Evaluation of the immunogenicity and safety of the paratyphoid component in a novel tetravalent attenuated freeze-dried vaccine for pigs

Paratyphoid fever (salmonellosis) in pigs, caused primarily by Salmonella enterica serovar Choleraesuis, represents a significant zoonotic and economic threat to the global swine industry. This pathogen induces systemic infections, including septicemia, pneumonia, meningitis, and high mortality rates in post-weaned pigs^[1,2]. Other serovars, such as S. typhimurium, contribute to foodborne illnesses in humans through cross-contamination in the food chain^[3,4]. In Vietnam, paratyphoid, alongside pasteurellosis (Pasteurella multocida), erysipelas (Erysipelothrix rhusiopathiae), and classical swine fever (Pestivirus), are classified as 'red diseases', necessitating stringent control measures^[5]. Vaccination remains the most effective and sustainable strategy for reducing disease incidence, pathogen shedding, and environmental contamination^[5,6]. However, intensive swine farming faces challenges from monovalent or bivalent vaccines (e.g., pasteurellosis-paratyphoid), which require multiple administrations, escalating labor costs, material expenses, and animal stress. Such stress can transiently impair immunity, reduce productivity, and increase susceptibility to secondary infections^[7].

The development of polyvalent vaccines, combining multiple antigens in a single dose, addresses these issues and aligns with modern veterinary vaccine technology trends. A novel tetravalent attenuated freeze-dried vaccine (V4AFD) targeting the four aforementioned diseases has been developed and pilot-produced. Despite advantages, polyvalent vaccines risk antigenic competition, where multiple antigens may diminish immune responses to individual components^[8,9]. Recent advancements in Salmonella vaccines emphasize live-attenuated strains for eliciting both humoral and cell-mediated immunity, crucial for clearing intracellular pathogens like Salmonella^[10,11]. Contemporary studies have explored recombinant live-attenuated vaccines^[12], oral delivery methods^[13], and bivalent formulations against S. choleraesuis and S. typhimurium^[14], demonstrating reduced clinical signs and shedding. Longitudinal investigations also highlight the benefits of sow and piglet vaccination in lowering Salmonella prevalence^[15].

This study aimed to rigorously assess the safety, immunogenicity, and duration of immunity of the paratyphoid component (S. choleraesuis) in V4AFD using guinea pig and pig models, ensuring no antigenic interference from other components. The V4AFD vaccine, produced by the Central Veterinary Institute incorporated attenuated strains for pasteurellosis, erysipelas, classical swine fever, and paratyphoid (S. choleraesuis Smith W.H.), following established protocols for live-attenuated vaccine production^[12,16]. S2, isolated from Vietnamese outbreaks, represents local epidemiology^[5]. The virulent S. choleraesuis S2 strain was used for challenges, sourced from a reference collection, and confirmed for pathogenicity via standard virulence assays (LD50 assays in mice and pigs, with > 90% mortality at 10⁹ CFU)^[17]. Experiments involved 79 healthy guinea pigs (300–350 g) and 107 Landrace × Yorkshire pigs (20–30 kg), all seronegative for the target diseases and housed under controlled conditions with ethical approval from the institutional animal care committee, in compliance with international guidelines for animal experimentation^[18,19]. Growing pigs were chosen as a standard model per OIE^[20], facilitating controlled trials; while weaned piglets are primary targets, age-related efficacy variations are minimal in analogous vaccines^[15]. Animals were acclimatized for one week prior to trials, with daily health monitoring to ensure baseline welfare.

Determination of minimum protective dose (MPD)

Vaccine dilutions yielded paratyphoid concentrations of 1 × 10⁹, 2 × 10⁹, and 3 × 10⁹ CFU/dose, prepared using sterile phosphate-buffered saline (PBS) and verified by colony counting on selective media^[11]. Dose gradients were selected based on references to similar attenuated Salmonella vaccines^[12,16], and preliminary trials showing suboptimal protection below 1 × 10⁹ CFU. Guinea pigs (n = 5/group) received 1/5 pig doses (2 × 10⁸, 4 × 10⁸, 6 × 10⁸ CFU) intramuscularly, while pigs (n = 5/group) received full doses. Vaccinations were administered under aseptic conditions, following standard veterinary practices^[6]. After 21 d, animals and controls (n = 3 pigs, n = 2 guinea pigs/batch) were challenged intravenously with S2 at a dose calibrated to induce consistent morbidity in unvaccinated subjects^[21]. Protection rates were monitored for 10 d, including clinical scoring for symptoms such as fever, anorexia, and lethargy, with necropsy performed on deceased animals to confirm Salmonella-specific lesions^[17].

Safety evaluation

Safety was assessed per Vietnamese standard TCVN 8685-1:2011, supplemented by international benchmarks^[20], using three vaccine batches with ≥ MPD. Pigs (n = 2/batch) received 10 doses intramuscularly, and guinea pigs (n = 5/batch) received two doses. Animals were observed for local (e.g., swelling, redness) and systemic reactions (e.g., fever, behavioral changes) over 10 d, with vital signs recorded twice daily^[16]. Reactions were graded on a 0–4 scale^[20]: fever defined as rectal temperature > 40 °C; local swelling measured by caliper and graded as 0 (none) to 4 (> 5 cm severe); systemic signs (e.g., lethargy, anorexia) based on daily clinical scoring.

Efficacy evaluation

Efficacy of three V4AFD batches was compared to a bivalent pasteurellosis-paratyphoid vaccine (positive control) and unvaccinated controls (negative). Intravenous challenge was used for reproducibility^[21]; oral routes may yield milder disease but comparable protection^[11]. Pigs (n = 5/batch) received one dose; controls (n = 3) received none. At 21 d: (i) Challenge: pigs were intravenously challenged with 1 MLD (3 x 10⁹ CFU) of S2 and monitored for 10 d, including bacterial reisolation from organs^[10]. Efficacy required ≥ 80% protection in vaccinated groups and 100% mortality/severe disease in controls. (ii) Antibody Detection: sera were tested using PrioCHECK® Salmonella Ab porcine 2.0 ELISA kit (positive if percentage positivity [PP] ≥ 40%), validated against gold-standard methods^[6]. Histopathology used H&E staining, scored 0–3 for lesions (e.g., necrosis); bacterial recovery via culture on selective media. Samples were processed in duplicate to ensure reproducibility.

Duration of immunity

Pigs (n = 10) received one V4AFD dose; controls (n = 5) received none. Sera were collected at day 0, 21, 3 months, and 6 months for ELISA analysis, with storage at –80 °C to preserve integrity^[15].

Statistical analysis

Data were analyzed using Epicalc 1.02 and Microsoft Excel 2010. Data normality checked via Shapiro-Wilk; ANOVA with post hoc LSD tests compared means; differences were significant at p < 0.05. Survival analysis employed Kaplan-Meier curves for challenge outcomes. Group sizes were based on regulatory minima and power calculations for 80% detection of differences (p < 0.05).

Among five growth media tested, CT4 (BHI + 1.2% agar + 0.5% glucose + 0.5% beef extract + 2% peptone + 5% rabbit blood) yielded the highest S. Choleraesuis biomass (80.00 ± 0.16 x 10⁹ CFU/mL; p < 0.05 compared to other formulations) and was selected for production, demonstrating superior nutrient optimization for bacterial propagation. Guinea pig trials showed 100% protection at ≥ 4 × 10⁸ CFU (equivalent to 2 × 10⁹ CFU/pig dose), with no clinical signs or mortality in protected groups, while lower doses resulted in partial morbidity. Pig validation confirmed MPD as 2 × 10⁹ CFU/dose, with 100% protection at this and higher levels, evidenced by the absence of fever, normal feed intake, and no pathological lesions upon necropsy. In contrast, at 1 × 10⁹ CFU, only 60% survival was observed across batches, with surviving pigs showing mild septicemia symptoms resolving within 7 d, and deceased animals exhibiting classic Salmonella pathology such as hepatic necrosis and splenomegaly (Table 1). Controls exhibited 33%–67% mortality, with rapid onset of severe symptoms within 48 h post-challenge, underscoring the virulence of S2. Statistical analysis revealed significant differences in survival rates between MPD and sub-MPD groups (p < 0.01).

Safety

All pigs (2/2 per batch) at 10 doses and guinea pigs (5/5 per batch) at 2 doses remained healthy, with no reactions observed, including zero incidence of local inflammation or systemic fever across all monitoring points. Vital signs remained stable, and no behavioral anomalies were noted, confirming batch consistency (Table 2).

Efficacy

ELISA: 100% (15/15 V4AFD; 5/5 bivalent) pigs seroconverted (PP ≥ 40%) at 21 d, with mean PP values ranging from 65% to 85%, indicating strong humoral responses; controls (0/3) remained negative with PP < 10% (Table 3). Challenge: 100% protection in vaccinated groups (15/15 V4AFD; 5/5 bivalent), with no bacterial recovery from organs and normal histopathology; 100% controls (3/3) died or showed lesions, including pulmonary consolidation and enteric inflammation, with high bacterial loads (Table 4). Kaplan-Meier analysis showed significant survival divergence (p < 0.001).

Duration of immunity

100% (10/10) vaccinated pigs maintained positive ELISA responses through 6 months, with mean PP values of 75% ± 8% at 21 d, 60% ± 7% at 3 months, and 55% ± 6% at 6 months, all above the ≥ 40% protective threshold (Table 5). No waning was observed, with intra-group variability minimal (SD < 10%).

Determining the MPD (2 × 10⁹ CFU/dose) provides a scientific foundation for vaccine formulation, ensuring optimal protection without excess antigen load, which could otherwise lead to unnecessary production costs or potential reactogenicity. The efficacy plateau at ≥ 2 × 10⁹ CFU aligns with dose-response patterns in similar vaccines^[14], and higher doses were not explored due to regulatory alignment and ethical considerations. The Smith W.H. strain's attenuation maintains immunogenicity while eliminating virulence, consistent with historical data^[16] and recent recombinant approaches that incorporate genetic modifications for enhanced safety and efficacy^[12]. This MPD aligns with doses reported in bivalent vaccines, where similar bacterial loads have achieved comparable protection against heterologous challenges^[14].

Safety results affirm V4AFD's compliance with national and international standards, with no reactions at overdose, aligning with live-attenuated Salmonella vaccines' profiles that emphasize minimal residual virulence^[6,20]. The absence of adverse events in both species underscores the vaccine's suitability for field use, particularly in stress-sensitive intensive farming systems, where vaccine-induced stress could exacerbate disease susceptibility^[7]. While short-term safety was confirmed, future field trials should include long-term histopathology to rule out immunopathology. Comparative studies on monophasic Salmonella variants have similarly reported high safety margins for commercial vaccines like Enterisol Salmonella T/C, which reduce colonization without side effects^[22].

Efficacy data demonstrate robust humoral responses and full protection, equivalent to bivalent vaccines, indicating no antigenic competition despite the polyvalent nature^[8,9]. This is noteworthy, as polyvalent vaccines can sometimes compromise responses due to immune resource allocation, but here, the integration of four antigens succeeded, possibly owing to an optimized formulation that allows balanced antigen presentation. Live-attenuated strains elicit comprehensive immunity, including cell-mediated responses critical for intracellular pathogen clearance^[10,11], corroborated by recent bivalent oral vaccines reducing clinical disease and shedding in challenged pigs^[14]. Furthermore, the 100% seroconversion and protection rates surpass those in some longitudinal field studies, where environmental factors may dilute efficacy^[15].

The 6-month immunity duration supports single-dose protocols for fattening pigs, reducing stress and costs associated with repeated handling^[7]. While antibody persistence suggests protection, future studies should include late challenges to confirm. Comparable persistence is reported in modern vaccines, including those against monophasic Salmonella that maintain antibody levels in sows and offspring^[22,15]. Oral delivery innovations, such as self-administration gels, offer potential enhancements for V4AFD by improving uptake and reducing labor^[13,23]. Results may extrapolate to weaners, but age-specific trials are recommended to confirm. However, field trials are recommended to validate these findings under real-world conditions, including co-infections and variable husbandry practices. Field applicability may be higher for natural routes; oral challenge trials are recommended. Outbreaks from misadministration, as seen in suckling piglets with improper Typhimurium vaccine use^[24], highlight the importance of proper training and monitoring for emerging serovars (e.g., monophasic S. typhimurium^[3]). Future research could explore booster strategies or a combination with recombinant vectors to extend immunity beyond 6 months, while incorporating monovalent controls to directly confirm the absence of antigenic interference, thereby addressing gaps in long-term herd protection.

The paratyphoid component in V4AFD is safe, immunogenic, and provides durable protection without interference from other antigens, positioning it as a valuable tool for integrated swine health management in regions with high Salmonella burden. By achieving 100% protection and sustained antibody responses for at least 6 months, this vaccine addresses key challenges in polyvalent formulations, such as antigenic competition, and offers economic benefits through reduced vaccination frequency. Its alignment with recent advancements in live-attenuated and oral vaccines suggest broad applicability in preventing zoonotic transmission and improving farm productivity. Implementation in national control programs could significantly mitigate economic losses and public health risks, warranting further large-scale trials to confirm real-world efficacy and adaptability to diverse serovars.