 400×g for 30 minutes at 18°C to separate the buffy coat layer. The PBMCs were collected from the interface and then washed twice with PBS to remove residual Ficoll and plasma. The harvested cells were resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics. The cells were then seeded in 96-well plates at a density of 1×10⁵ cells/well and stimulated with 400 ng/well of crude soluble antigen extracted from the SG wild-type strain for 72 hours in a 5% CO₂ incubator at 37°C. Following antigen stimulation, cells were collected and incubated with fluorescently labeled antibodies: anti-CD3-FITC (Cat: 8200-02, SouthernBiotech, Birmingham, AL, USA), anti-CD8-PE (Cat: 8220-09, SouthernBiotech), and anti-CD4-AF700 (Cat: 8210-31, SouthernBiotech) (each at a concentration of 8 μg/mL) at 4°C for 30 minutes in the dark. After incubation, cells were washed with FACS buffer (PBS containing 2% FBS and 0.1% sodium azide) to remove unbound antibodies. The stained cells were then analyzed using a Macsquant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). Data acquisition was performed, and T-cell subsets (CD3⁺, CD4⁺, and CD8⁺) were quantified. The results were analyzed using Macsquant analysis software (version 2.6), allowing for a detailed assessment of the cell-mediated immune response elicited by the immunization.
Statistical analysis
Statistical analysis was performed using Student's t-test and ANOVA to evaluate statistical differences. A p-value <0.05 was considered significant. All analyses were done in GraphPad Prism 9.00 software (San Diego, CA, USA).