Salmonella chitosan nanoparticle vaccine administration is protective against Salmonella Enteritidis in broiler birds

Abstract

Salmonella control strategies include vaccines that help reduce the spread of Salmonella in poultry flocks. In this study we evaluated the efficacy of administering a live Salmonella vaccine followed by a killed Salmonella chitosan nanoparticle (CNP) vaccine booster on the cellular and humoral immunity of broilers. The CNP vaccine was synthesized with Salmonella Enteritidis (S. Enteritidis) outer-membrane-proteins (OMPs) and flagellin-proteins. At d1-of-age, one-hundred-sixty-eight chicks were allocated into treatments: 1) No vaccine, 2) Live vaccine (Poulvac®ST), 3) CNP vaccine, 4) Live+CNP vaccine. At d1-of-age, birds were orally vaccinated with PBS, Live vaccine, or CNP. At d7-of-age, the No vaccine, Live vaccine and CNP vaccine groups were boosted with PBS and the Live+CNP vaccine group was boosted with CNP. At d14-of-age, birds were challenged with 1×109 CFU/bird S. Enteritidis. There were no significant differences in body-weight-gain (BWG) or feed-conversion-ratio (FCR). At 8h-post-challenge, CNP and Live+CNP-vaccinated birds had 17% and 24% greater levels (P<0.05) of anti-Salmonella OMPs IgA in bile, respectively, compared to control. At d28-of-age, CNP, Live, and Live+CNP-vaccinated birds had 33%, 18%, and 24% greater levels (P<0.05) of anti-Salmonella OMPs IgA in bile, respectively, compared to control. At d14-of-age, Live+CNP-vaccinated birds had 46% greater levels (P<0.05) of anti-Salmonella OMPs IgY in serum, compared to control. At d21-of-age, splenocytes from CNP and Live-vaccinated birds had increased (P<0.05) T-lymphocyte proliferation at 0.02 mg/mL OMPs stimulation compared to the control. At d28-of-age, CNP and Live+CNP-vaccinated birds had 0.9 Log10 CFU/g and 1 Log10 CFU/g decreased S. Enteritidis cecal loads (P<0.05), respectively, compared to control. The CNP vaccine does not have adverse effects on bird's BWG and FCR or IL-1β, IL-10, IFN-γ, or iNOS mRNA expression levels. It can be concluded that the CNP vaccine, as a first dose or as a booster vaccination, is an alternative vaccine candidate against S. Enteritidis in broilers.

Fig 1. Effects of Salmonella CNP vaccine on anti-OMPs specific IgY antibodies of vaccinated birds.

At d1 of age birds were allocated into treatment groups: 1) No vaccine, 2) Live vaccine, 3) CNP vaccine, or 4) Live+CNP vaccine. At d1 of age, birds were orally vaccinated with PBS, Live vaccine, or CNP. At d7 of age, the No vaccine, Live vaccine and CNP vaccine groups were boosted with PBS and the Live+CNP vaccine group was boosted with CNP. At d14 of age, birds were orally challenged with 1 × 10⁹ CFU/bird of S. Enteritidis. Blood samples were collected pre- and post-challenge and analyzed for anti-Salmonella OMPs-specific IgY levels by ELISA (n = 6). (A) Pre-challenge; (B) Post-challenge. Results were reported as average optical density (OD) values. Significant differences between the groups were determined by Kruskal-Wallis Test, followed by Dunn post-hoc test. Bars (+SE) with no common superscript differ (P<0.05). No vaccine: mock PBS; Live vaccine: POULVAC^® ST vaccine; CNP vaccine: 10μg killed CNP vaccine; Live+CNP vaccine: POULVAC^® ST vaccine + 10μg killed CNP vaccine booster.

Fig 2. Effects of Salmonella CNP vaccine on anti-OMPs specific IgA antibodies of vaccinated birds.

At d1 of age birds were allocated into treatment groups: 1) No vaccine, 2) Live vaccine, 3) CNP vaccine, or 4) Live+CNP vaccine. At d1 of age, birds were orally vaccinated with PBS, Live vaccine, or CNP. At d7 of age, the No vaccine, Live vaccine and CNP vaccine groups were boosted with PBS and the Live+CNP vaccine group was boosted with CNP. At d14 of age, birds were orally challenged with 1 × 10⁹ CFU/bird of S. Enteritidis. Cloacal swabs samples were collected pre- and post-challenge and analyzed for anti-Salmonella OMPs-specific IgA levels by ELISA (n = 6). (A) Pre-challenge; (B) Post-challenge. Results were reported as average optical density (OD) values. Significant differences between the groups were determined by Kruskal-Wallis Test, followed by Dunn post-hoc test. Bars (+SE) with no common superscript differ (P<0.05). No vaccine: mock PBS; Live vaccine: POULVAC^® ST vaccine; CNP vaccine: 10μg killed CNP vaccine; Live+CNP vaccine: POULVAC^® ST vaccine + 10μg killed CNP vaccine booster.

Fig 3. Effects of Salmonella CNP vaccine on anti-OMPs specific IgA antibodies of vaccinated birds.

At d1 of age birds were allocated into treatment groups: 1) No vaccine, 2) Live vaccine, 3) CNP vaccine, or 4) Live+CNP vaccine. At d1 of age, birds were orally vaccinated with PBS, Live vaccine, or CNP. At d7 of age, the No vaccine, Live vaccine and CNP vaccine groups were boosted with PBS and the Live+CNP vaccine group was boosted with CNP. At d14 of age, birds were orally challenged with 1 × 10⁹ CFU/bird of S. Enteritidis. Bile samples were collected post-challenge and analyzed for anti-Salmonella OMPs-specific IgA levels by ELISA (n = 6). Results were reported as average optical density (OD) values. Significant differences between the groups were determined by Kruskal-Wallis Test, followed by Dunn post-hoc test. Bars (+SE) with no common superscript differ (P<0.05). No vaccine: mock PBS; Live vaccine: POULVAC^® ST vaccine; CNP vaccine: 10μg killed CNP vaccine; Live+CNP vaccine: POULVAC^® ST vaccine + 10μg killed CNP vaccine booster.

Fig 4. Ex-vivo recall-response of splenic PBMCs of vaccinated birds.

At d1 of age birds were allocated into treatment groups: 1) No vaccine, 2) Live vaccine, 3) CNP vaccine, or 4) Live+CNP vaccine. At d1 of age, birds were orally vaccinated with PBS, Live vaccine, or CNP. At d7 of age, the No vaccine, Live vaccine and CNP vaccine groups were boosted with PBS and the Live+CNP vaccine group was boosted with CNP. At d14 of age, birds were orally challenged with 1 × 10⁹ CFU/bird of S. Enteritidis. Splenocytes PBMCs of broilers at d21 of age were harvested and stimulated with either 0.05 mg/mL, 0.1 mg/mL, 0.20 mg/mL OMPs or 0.05 mg/mL, 0.1 mg/mL flagellin proteins for 3 days. As a positive control, splenocytes stimulated with 0.0 mg/mL of antigen were used. Recall response was measured using MTT assay and absorbance was measured at OD570nm. (A) OMPs; (B) Flagellin. Results were reported as average optical density (OD) values. Significant differences between the groups were determined by one-way ANOVA, followed by Tukey post-hoc test. Bars (+SE) with no common superscript differ (P<0.05). No vaccine: mock PBS; Live vaccine: POULVAC^® ST vaccine; CNP vaccine: 10μg killed CNP vaccine; Live+CNP vaccine: POULVAC^® ST vaccine + 10μg killed CNP vaccine booster.

Fig 5. Salmonella loads in the ceca of vaccinated birds.

At d1 of age birds were allocated into treatment groups: 1) No vaccine, 2) Live vaccine, 3) CNP vaccine, or 4) Live+CNP vaccine. At d1 of age, birds were orally vaccinated with PBS, Live vaccine, or CNP. At d7 of age, the No vaccine, Live vaccine and CNP vaccine groups were boosted with PBS and the Live+CNP vaccine group was boosted with CNP. At d14 of age, birds were orally challenged with 1 × 10⁹ CFU/bird of S. Enteritidis. Ceca samples were collected from 1 birds/pen, stomached, and enriched for enumeration by plating on XLT-4 agar. Salmonella enumeration data were recorded as CFU/g of ceca and then transformed to Log 10 CFU/g of ceca for statistical analysis. Significant differences between the groups were determined by one-way ANOVA, followed by Tukey post-hoc test. Bars (+SE) with no common superscript differ (P<0.05). No vaccine: mock PBS; Live vaccine: POULVAC^®ST vaccine; CNP vaccine: 10μg killed CNP vaccine; Live+CNP vaccine: POULVAC^® ST vaccine + 10μg killed CNP vaccine booster.

Fig 6. Cytokine gene expression in the cecal tonsils of vaccinated birds.

At d1 of age birds were allocated into treatment groups: 1) No vaccine, 2) Live vaccine, 3) CNP vaccine, or 4) Live+CNP vaccine. At d1 of age, birds were orally vaccinated with PBS, Live vaccine, or CNP. At d7 of age, the No vaccine, Live vaccine and CNP vaccine groups were boosted with PBS and the Live+CNP vaccine group was boosted with CNP. At d14 of age, birds were orally challenged with 1 × 10⁹ CFU/bird of S. Enteritidis. Cecal tonsil samples were collected at d28 and analyzed for cytokine mRNA amounts by RT-PCR. Data represented fold change compared to control. (A) IL-1β mRNA; (B) IL-10 mRNA; (C) IFN-γ; (D) iNOS mRNA. Significant differences between the groups were determined by one-way ANOVA, followed by Tukey post-hoc test. Bars (+SE) with no common superscript differ (P<0.05). No vaccine: mock PBS; Live vaccine: POULVAC^®ST vaccine; CNP vaccine: 10μg killed CNP vaccine; Live+CNP vaccine: POULVAC^® ST vaccine + 10μg killed CNP vaccine booster.