Combined Salmonella and Pasteurella multocida Antigens, Single-Dose and Adsorbed in Aluminum Hydroxide-Chitosan, Protect Swiss Mice Against Lethal Challenge

1. Introduction

Fowl cholera and fowl typhoid are bacterial diseases with major economic impact, causing significant losses to poultry production in various regions of the world. Fowl cholera is caused by Pasteurella multocida, a pathogen capable of infecting different animal species; it causes hepatic lesions and sudden death in infected chickens, posing a serious threat to the poultry industry. It is classified into five capsular serogroups (A, B, D, E, and F), with serogroups A and F (and more rarely D) primarily associated with disease cases [1]. On the other hand, Salmonella spp. infections remain a persistent challenge, as they can lead to secondary mutations and virulence reversion, as well as severe reactions in immunocompromised animals [2,3]. The persistence and dissemination of these agents within poultry production systems present additional control challenges. In the case of fowl cholera, wild birds act as important reservoirs for P. multocida, facilitating the spread of the agent to commercial flocks [4]. Furthermore, once introduced into a farm, the bacterium can establish persistent infections and become endemic, hindering eradication efforts and fostering recurrent outbreaks [5]. Similarly, fowl typhoid remains endemic in various regions of Central America, South America, Africa, and Southeast Asia, posing an obstacle to the sustainability of poultry production and to the international trade of poultry and poultry products [2]. Inactivated vaccines are generally well-tolerated by host organisms and serve as key strategies for controlling infectious diseases in poultry farming, contributing to reduced mortality, improved production metrics, and decreased economic losses associated with infectious outbreaks [3]. However, commercially available inactivated vaccines still face limitations regarding the breadth and duration of the induced immune response [6]. Strategies involving antigen selection and adsorption methods utilizing novel immunomodulatory adjuvants show promise for enhancing vaccine efficacy. Adjuvants can boost antigen immunogenicity, prolong the immune response, stimulate cellular and humoral immunity mechanisms, and reduce antigenic competition in combination vaccines [7,8,9]. Research into novel adjuvants highlights the use of chitosan, valued for its mucoadhesive properties and ability to facilitate interactions between antigens and immune system cells. Saponins enhance the simultaneous stimulation of cellular and humoral responses, contributing to mechanisms associated with both Th1 and Th2 profiles when combined with aluminum hydroxide matrixes; the latter are predominantly used in commercial vaccines due to their role in inducing Th2-type responses [10,11,12,13,14,15].

Therefore, this study proposes an experimental vaccine utilizing inactivated Salmonella spp. antigens and P. multocida adsorbed onto a matrix composed of chitosan, aluminum hydroxide, and saponin, aiming to enhance the immune response and extend the protection conferred by immunization in murine models. The proposal seeks to contribute to the development of more efficient vaccination strategies for the poultry industry, with the potential to reduce the handling associated with repeated administrations and optimize immunization programs for poultry.

2. Materials and Methods

2.1. Bacterial Strains and LD₅₀ Determination

The P. multocida and Salmonella spp. bacterial strains used in this study were provided by the Avian Medicine Laboratory at the State University of Londrina, Brazil. The isolates were cultured in Brain Heart Infusion (BHI) broth (Merck^®) in an orbital shaking incubator for 12 h at 37 °C and 180 rpm. Cells were harvested using a benchtop centrifuge (model 80-2B) at 3,000 rpm for 10 min; the cells were collected and resuspended in phosphate-buffered saline (PBS, pH 7.2), a process repeated three times. The median lethal dose (LD₅₀) of P. multocida and Salmonella spp. was determined according to the Reed and Muench method [16,17]. To determine the LD₅₀ of P. multocida, mice (n = 6 per group) were inoculated intraperitoneally with 200 µL of active bacterial suspension at concentrations of 7.50×10¹³; 8.37×10¹²; 2.17×10¹¹; 1.16×10¹⁰; 1.30×10⁹; and 6.26×10⁸ CFU/mL. For the LD₅₀ determination of Salmonella spp., animals were distributed into five groups (n = 6 per group) and inoculated intraperitoneally with the same volume (200 µL) of active suspension at concentrations of 1.37×10¹⁵; 4.90×10¹²; 5.41×10¹⁰; 1.84×10⁹; and 5.97×10⁸ CFU/mL. The animals were monitored for 48 hours, with events and clinical signs recorded, to determine the LD₅₀.

2.2. Ethical/legal Requirements and Use of Animals in Experiments

Mice weighing 20–24 g were obtained from the Federal University of Goiás (UFG) and housed in the preclinical testing room of the Biomolecules and Vaccines Laboratory (LaBVac) at the Federal University of Tocantins (UFT), Brazil. The animals were kept in pathogen-free primary enclosures with ad libitum access to food and water, maintained at a controlled temperature (24 °C), with odor and lighting controls adjusted to a 12-hour light/dark cycle. All animals used in this experiment were previously dewormed via access to an Ivermectin^® solution (1:20 dilution) for 7 days. The experiments were conducted in accordance with ethical recommendations established by the law governing procedures for the scientific use of animals. They were approved by the Ethics Committee on Animal Use (CEUA) of the Federal University of Tocantins (protocol no. 23101.002359/2020-31). Every effort was made to avoid undue suffering or pain; the animals were monitored for clinical status, changes in body weight, and clinical signs such as lethargy, hypothermia, and/or respiratory distress. The onset of such clinical signs was treated as a death event and recorded to determine the cumulative mortality for each experimental group and subsequently calculate the LD₅₀. At the end of the observation period, the animals were euthanized using ketamine (300 mg/kg) (Vetbrands^®, Brazil) and xylazine hydrochloride (22.5 mg/kg) (Syntec^®, Brazil).

2.3. Antigen Production, Inactivation, and Vaccine Formulation

The vaccine antigen was obtained from inactivated P. multocida and Salmonella spp. bacterial cells derived from pathogen cultures in BHI broth. Inactivation was achieved by adding a 0.6% (v/v) formalin solution under agitation at 150 rpm for 48 hours at 37 °C. Inactivation efficacy was verified by plating 1 mL of each inactivated sample onto BHI agar, followed by incubation for 24 hours at 37 °C. The adjuvant used was prepared by first solubilizing chitosan powder (Polymar Ltd.a, 81% degree of deacetylation) in 0.8% (v/v) acetic acid and 0.9% (w/v) saline solution, using a 2:1 ratio of chitosan to aluminum hydroxide (DEUSDARÁ et al., 2023), supplemented with saponin at a final concentration of 500 ng/mL. The experimental vaccine was formulated with both antigens at a concentration of 10⁹ cells/mL, using a ratio of 25% (v/v) adjuvant and 10% (v/v) saponin.

2.4. Definition of Vaccine Dose

To determine the dose of the experimental vaccine, mice were assigned to four experimental groups of 12 animals each, designated GC1, GC2, GC3, and PBS. Groups GC1, GC2, and GC3 received a single intramuscular dose of 250 µL, 125 µL, and 50 µL of the experimental vaccine, respectively. The PBS group received a single 250 µL dose of PBS and served as the negative control. Animals were monitored daily from day 1 to day 21 post-immunization and evaluated for clinical signs including changes in feed intake, lethargy, prostration, and diarrhea as well as variations in body weight (Figure 1-A). Following this analysis period, the groups were subdivided and challenged intraperitoneally with 200 µL of a suspension containing the respective viable bacteria at a concentration corresponding to the median lethal dose (LD₅₀) previously determined for each pathogen. A second study was designed based on data obtained from the standardization of the experimental vaccine dose. Thus, the vaccine dose defined in the previous study was compared with the vaccination protocol used by the manufacturer of a commercial vaccine employed in the poultry industry against both pathogens (Figure 1-B). Mice were assigned to four experimental groups of 12 animals each: experimental vaccine (single 250 µL dose); commercial vaccine (two 250 µL doses with a 14-day interval); adjuvant (single 250 µL dose of the formulation without bacterial antigen); and negative control (250 µL of PBS). Twenty-one days after immunization, the animals were challenged intraperitoneally with 200 µL of a virulent bacterial suspension of P. multocida and Salmonella spp. Following the challenge, the animals were monitored daily for seven days, with clinical signs and mortality recorded.

2.5. Cytokine Determination Assay

Cytokine analysis of animals was carried out and distributed into six experimental groups: G1 and G2, composed of animals immunized, respectively, with a single dose of the experimental vaccine and two doses of the commercial vaccine, without subsequent challenge; G3 and G4, immunized with a single dose of the experimental vaccine and challenged with P. multocida and Salmonella spp., respectively; and G5 and G6, immunized with two doses of the experimental vaccine and subsequently challenged with P. multocida and Salmonella spp., respectively. Blood plasma samples from the unchallenged groups were collected 21 days after the first immunization. In contrast, in the groups subjected to the experimental challenge, collection was carried out three days after inoculation of the pathogens. Plasma was obtained by centrifuging whole blood in a microcentrifuge (Microcentrifuge G-L, IKA^®) at 15,000 rpm for 30 minutes. After separation, the samples were stored at 2 °C until flow cytometry analysis. The concentrations of interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-17, Tumor Necrosis Factor alpha (TNF-α), and interferon-gamma (IFN-γ) were determined using Cytometric Bead Array (CBA) technology. For this analysis, the BD™ CBA Mouse Th1/Th2/Th17 kit was used, following the manufacturer’s instructions. Data were acquired on a flow cytometer (FACSCanto™ II), and results were expressed as mean fluorescence intensity (mean fluorescence intensity—MFI).

2.6. Statistical Analysis

Statistical tests were performed using GraphPad Prism 8.0 software. The log-rank test was used to compare two or more groups with censored data, defined as individual death, evaluated via Kaplan-Meier analysis. Results are presented as mean values ± SD. ANOVA was used to assess statistical differences between groups. Statistical significance was set at p < 0.05.

3. Results

3.1. Determination of the Lethal Dose of Salmonella Spp. and P. Multocida in Murine Models

The virulence of P. multocida strains in Swiss mice resulted in clinical signs consistent with avian cholera at concentrations ranging from 7.5×10¹³ to 2.17×10¹¹, leading to the most acute form of the disease and 100% mortality in the group (Figure 2-A). Conversely, animals inoculated with concentrations equal to or lower than 1.16×10¹⁰ included survivors throughout the experimental period (Figure 2-A), indicating a lethal infection dose corresponding to 95 LD₅₀ (1×10¹⁰ CFU/mL). Regarding the LD₅₀ of Salmonella spp., all animals inoculated with a concentration of 1.37×10¹⁵ died within 12 hours of observation; lower doses showed progressively higher survival rates as the bacterial infection concentration was reduced (Figure 2-B), indicating a lethal infection dose corresponding to 40 LD₅₀ (5×10¹⁰ CFU/mL).

3.2. Evaluation of Vaccine Dosage Using Murine Models

In the preliminary study of vaccine dosages GC1 (250 µL), GC2 (125 µL), and GC3 (50 µL), the evaluation of the experimental animals’ mean body mass revealed no significant changes in weight gain throughout the 21-day post-immunization period. At the end of this period, a statistically significant difference (p < 0.05) was observed only between the GC2 group and the control group. In contrast, the GC1 and GC3 groups showed no differences, thereby demonstrating the formulation’s safety and the maintenance of the animals’ zootechnical performance (Figure 3-A). Regarding the survival rate of animals immunized and challenged with the P. multocida LD₅₀, the GC1 group exhibited 100% survival, representing the highest observed survival rate. In contrast, animals in the GC2 and GC3 groups showed survival rates of 83% and 66%, respectively (p < 0.05) (Figure 3-B). Meanwhile, the survival rate of animals immunized and challenged with Salmonella spp. showed a progressive decline over the monitoring period, varying according to the administered dose of the experimental vaccine; the GC1 group demonstrated the highest survival rate (83%), whereas the GC2 and GC3 groups showed a more marked reduction over time, resulting in 33% survival in both groups (p < 0.05) (Figure 3-C).

With the vaccine dosage established, the experimental vaccine provided 100% survival for animals challenged with the LD₅₀ of P. multocida and Salmonella spp, during the seven-day post-challenge observation period. In contrast, animals immunized with the commercial vaccine showed a survival rate of 60%, whereas those that received only the adjuvant formulation showed a survival rate of 16%. No surviving animals were observed in the control group inoculated with PBS (p < 0.05) (Figure 4A,B).

3.3. Determination of the Cytokine Profile of Animals Immunized with Experimental and Commercial Vaccines, with and Without Challenge, in Murine Models

Evaluation of the cytokine profile post-immunization and before experimental challenge (Figure 5-A) revealed slight differences between the single-dose immunization group (G1) and the commercial double-dose vaccine group (G2) (Figure 5-B); however, among the cytokines evaluated, IL-6 showed a significant difference (p<0.05), with higher levels in the single-dose group (G1). We also investigated the effect of bacterial challenge with either P. multocida (G3) or Salmonella spp. (G4) on the immune response induced by immunization. Data showed no significant differences for most of the cytokines evaluated (Figure 5-C). However, it was again observed that IL-6 levels were significantly higher in immunized, unchallenged animals compared to the group immunized and subsequently challenged with P. multocida (p < 0.05), whereas Salmonella spp. (G4) did not interfere with cytokine stimulation before or after experimental challenge; this indicates that the primary stimulus was driven by immunization, without further stimulation interference following the experimental challenge (Figure 5-D). We also conducted a comparative study of immunization protocols and cytokine stimulation data using single-dose (G3 and G4) and double-dose (G5 and G6) experimental vaccines, assessing the period post-immunization and following the respective bacterial challenges to contribute to the understanding of the protective immune response against both pathogens (Figure 5-D). Here, we observed that animals immunized with either the single-dose or double-dose experimental vaccine protocols were protected following the challenge test, with 100% survival; this demonstrates that the stimulated cytokine levels conferred a protective response even when the animals were challenged with either pathogen. Following challenge, animals that received two vaccine doses exhibited enhanced cytokine stimulation. Of note were the groups challenged with Salmonella spp., where the two-dose regimen resulted in significantly higher (p < 0.05) concentrations of IFN-γ, TNF-α, and IL-10 compared to the single-dose group; this indicates greater activation of cellular and regulatory immunological mechanisms associated with the response to intracellular pathogens. In contrast, no significant differences were observed between the evaluated vaccination protocols in the groups challenged with P. multocida.

4. Discussion

Inactivated vaccines serve as an alternative that enables broader coverage against infectious agents through simultaneous antigen exposure, stimulates the herd’s immune system, offers logistical efficiency and reduced transport costs, and improves animal management. However, in some cases, they may induce weaker immune responses; therefore, it is important to devise strategies, such as using adjuvants to enhance immunogenicity [18]. Commonly used adjuvants include saponin [19], chitosan [20], and aluminum hydroxide [21]. In this study, we developed a single-dose bivalent inactivated vaccine targeting Salmonella spp. and P. multocida, pathogens that cause significant economic losses in the global poultry industry. Data from a murine model demonstrated the high efficacy of the experimental vaccine, ensuring complete protection following challenge with virulent strains of Salmonella spp. (Figure 4-A) and P. multocida (Figure 4-B), and showing superior performance compared to the reference commercial vaccine, which requires a two-dose regimen.

Given the sanitary and economic significance of these diseases, developing effective vaccination strategies is crucial for controlling fowl typhoid and fowl cholera in poultry production systems. Various approaches, such as vaccines using Salmonella spp. strains attenuated via SptP gene deletion, radiation-inactivated vaccines, inactivated vaccines combined with L-proline supplementation, and the use of adjuvants derived from bacterial DNA have been reported to enhance humoral and cellular immune responses in murine models [22,23,24,25]. Consequently, data from numerous studies reinforce the potential of inactivated vaccines as a cost-effective disease control strategy, corroborating the findings of the present study.

In this context, the route of administration can also provide insight into the immune response. The intramuscular immunization route adopted in this study may facilitate local antigen retention at the injection site, promoting the recruitment of antigen-presenting cells and the activation of the systemic immune response in regional lymph nodes [26]. Conversely, the intraperitoneal challenge widely used in experimental models of bacterial infection mimics the systemic dissemination of pathogens; this makes the systemic immunity induced by intramuscular vaccination biologically relevant and capable of conferring protection in this experimental model [27]. Regarding the cytokine profile, IFN-γ levels increased significantly in the group immunized with a double dose of the experimental vaccine and subsequently challenged with Salmonella spp. (G6) (Figure 5-C); this pattern mirrored the TNF-α levels observed in the immunized animals of group G6, suggesting the involvement of immunological mechanisms characteristic of a Th1 response. Such mechanisms are crucial for containing intracellular pathogens, as they promote macrophage activation and enhance cell-mediated immunity [28]. Activation of this immunological pathway is recognized as a key mechanism in the elimination of intracellular pathogens, contributing to the control of bacterial replication and the establishment of a more efficient protective response against Salmonella spp. Infection [29].

IL-17 levels in group G6 do not allow us to confirm the involvement of the Th17 response in the conditions evaluated (p > 0.05), although the observed pattern suggests a possible role for this immunological mechanism. This response has been linked to the recruitment of inflammatory cells to sites of infection and the amplification of immunity against invasive bacteria [28]. The increase in IL-10 observed in group G6 compared to G4 suggests that administering double doses of the experimental vaccine promoted a more pronounced modulation of the immune response following the Salmonella spp. challenge. IL-10 plays a key regulatory role, contributing to the control of inflammation and limiting tissue damage resulting from the immune response [30].

Although this profile indicates active engagement of the adaptive immune response, it is worth noting that the group immunized with a single dose (G4) despite showing no statistically significant changes in cytokine levels after the challenge (p > 0.05) (Figure 5-B) achieved a survival rate equivalent to that of the double-dose group, with both groups showing 100% protection. This finding suggests that the single-dose was capable of establishing sufficient protective immunity to control the Salmonella spp. infection without requiring intense systemic inflammatory activation; this can be interpreted as a more efficiently modulated immune response, wherein the immunity established by vaccination obviated the need for extensive recruitment of pro-inflammatory mediators following the challenge [31]. From this perspective, cytokine stability in the single-dose group does not indicate an absence of immune response but rather evidence of effective infection control with a lower inflammatory cost—a desirable characteristic for vaccine formulations, especially considering that single-dose regimens reduce animal stress, operational costs, and vaccination coverage failures associated with multidose schedules in the field [26]. In the group challenged with Salmonella spp., levels of IL-6 and the other cytokines evaluated (IL-2, IL-4, IL-10, IL-17, TNF-α, and IFN-γ) remained stable relative to pre-challenge levels (p > 0.05); this is biologically consistent with an immune response efficiently modulated by vaccination, wherein previously established immunological memory obviated the need for secondary systemic inflammatory activation, as described in murine models of vaccine-mediated protection against Salmonella [31,32]. The stability of plasma cytokines post-challenge observed in vaccinated animals aligns with studies demonstrating distinct immunological profiles between immunized and non-immunized animals following a DL50 challenge, often characterized by the absence of exacerbated systemic inflammatory amplification in protected groups [33].

Immunization against P. multocida and the resulting cytokine profile indicate stimulation characteristic of a response to a predominantly extracellular pathogen; this suggests that mechanisms linked to the humoral response may have contributed to animal protection, given the high survival rate and effective protection observed during the experimental challenge [34]. In non-challenged animals, the group receiving the experimental vaccine (G1) exhibited basal plasma IL-6 levels significantly higher than those of the group receiving the commercial vaccine (G2) (p < 0.05) (Figure 5-B). This effect is attributed to the immunostimulatory potential of the adjuvant used—composed of chitosan, aluminum hydroxide, and saponin corroborating findings by Zhao et al. [35], who observed elevated IL-6 levels in mice immunized with a bivalent pasteurellosis vaccine delivered via chitosan nanoparticles.

The data demonstrate that the evaluated bivalent vaccine successfully induced an immune response and provided effective protection against Salmonella spp. and P. multocida in the study model; this protection is associated with the modulation of immunological mechanisms linked to cellular and regulatory responses. Such findings reinforce the potential of this experimental vaccine as a promising strategy for developing bivalent vaccines aimed at minimizing the impact of poultry diseases. Future studies in the target species will be necessary to expand knowledge regarding the efficacy and safety of the vaccine developed in this pilot study. Additionally, further characterization of the vaccine-induced immune response, potentially including comparative studies of specific antibodies in murine and avian models, is warranted to assess its direct application for disease control in the poultry industry.

5. Conclusions

The results demonstrate that the single-dose bivalent inactivated vaccine developed in this study possesses high protective potential against Salmonella spp. and P. multocida challenges in a murine model, conferring 100% survival to immunized animals. Although the two-dose regimen increased cellular and regulatory cytokines post-challenge with Salmonella spp., the single-dose immunization was sufficient to activate the immunological mechanisms required for complete protection. Among the formulations evaluated, the 250 µL dose exhibited the best protective performance and an excellent safety profile, without affecting animal weight gain. Taken together, the data highlight the potential of combining chitosan, aluminum hydroxide, and saponin as a promising adjuvant platform, providing a basis for future studies in the target avian species—an essential step to bridge the immunological differences between the models.