Comparative evaluation of the immunogenicity and protective efficacy of gamma-irradiated Salmonella Gallinarum (γ-SG), SG ΔpurB mutant, and their combined vaccine formulations in chicken

Abstract

Salmonella Gallinarum (SG), continues to impose economic losses in poultry, highlighting the need for better vaccination strategies to improve immunogenicity and protection. This study comparatively evaluated the immunogenicity and protective efficacy of a γ-SG vaccine, an SG ΔpurB live attenuated mutant, and their combined vaccine formulations in chickens. Five groups of chickens (A-E) were administered with SG ΔpurB IM (2.0 × 10⁸ CFU), γ-SG Oral (2.0 × 10⁸ CFU), SG ΔpurB + γ-SG Oral (2.0 × 10⁸ CFU), SG ΔpurB + γ-SG IM (2.0 × 10⁸ CFU), and an unvaccinated control (200 μL PBS), respectively. Combination vaccine doses were prepared using a 1:1 formulation, with each vaccine administered at 1.0 × 10⁸ CFU per bird to achieve a total dose of 2.0 × 10⁸ CFU. Comparative analysis of the vaccinated groups revealed that the SG ΔpurB + γ-SG Oral formulation induced the strongest early immune responses with significantly elevated CD4⁺ (31%) and CD8⁺ (19%) T cell populations at 1 WPV (week post vaccination) compared with other vaccinated and unvaccinated control groups (p < 0.0001). At 2 and 3 WPV, the highest levels of CD4⁺ and CD8⁺ T cells were observed in the γ-SG Oral group, whereas a transient decline in these T cell populations was observed in the other vaccinated groups compared to the γ-SG Oral group (p < 0.0001). Humoral IgG responses followed a similar trend, SG ΔpurB + γ-SG Oral group attaining the highest titers at 1 WPV (p < 0.0001). At 2 and 3 WPV, antibody titers were highest in the γ-SG Oral group, while a significant decline was observed in all other vaccinated groups compared to the γ-SG Oral group (p < 0.0001). Although the other vaccinated groups also demonstrated protection against virulent SG challenge, but the highest protective efficacy was achieved by the γ-SG Oral group, which provided 100% survival. However further dose optimization of combined vaccine formulation is required to obtain sustained immunity.

1. Introduction

Fowl typhoid is a septicemic disease of adult birds caused by Salmonella Gallinarum and characterized by variable morbidity and substantial mortality [1]. In developing countries, fowl typhoid remains a major cause of economic losses in poultry industries [2]. Many control measures have been implemented to reduce Salmonella infection in chickens, including improvements in hygiene, better husbandry management, and the use of antibiotics and vaccines [3,4]. In countries where Salmonella Gallinarum is endemic, vaccination represents the most practical and effective strategy for controlling fowl typhoid [5,6]. The rough mutant strain SG9R, a live attenuated vaccine strain, is developed to prevent fowl typhoid caused by Salmonella Gallinarum [7]. However, the use of the live SG9R vaccine is limited to layer breeds older than six weeks of age and is associated with suboptimal protective efficacy and residual virulence in internal organs [2,8,9]. The major limitation of live attenuated vaccines is the potential risk of reversion to virulence [10].

A novel attenuated mutant, SG ΔpurB, has recently been developed by targeted deletion of the purB gene, disrupting purine biosynthesis and leading to reduced growth and virulence, thereby highlighting its potential as a vaccine candidate against fowl typhoid in chickens [11]. In comparison to attenuated vaccines, inactivated vaccines are preferred, as they remove the risk of the vaccine strain reverting to a pathogenic form [12]. Among the inactivated vaccine approaches, gamma irradiation has been shown to inactivate bacteria without causing damage to their cell membranes. Moreover, substantial evidence indicates that cells exposed to lethal doses of ionizing radiation can retain residual metabolic activity following treatment [13]. A study investigating gamma-irradiated Salmonella Gallinarum in a chicken model demonstrated that vaccination by oral route elicited robust humoral and cell-mediated immune responses, underscoring the potential of gamma irradiation as an effective strategy for vaccine development [14].

In a novel combination approach a trivalent inactivated vaccine comprising S. Enteritidis, S. Typhimurium, and S. Infantis, with or without prior live SG9R vaccination, effectively induced antibody responses and markedly reduced S. Enteritidis shedding, underscoring its potential for broad Salmonella control in poultry [15]. This study is designed to assess the immunogenicity and protective efficacy of gamma-irradiated Salmonella Gallinarum (γ-SG) and live attenuated SG ΔpurB mutant and their combined vaccine formulations in chicken model.

2. Materials and methods

2.1. Ethical statement

The animal study protocol was reviewed and approved by the Ethical Review Committee of the Office of Research, Innovation and Commercialization (ORIC), UVAS, Lahore, Pakistan (Approval No. DR/93, March 6, 2025).

2.2. Bacterial strains

A well-characterized Salmonella enterica serovar Gallinarum strain (Accession no. CP150644.1) and its derived vaccine candidates used in this study are summarized in Table 1.

Table 1.

Bacterial strains and vaccine candidates used in this study.

Bacterial Strain	Characteristics	References
Wild-type Salmonella enterica serovar Gallinarum	Well-characterized field strain isolated from an outbreak in the district Sheikhupura, Province Punjab, Pakistan	[11]
Vaccine candidates
SG ΔpurB	Live attenuated S. Gallinarum mutant	[11]
γ-SG	Gamma-irradiated inactivated S. Gallinarum	This study

2.3. Vaccination dose preparation and formulations

SG ΔpurB mutant was grown overnight in Luria-Bertani (LB) broth at 37 °C. Bacterial growth was adjusted to an optical density at 630 nm (OD₆₃₀) of 0.5, equivalent to approximately 1.0 × 10⁸ colony forming units (CFU)/mL. Cells were harvested by centrifugation, washed with sterile phosphate-buffered saline (PBS) and resuspended in PBS to obtain a final concentration of 1.0 × 10⁸ CFU/mL.

γ-SG was prepared as inactivated Salmonella Gallinarum vaccine candidate. For this Salmonella Gallinarum was cultured overnight into Luria Bertani broth at 37 °C. Bacterial growth was adjusted to an optical density at 630 nm (OD₆₃₀) of 0.5, equivalent to approximately 1.0 × 10⁸ (CFU)/mL. Cells were harvested by centrifugation, washed with sterile phosphate-buffered saline (PBS) and resuspended in PBS to obtain a final concentration of 1.0 × 10⁸ CFU/mL prior to irradiation. Inactivation of bacterial cells was performed using a cobalt-60 gamma irradiator at a dose of 7 kGy at Pakistan Radiation Services (PARAS), as previously optimized by Ref. [14]. Complete inactivation was confirmed by the absence of growth on Brilliant Green Agar following 24 h incubation at 37 °C, in accordance with [16]. Residual metabolic activity after irradiation was assessed as described previously by Ref. [14].

For vaccine groups, the live SG ΔpurB mutant and γ-SG vaccines were prepared at 1.0 × 10⁸ CFU/mL and adjusted to a final administered dose of 2.0 × 10⁸ CFU per 200 μL. In the combination vaccine groups (SG ΔpurB + γ-SG) the total administered dose was also maintained at 2.0 × 10⁸ CFU per 200 μL. For this purpose, both the SG ΔpurB mutant and γ-SG vaccines were mixed in a 1:1 ratio, with each vaccine component adjusted to 1.0 × 10⁸ CFU per 100 μL, resulting in a final combined dose of 2.0 × 10⁸ CFU per 200 μL.

2.4. Chicken immunization study design

A total of 50 day-old Salmonella free broiler chickens (Ross 308) obtained from a commercial farmhouse and raised in the poultry houses of the Institute of Microbiology (IOM), UVAS, Lahore. Chickens were provided ad libitum access to water and feed throughout the experimental period and were randomly allocated into five groups (A-E), each group comprising on ten birds (n = 10). At 7days of age, Group A was vaccinated intramuscularly with (SG ΔpurB at a dose of 2.0 × 10⁸ CFU/200 μL PBS), while Group B was vaccinated orally with (γ-SG at a dose of 2.0 × 10⁸ CFU/200 μL PBS), Group C received the combined vaccine orally with (SG ΔpurB + γ-SG at a dose of 2.0 × 10⁸ CFU/200 μL PBS), consisting of (SG ΔpurB mutant; 1.0 × 10⁸ CFU/100 μL) and (γ-SG; 1.0 × 10⁸ CFU/100 μL). Group D was administered with the respective combination dose intramuscularly. Group E served as the unvaccinated control group, received 200 μL of sterile PBS orally. A booster dose was administered at one week post primary vaccination. A total of five randomly selected whole-blood samples from each group were collected on weekly basis for flow cytometry analysis and serum harvested for IgG antibody response using enzyme-linked immunosorbent assay (ELISA) using wing vein. Two weeks post-booster immunization, all birds were challenged with wild-type SG (2.0 × 10¹⁰ CFU in 200 μL PBS). Schematic illustration of the experimental groups is shown in Fig_1.

Fig_1.

Schematic illustration of the experimental groups used to evaluate the immunogenicity and protective efficacy of the vaccine candidates against Salmonella Gallinarum. The illustration was created using Servier Medical Art, licensed under the Creative Commons Attribution 4.0 International License (CC BY 4.0).

2.5. Evaluation of cellular immune response by flow cytometry analysis

2.5.1. Isolation of Peripheral Blood Mononuclear Cells (PBMCS) from whole chicken blood

Blood samples were collected from the wing vein of randomly selected chickens from each experimental group using EDTA-coated vacutainer tubes. Randomly selected blood samples from each group were pooled and prepared for flow cytometry analysis. Each whole blood sample was mixed with an equal volume of phosphate-buffered saline (1:1) and gently layered over the same volume of lymphocyte separation medium (LSM^™) for PBMCS isolation. Following centrifugation at 420 × g for 15 min at 4 °C, the mononuclear cell layer (buffy coat) was carefully aspirated, transferred to a fresh tube and washed with PBS. Cell viability and counts were determined using the trypan blue exclusion method with minor modifications and cell suspension was adjusted to a final concentration of 5.0 × 10⁵ cells/mL prior to analysis [17].

2.5.2. Staining of cells and flow cytometry analysis

Following cell counting, aliquots containing 5.0 × 10⁵ cells/mL were dispensed into individual tubes for each sample. Cells were blocked by adding 200 μL of 10% fetal bovine serum (FBS) to each tube, followed by incubation for 30 min at 37 °C. After incubation, the samples were centrifuged at 18,000 rpm for 10 min, and the resulting cell pellets were resuspended in 100 μL of 10% FBS. For immunostaining, 2 μL of monoclonal antibodies were added to each tube, and the samples were incubated in the dark at room temperature for 30 min. Mouse anti-chicken monoclonal antibody CD4 (CT-4), FITC (Ref# MA5-28685; Invitrogen, Thermo Fisher Scientific, USA) was used for the detection of chicken CD4⁺ T cells, while mouse anti-chicken alpha monoclonal antibody CD8 (CT-8), PE (Ref# MA5-28726; Invitrogen, Thermo Fisher Scientific, USA) was employed for the identification of chicken CD8⁺ T cells. Isotype control, mouse IgG1-FITC conjugate (Ref# GM4992; Invitrogen, Thermo Fisher Scientific, USA) Following staining, cells were fixed with 200 μL of 2% paraformaldehyde (Sigma-Aldrich^®, St. Louis, MO, USA) prior to flow cytometric analysis, according to method with minor modifications as described by Ref. [17]. Data acquisition was performed using a BD FACS Melody^™ flow cytometer (BD Biosciences) and subsequently results were analyzed using BD FACSDiva 8.0.1 Software.

2.6. Evaluation of humoral immune response by IgG ELISA

IgG antibody levels were measured to evaluate humoral immune response. Blood samples were randomly collected from each experimental group at 0, 1, 2, and 3 weeks post-vaccination (WPV). Blood samples were allowed to clot at room temperature, and serum was separated from pooled blood samples by centrifugation at 4000 × g for 7 min at 4 °C, as previously described by Ref. [18]. Antibodies in serum samples were measured using the ID Screen^® Avian Salmonella Indirect Groups B and D ELISA kit (IDvet, Grabels, France; Cat. No. SALS-5P) according to the manufacturer’s instructions. Prior to use, all kit reagents were allowed to reach room temperature and were gently mixed by vortexing to ensure proper homogenization. The positive and negative controls provided with the kit were ready to use and were included in each assay. Serum samples were pre-diluted by adding 5 μL of serum to 245 μL of Dilution Buffer 14 (1:50 dilution). Subsequently, 50 μL of the pre-diluted serum was added to 50 μL of Dilution Buffer 14 in the ELISA plate, resulting in a final serum dilution of 1:100. Briefly, diluted serum samples were added to microplate wells and incubated for 30 ± 3 min at 21 ± 5 °C according to the manufacturer’s instructions. After incubation, plates were washed, and anti-chicken horseradish peroxidase (HRP) conjugate solution was added to each well, followed by incubation for 30 ± 3 min at 21 ± 5 °C. Following subsequent washing, tetramethylbenzidine (TMB) substrate solution was added and incubated for 15 ± 2 min at room temperature in the dark. The reaction was stopped using the stop solution, and the color changed from blue to yellow. Absorbance values were measured at 450 nm using a microplate reader.

2.7. Challenge dose administration

Two weeks post booster vaccination, birds in all experimental groups were challenged via the oral route with wild-type SG (2.0 × 10¹⁰ CFU/mL) in 200 μL PBS. The administered challenge dose was equivalent to 100x lethal dose (LD₅₀), as mentioned by Ref. [19].

2.8. Statistical analysis

Data are presented in mean ± SEM. Flow cytometry based quantification of CD4⁺ and CD8⁺ T cell populations in each experimental group at different time interval post vaccination were analyzed by using one-way ANOVA and repeated-measures two-way ANOVA followed by Tukey’s multiple comparisons test. Humoral immune response was analyzed using two-way ANOVA. The Survival rate was analyzed using Kaplan Meier survival analysis. All statistical analyses were performed using GraphPad Prism software (version 8.0.2). Differences were considered statistically significant at p < 0.05.

3. Results

3.1. Baseline quantification of CD4⁺ and CD8⁺ T-cells

To quantify baseline T-lymphocyte populations prior to immunization, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples and analyzed using a BD FACS Melody^™ flow cytometer (BD Biosciences). Acquisition and analysis of data were performed using BD FACSDiva 8.0.1 Software. At 0WPV, percentages of CD4⁺ and CD8⁺ T cells were comparable across all experimental groups, with no statistically significant differences were observed (Fig. 2A and B). Data were analyzed using one-way ANOVA, performed in GraphPad Prism software (version 8.0.2).

Fig_2.

Pre-vaccination flow cytometric analysis of CD4⁺ and CD8⁺ T cell populations across all experimental groups. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood collected from randomly selected chickens before vaccine administration at 0WPV. T-cell populations were quantified by flow cytometry using a BD FACS Melody^™ flow cytometer (BD Biosciences) (A) Bar graph illustrates the percentages of CD4⁺ T cells across all experimental groups and unvaccinated control group. Flow cytometric analysis showed comparable baseline CD4⁺ T-cell levels among all groups, with no statistically significant differences were observed between groups (ns = non-significant). (B) Bar graph showing percentages of CD8⁺ T cells across all experimental groups and unvaccinated control group with no statistically significant differences were observed between groups (ns = non-significant).

3.2. Transient CD4⁺ and CD8⁺ T cells induction following vaccination

To evaluate CD4⁺ and CD8⁺ T cell populations, flow cytometry analysis was performed at 1, 2, and 3 weeks post-vaccination (WPV) across all experimental groups. At 1 WPV, Group C exhibited the highest CD4⁺ T cell percentage (31.1%) compared to other vaccinated groups; Group A (21.1%), Group D (16.1%), Group B (11.7%) and Group E (unvaccinated control; 11.5%) (All p < 0.0001). By 2 WPV, CD4⁺ T cell percentage in Group C (20.2%) remained higher than other vaccinated groups; Group A (13.0%), Group D (12.5%) and in Group E (unvaccinated control) (11.7%) (All p < 0.0001). However, a decline in CD4⁺ T cell percentage was observed in the Group C relative to Group B, which exhibited significantly higher CD4⁺ T cell levels (31.5%, p < 0.0001). Whereas at 3 WPV, the Group C exhibited higher CD4⁺ T cell percentages (16.1%) than other vaccinated groups; Group A (14.2%; p = 0.04), Group D (13.3%; p = 0.0003), and Group E (unvaccinated control) (11.2%; p < 0.0001). By contrast, a relative decline in CD4⁺ T cell percentage was observed compared with the Group B, which exhibited markedly higher CD4⁺ T-cell levels (38.2%; p < 0.0001). By 3WPV, all vaccinated groups showed significantly higher CD4⁺ T cell responses than the Group E (unvaccinated control); (Group A, p = 0.0189), (Group B, p < 0.0001), (Group D, p = 0.0009) and (Group C, p < 0.0001). These findings are illustrated in (Fig. 3A).

Fig_3.

Quantification of CD4⁺ and CD8⁺ T-cell populations across all experimental groups at different time intervals post-vaccination. (A) Bar graph represents CD4⁺ T-cell populations following immunization. At 1 WPV, Group C (SG ΔpurB + γ-SG Oral) exhibited a highest CD4⁺ T cells percentage compared with all other vaccinated and unvaccinated control groups. At later time points (2 and 3 WPV), highest CD4⁺ T cells percentage exhibited by Group B (γ-SG Oral) compared to other vaccinated and unvaccinated control groups. Statistical significant differences are shown as*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Bar graph represents CD8⁺ T-cell percentage among vaccinated and unvaccinated control groups. At 1 WPV Group C (SG ΔpurB + γ-SG Oral) exhibited highest CD8⁺ T-cells compared with all other vaccinated and unvaccinated control groups. At later time points (2 and 3 WPV), highest CD8⁺T cells percentage exhibited by Group B (γ-SG Oral) compared to other vaccinated and unvaccinated control groups. Statistical significant differences are shown as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns = not significant.

Similarly, at 1 WPV, Group C exhibited the highest CD8⁺ T cell (19.1%) compared with all other vaccinated groups; with Group A (15.3%) Group D (12.2%), Group B (6.7%) and Group E (unvaccinated control) (6.8%) (All p < 0.0001). While at 2 WPV, CD8⁺ T cell in Group C (13.5%) remain higher than other vaccinated groups; Group A (10.9%), Group D (9.6%) and Group E (unvaccinated control) (6.5%) (All p < 0.0001). Whereas, a decline in CD8⁺ T cell percentages was observed in the Group C relative to the Group B (14.2%, p = 0.04), which exhibited higher CD8⁺ T cell levels. By 3 WPV, Group C (13.2%) remained higher than other vaccinated groups; Group A, Group D (8.2%) and Group E (unvaccinated control) (6.2%) (All p < 0.0001). While a decline in CD8⁺ T cell response in Group C was observed relative to Group B (25%, p < 0.0001) which exhibited higher CD8⁺ T cell levels. Whereas at 3WPV, all vaccinated groups remained significantly higher than unvaccinated control group; (Group C, p < 0.0001), (Group D, p = 0.002), (Group A, p = 0.0002) and (Group B, p < 0.0001). These findings are illustrated in (Fig. 3B). Data were analyzed by using repeated measures two-way ANOVA, followed by Tukey’s test, performed in GraphPad Prism 8.0.2.

3.3. Transient systemic IgG response following vaccination

Randomly selected five serum samples from each experimental group were used to perform indirect ELISA to evaluate antibody titers against group D Salmonella. Absorbance values at (OD₄₅₀) are presented in Mean ± SEM and analyzed by using GraphPad Prism (version 8.0.2). A significant increase in antibody levels were observed at 1WPV across all vaccinated groups compared with the unvaccinated control group (all p ≤ 0.0001). At 1 WPV, Group C exhibited significantly highest mean OD₄₅₀ values compared with other vaccinated groups and unvaccinated control (p < 0.0001), Group A (p < 0.0001), Group B (p = 0.0002), and Group D (p = 0.0056). At 2 and 3 WPV highest antibody response was exhibited by Group B compared to other vaccinated and unvaccinated control groups (p < 0.0001). These findings are shown in Fig. 4. Mean absorbance values for all groups are summarized in Table 2.

Fig_4.

Serum IgG antibody response following vaccination. Serum samples were collected from chickens of each experimental group at 1, 2, and 3 weeks post-vaccination (WPV). IgG levels were quantified using a Salmonella Group D indirect ELISA, and absorbance value was measured at O.D₄₅₀. At 1 WPV, Group C (SG ΔpurB + γ-SG Oral) showed the highest antibody response compared with all other vaccinated groups, and the unvaccinated control groups. At 2 and 3 WPV, the highest antibody response exhibited by Group B (γ-SG Oral) compared with all other vaccinated groups, and the unvaccinated control groups. Statistical significance results are shown as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns = not significant.

Table 2.

Mean O.D450 values at different weeks post-vaccination (WPV).

Groups	Vaccine Components	Dose (CFU/200 μl PBS)	0 WPV	1 WPV	2 WPV	3 WPV
Group A	SG ΔpurB (IM)	2.0 × 108/200 μl PBS	0.28 ± 0.006	0.64 ± 0.007	0.58 ± 0.007	0.34 ± 0.006
Group B	γ-SG (Oral)	2.0 × 108/200 μl PBS	0.24 ± 0.005	0.69 ± 0.004	1.04 ± 0.007	1.38 ± 0.006
Group C	SG ΔpurB + γ-SG (Oral)	2.0 × 108/200 μl PBS	0.29 ± 0.005	0.92 ± 0.006	0.84 ± 0.006	0.67 ± 0.006
Group D	SG ΔpurB + γ-SG (IM)	2.0 × 108/200 μl PBS	0.29 ± 0.005	0.82 ± 0.009	0.74 ± 0.004	0.62 ± 0.005
Group E	Control (Unvaccinated)	200 μl PBS	0.27 ± 0.009	0.38 ± 0.008	0.39 ± 0.003	0.36 ± 0.003

3.4. Post-challenge survival analysis

All chickens from both vaccinated and unvaccinated control groups were challenged with a wild-type Salmonella Gallinarum (SG) strain at a dose of 2.0 × 10¹⁰ CFU/mL in 200 μL PBS. Following challenge, birds were monitored daily for a period of 14 days to assess survival outcomes. Group B exhibited complete protection, with a survival rate of 100%. In comparison, Group C showed an 80% survival rate, while Group D and Group A each group exhibited a survival rate of 70%. Survival rate with only 30% was observed in the Group E (unvaccinated control) during the challenge period. (Fig. 5).

Fig_5.

Survival analysis of vaccinated and unvaccinated control group chickens following wild-type SG challenge. Chickens from all vaccinated and unvaccinated control groups were monitored daily for 14 days post-challenge to evaluate survival outcomes. Group B (γ-SG Oral) exhibited 100% complete protection against SG infection, Group C (SG ΔpurB + γ-SG Oral) exhibited 80% survival, whereas both Group A (SG ΔpurB IM) and Group D (SG ΔpurB + γ-SG IM) exhibited 70% survival. In contrast, the unvaccinated control group showed the lowest survival rate, with only 30% of birds surviving following the challenge dose.

4. Discussion

In the present study we evaluated the immunogenicity and protective efficacy of vaccines comprising on a previously developed SG ΔpurB mutant [11] and γ-SG [14] and their combined formulations. Vaccination strategies combining live and inactivated Salmonella vaccines have been reported to be effective [20]. In a prior study, a combination approach was used in which laying hens immunized with a live attenuated Salmonella Enteritidis (SE) vaccine together with an inactivated SG-SE vaccine achieved significantly higher protection against fowl typhoid compared with inactivated vaccination alone [21].

Previous studies have demonstrated the critical role of CD4⁺ and CD8⁺ T cells in protective immunity against Salmonella infection. Depletion of both T cell subsets markedly reduces host resistance, increasing susceptibility to infection [22,23].

In the present study, all vaccinated groups exhibited elevated CD4⁺ and CD8⁺ T cell responses compared with the unvaccinated control group. Significantly, Group C (SG ΔpurB + γ-SG Oral) elicited the highest CD4⁺ and CD8⁺ T cell activation at 1 WPV, likely mediated by SG ΔpurB mutant, which induces early T-lymphocyte activation as previously described by Ref. [19].

In addition, this enhanced early CD4⁺ and CD8⁺ T cell activation in the combination group likely reflects GALT targeting via oral administration, promoting stronger immune responses than intramuscular delivery of the combination vaccine [24]. However, at 2 and 3WPV a gradual decline in CD4⁺ and CD8⁺ T cell was observed in the combination vaccine groups is due to, the rapid clearance of SG ΔpurB mutant from systemic organs such as the liver and spleen, as reported in the safety evaluation study of SG ΔpurB mutant [11]. In contrast, Group B (γ-SG Oral) exhibited highest CD4⁺ and CD8⁺ T cell response at 2 and 3 WPV, also these findings are consistent with previous study [14].

In Consistent with these observation rapid antigen clearance reduces antigen availability and effector T cell stimulation [25]. In the present study, limited antigen persistence in Group C and D (SG ΔpurB + γ-SG) likely impaired durable memory T cell development, leading to reduced booster effects, highlighting the necessity of sustained antigen for long-term immunity [26].

In the present study a similar pattern was observed for humoral immune response, showed that at 1 WPV all vaccinated groups exhibited elevated IgG antibody levels, with the highest antibody response observed in the Group C (SG ΔpurB + γ-SG Oral). However, at 2 and 3WPV a transient decline in IgG levels was evident in the Group C and D (SG ΔpurB + γ-SG) and the highest antibody response observed in the Group B (γ-SG Oral). This reduction in humoral immunity in Group C and D (SG ΔpurB + γ-SG) is likely attributable to the rapid clearance SG ΔpurB mutant which results in reduced antigen availability. Because in the absence of sustained antigen availability, continued activation of T and B lymphocytes remain no longer, leading to reduced protective antibody levels in serum [27].

Moreover, the observed transient decline at 2 and 3 WPV in both cellular and humoral immune responses in the Group C and D (SG ΔpurB + γ-SG) underscores the importance of antigenic load, as both vaccine candidates were administered at a reduced dose relative to the previously standardized dose [14,19]. A similar dose-dependent effect has been reported in a previous study evaluating the concurrent administration of the Brucella abortus RB51 vaccine with a modified live viral vaccine (vMLV), where RB51 doses below the recommended threshold failed to induce optimal cell-mediated immunity [28].

Although the combination vaccine strategy elicited early activation of both cellular and humoral immune responses but unable to confer complete protection against the field wild-type SG strain. In the present study group B (γ-SG) demonstrating 100% survival following wild-type SG challenge among all experimental groups. While the combination vaccine group C (SG ΔpurB + γ-SG Oral) conferred 80% survival, whereas the combination vaccine group D (SG ΔpurB + γ-SG IM) resulted in 70% survival, demonstrating moderate protective efficacy without complete protection. Despite eliciting measurable cellular and humoral immune responses, the combined SG ΔpurB and γ-SG formulations did not provide greater protection than γ-SG vaccination alone. These findings indicate that the combined formulation of the two vaccine candidates did not result in synergistic enhancement of protective efficacy under the experimental conditions evaluated.

5. Conclusion

Our findings demonstrated that, the combination of SG ΔpurB mutant and γ-SG vaccine candidates elicited an early and enhanced cellular and humoral immune responses, however this response is not sustained over time and unable to confer complete (100%) protection against SG field challenge. These findings indicate that further dose optimization of the combined vaccine formulation is required to enhance its applicability for commercial broiler immunization programs. Moreover, evaluation of alternative attenuated Salmonella Gallinarum mutants with enhanced in vivo persistence and prolonged antigen presentation ability may potentiate immune responses when used in combination with γ-SG, ultimately leading to improved protective efficacy against fowl typhoid.

6. Limitation

The immunogenicity and protective efficacy of the combined vaccine were evaluated over a relatively short experimental period which may not fully reflect long-term immunity. Moreover, key immunological parameters, including cytokine responses (e.g., IFN-γ), were not assessed in the present study, which could have provided further insight into the cellular immune mechanisms induced by vaccination. In addition the experiment was conducted under controlled conditions that may limit extrapolation to commercial poultry production systems.

7. Recommendation

Future studies should focus on optimizing the vaccine dose to enhance the protective efficacy of the combined vaccine formulation. Additionally, comprehensive assessment of immune responses, including cytokine profiling such as IFN-γ production, is recommended to better understand the immune mechanisms of vaccination. Finally, field trials under commercial poultry production conditions are necessary to determine the practical applicability of the vaccine candidate for controlling fowl typhoid.

Data availability

All data generated and analyzed during this study are included in this article.