Chitosan nanoparticle vaccine loaded with crude extracellular proteins of C. perfringens and Salmonella flagella is partially protective against necrotic enteritis in broiler chickens

Necrotic enteritis (NE) causes significant economic losses and food shortages world-wide. There are currently no licensed commercial vaccines against NE in broilers. Chitosan nanoparticles were formulated with extracellular proteins of C. perfringens surface-tagged with Salmonella flagellar proteins. One-day-old male broiler chicks were completely randomized to 3 treatments: Non-vaccinated non-challenged as negative control, Vaccinated-challenged, and non-vaccinated challenge as positive control. On day of hatch, d7, and d14 post-hatch, vaccinated-challenged birds were orally gavage with 50μg vaccine in 0.5ml PBS while positive control birds were gavage with 0.5ml PBS only. Birds in the vaccinated-challenged and positive control groups were orally infected on d14 post-hatch, with 5,000 oocysts/bird of E. maxima, followed by log 8 CFU of a virulent strain of C. perfringens on d19, d20, and d21 post-hatch. From d14 to 21 and d14 to 28 post-hatches, mortality in the vaccinated-challenged group was higher than that in the positive control group, approaching statistical significance (p=0.07). On d21 post-hatch, the mean lesion score of 3 birds/cage in the vaccinated-challenged group was higher than the positive control group, approaching statistical significance (p = 0.05). From d 14 to 28 post-hatch, the feed intake was higher and feed conversion ratio lower in the vaccinated-challenged group compared to the positive control group (p<0.05). On d21 post-hatch, antigen specific recall proliferation in the vaccinated-challenged group was higher than that in the negative and positive control groups (p<0.05). On d21 post-hatch, cecal tonsils CD8+ T lymphocytes expression in the vaccinated-challenged group was similar to the negative control group (p>0.05) but higher than that in the positive control group (p<0.05). Finally, vaccination resulted in an increase in ileal mRNA levels of zonula occluding on d21 post-hatch. In conclusion, there were numerical but not statistically significant decrease in NE lesions and mortality in vaccinated and challenged broilers. Further studies are needed to improve the efficacy of the vaccine and understand the mechanism underlying protection in vaccinated birds.

219 Wallis chi-square test. Significance was determined at P < 0.05 and/or at P < 0.01. P values less 220 than 0.1 were determined as approaching significance.

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Birds in the vaccinated-challenged group had comparable mortality to that in the negative 225 control group, from d 14 to d 21 (one-week post challenge) and d 14 to d 28 post-hatch (two weeks 11 226 post challenge), but had lower mortality than that in the positive control group which approached 227 significance (P = 0.07, Table 1).

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Birds in the vaccinated-challenged group had higher lesion scores (  (Table 3). However, the vaccinated-challenged group had numerically lower feed intake than that 236 in the negative control group which approached significance (P = 0.08, Table 3), but had 237 comparable feed intake to the positive control group from d 14 to d 21 post-hatch (Table 3).

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The vaccinated-challenged group also had comparable FI to the negative control group 239 (Table 3) but had statistically significantly higher FI than that in the positive control group from d 240 14 to d 28 post-hatch (p < 0.05, Table 3).

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The vaccinated-challenged group had comparable FCR to the negative control group 242 (Table 3)  247 stimulated with 0 g ECP, 50 g ECP and 0.1 g Con A had comparable proliferation ( Fig 1A).
248 Splenic mononuclear cells obtained from the vaccinated-challenged group on d 18 post-hatch 12 249 and stimulated with 25 g ECP had comparable proliferation to that in the negative control group 250 ( Fig 1A) but had lower proliferation than that in the positive control birds approaching 251 significance (p = 0.09, Fig 1A). Splenic mononuclear cells obtained from the vaccinated-252 challenged group on d 18 post-hatch and stimulated with 100g ECP had comparable 253 proliferation to that in the negative control group (Fig 1A), but statistically significantly lower 254 proliferation than that in the positive control group (p < 0.01, Fig 1A).

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Splenic mononuclear cells obtained from all 3 treatment groups on d 21 post-hatch, and 256 stimulated with 0g ECP, 50g ECP, 100g ECP, and 0.1g Con A had comparable 257 proliferation (Fig 1B). Splenic mononuclear cells obtained from the vaccinated-challenged group 258 on d 21 post-hatch and stimulated with 25g ECP had statistically significantly higher 259 proliferation than that in the negative control and positive control groups (p < 0.05, Fig 1B).

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Splenic mononuclear cells obtained from all 3 treatment groups on d 28 post-hatch and 261 stimulated with 0g ECP, 25g ECP, and 50g ECP had comparable proliferation ( Fig 1C).
262 Splenic mononuclear cells obtained from chickens in the vaccinated-challenged group on d 28 263 post-hatch and stimulated with 100g ECP had statistically significantly lower proliferation than 264 that in the negative control group (p < 0.05, Fig 1C) but had comparable proliferation to the 265 positive control group ( Fig 1C). Splenic mononuclear cells obtained from chickens in the 266 vaccinated-challenged group on d 28 post-hatch, and stimulated with 0.1g ConA had comparable 267 proliferation to that in the negative control group but had statistically significantly lower 268 proliferation than that in the positive control group (p < 0.05, Fig 1C).

CD4 + , CD8 + cells, double positive CD4 + -CD8 + cells and CD4 + :CD8 + ratio
282 Splenic mononuclear cells obtained from all treatment groups on d 18, 21 and 28 post-283 hatch had comparable proportion of CD4 + cells (Fig 2A). Cecal tonsil mononuclear cells 284 obtained from all treatment groups on d 18 and 28 post hatch also had comparable proportion of 285 CD4 + cells (Fig 2A). Cecal tonsil mononuclear cells obtained from the vaccinated-challenge 286 group on d 21 post-hatch had a comparable proportion of CD4 + cells to that in the negative 287 control group (Fig 2A), but had a lower proportion of CD4 + cells than that in the positive control 288 group, approaching statistical significance (p = 0.07, Fig 2A).

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Splenic mononuclear cells obtained from all treatment groups on d 18 and 28 post-hatch 290 had a comparable proportion of CD8 + cells (Fig 2B). Splenic mononuclear cells obtained from 291 the vaccinated-challenged group on d 21 post-hatch had a numerically lower proportion of CD8 + 292 cells than that in the negative control group approaching statistical significance (p = 0.06, Fig   293 2B) but had a comparable proportion of CD8 + cells to that in the positive control group (Fig 2B).
14 294 Cecal tonsil mononuclear cells obtained from the vaccinated-challenge group on d 18 post-hatch 295 had a comparable proportion of CD8 + cells to that in the negative control group (Fig 2B), but had 296 a statistically significantly lower proportion of CD8 + cells than that in the positive control group 297 (p < 0.01, Fig 2B). Cecal tonsil mononuclear cells obtained from the vaccinated-challenge group 298 on d 21 post-hatch had a comparable proportion of CD8 + cells to that in the negative control 299 group but had a numerically lower proportion of CD8 + cells than that in the positive control 300 group, approaching statistical significance (p = 0.07, Fig 2B). Cecal tonsil mononuclear cells 301 obtained from all treatment groups on d 28 post-hatch had comparable percentages of CD8 + cells 302 (Fig 2B). 314 CD4 + CD8 + cells (Fig 2C).

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Splenic mononuclear cells obtained from the vaccinated-challenged group on d 18 post-316 hatch had a numerically higher CD4 + :CD8 + ratio than that in the negative control group 15 317 approaching significance (p = 0.05, Fig 2D) but had a comparable CD4 + :CD8 + ratio to that in 318 positive control group (Fig 2D). Splenic mononuclear cells obtained from all treatment groups on 319 d 21 and d 28 post-hatch had comparable CD4 + :CD8 + ratios (Fig 2D). Cecal tonsil mononuclear 320 cells obtained from all three treatment groups on d 18 and 21 post-hatch had comparable 321 CD4 + :CD8 + ratios (Fig 2D). Cecal tonsil mononuclear cells obtained from the vaccinated-322 challenge group on d 28 post-hatch had comparable CD4 + :CD8 + ratio to that in the negative 323 control group but had a statistically significantly lower CD4 + :CD8 + ratio than that in the positive 324 control group (p < 0.01, Fig 2D).