Efficacy of a nanoparticle vaccine administered in-ovo against Salmonella in broilers

Abstract

Salmonella is a zoonotic pathogen that persists in poultry. Salmonella vaccines that can be delivered in-ovo can be cost-effective and can decrease Salmonella load in poultry. This study evaluates the efficacy of a Salmonella chitosan-nanoparticle (CNP) vaccine, administered in-ovo, in broilers. CNP vaccine was synthesized with Salmonella Enteritidis (SE) outer-membrane-proteins (OMPs) and flagellin proteins. At embryonic-d18, one-hundred-thirty-six eggs were injected with 200μl PBS or 1000μg CNP into the amniotic cavity. At d1-of-age, 132 chicks were allocated in 6 pens/treatment with 11 chicks/pen. At d7, birds were orally challenged with 1×109 CFU/bird SE. At d1, 8h-post-challenge, d14, and d21, serum anti-SE-OMPs IgY were analyzed. At d14 and d21, cloacal swabs and bile anti-SE-OMPs IgA, CD4+/CD8+-T-cell ratios, and ceca SE loads were analyzed. At d21, cecal tonsil IL-1β, IL-10, and iNOS mRNA were analyzed. Body-weight-gain (BWG) and feed-conversion-ratio (FCR) were recorded weekly. Data were analyzed by Student's t-test at P<0.05. There were no significant differences in BWG or FCR between vaccinated birds compared to control. At d1, CNP-vaccinated birds had 5.62% greater levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 8h-post-challenge, CNP-vaccinated birds had 6.39% greater levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 2wk-post-challenge, CNP-vaccinated birds had 7.34% lower levels (P<0.05) of anti-SE-OMPs IgY, compared to control. At 1wk-post-challenge, CNP-vaccinated birds had 15.30% greater levels (P<0.05) of bile anti-SE-OMPs IgA, compared to control. At d14 and d21, CNP-vaccinated birds had 0.62 and 0.85 Log10 CFU/g, decreased SE ceca load (P<0.05), respectively, compared to control. There were no significant differences in CD4+/CD8+-T-cell ratios between vaccinated birds compared to control. There were no significant differences in IL-1β, IL-10, iNOS mRNA between vaccinated birds compared to control. Findings demonstrate that the in-ovo administration of CNP vaccine can induce an antigen-specific immune response against SE and can decrease SE cecal load in broilers.

Fig 1. Anti-SE OMPS IgY/IgA in serum, cloacal swab, and bile of chickens vaccinated with in-ovo CNP.

At embryonic d18, broiler eggs were vaccinated with either PBS (control) or 1000 μg CNP vaccine. At d7 of age, birds were challenged with live SE (1.0 × 10⁹ CFU/bird). Blood, cloacal swabs, and bile samples were collected pre- and post-challenge and analyzed for anti-Salmonella antigen-specific IgY and IgA levels by ELISA. Results were reported as average optical density (OD) values. A–Serum OMPs IgY; B–Cloacal swab OMPs IgA; C–Bile OMPs IgA. Data analyzed by parametric Student t-test; Means+SEM.Bars. n = 6. "*" means P<0.05.

Fig 2. CD4⁺/CD8⁺ cell percentages in cecal tonsils of chickens vaccinated with in-ovo CNP at 1wk-post-challenge.

At embryonic d18, broiler eggs were vaccinated with either PBS (control) or 1000 μg CNP vaccine. At d7 of age, birds were challenged with live SE (1.0 × 10⁹ CFU/bird). The CD4⁺/CD8⁺ T-cell percentage in cecal tonsils were analyzed by Flow Cytometry (Guava Eascyte; Millipore). The CD4⁺/CD8⁺ cell percentage was analyzed after gating cells based on forward-scatter and side-scatter plot for lymphocytes. Data analyzed by parametric Student t-test; Means+SEM.Bars. n = 6. "*" means P<0.05.

Fig 3. CD4⁺/CD8⁺ cell ratios in cecal tonsils of chickens vaccinated with in-ovo CNP at 2wk-post-challenge.

At embryonic d18, broiler eggs were vaccinated with either PBS (control) or 1000 μg CNP vaccine. At d7 of age, birds were challenged with live SE (1.0 × 10⁹ CFU/bird). The CD4⁺/CD8⁺ T-cell percentage in cecal tonsils were analyzed by flow cytometry (Guava Eascyte; Millipore). The CD4⁺/CD8⁺ cell percentage was analyzed after gating cells based on forward-scatter and side-scatter plot for lymphocytes. Data analyzed by parametric Student t-test; Means+SEM.Bars. n = 6. "*" means P<0.05.

Fig 4. Salmonella loads in the ceca of chickens vaccinated in-ovo with CNP at 1wk-post-challenge.

At embryonic d18, broilers egg were vaccinated with either PBS (control) or 1000 μg CNP vaccine. At d7 of age, birds were challenged with live SE (1.0 × 10⁹ CFU/bird). Ceca samples were collected from 3 birds/pen, pulled, and stomached 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. Data analyzed by parametric Student t-test; Means+SEM.Bars. n = 6. "*" means P<0.05.

Fig 5. Salmonella loads in the ceca of chickens vaccinated in-ovo with CNP at 2wk-post-challenge.

At embryonic d18, broiler eggs were vaccinated with either PBS (control) or 1000 μg CNP vaccine. At d7 of age, birds were challenged with live SE (1.0 × 10⁹ CFU/bird). Ceca samples were collected from 3 birds/pen, pulled, and stomached 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. Data analyzed by parametric Student t-test; Means+SEM.Bars. n = 6. "*" means P<0.05.

Fig 6. Cytokine gene expression in the cecal tonsils of chickens vaccinated in-ovo with CNP.

At embryonic d18, broiler eggs were vaccinated with either PBS (control) or 1000 μg CNP vaccine. At d7 of age, birds were challenged with live SE (1.0 × 10⁹ CFU/bird). Cecal tonsil samples were collected at 2wk post-challenge 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–iNOS mRNA. Data analyzed by parametric Student t-test; Means+SEM.Bars. n = 6. "*" means P<0.05.