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. 2023 Oct 26;13(21):3323.
doi: 10.3390/ani13213323.

Evaluation of the Immunoprotective Capacity of Five Vaccine Candidate Proteins against Avian Necrotic Enteritis and Impact on the Caecal Microbiota of Vaccinated Birds

Sara Heidarpanah  1   2 , Alexandre Thibodeau  1   2   3 , Valeria R Parreira  4 , Sylvain Quessy  1 , Mariela Segura  2   3 , Marcelo Gottschalk  2   3 , Annie Gaudreau  2   3 , Tristan Juette  5 , Marie-Lou Gaucher  1   2   3
Affiliations

Evaluation of the Immunoprotective Capacity of Five Vaccine Candidate Proteins against Avian Necrotic Enteritis and Impact on the Caecal Microbiota of Vaccinated Birds

Sara Heidarpanah et al. Animals (Basel). .

Abstract

Avian necrotic enteritis is an enteric disease of broiler chickens caused by certain pathogenic strains of Clostridium perfringens in combination with predisposing factors. A vaccine offering complete protection against the disease has not yet been commercialized. In a previous study, we produced five recombinant proteins predicted to be surface-exposed and unique to necrotic enteritis-causing C. perfringens and the immunogenicity of these potential vaccine candidates was assessed in broiler chickens. In the current work, the relative contribution of the antibodies raised by these putative antigens to protect broiler chickens was evaluated using an experimental necrotic enteritis induction model. Additionally, the link between the immune response elicited and the gut microbiota profiles in immunized birds subjected to infection with virulent C. perfringens was studied. The ELISA results showed that the IgY antibody titers in vaccinated birds on days 21 and 33 were significantly higher than those on days 7 and 14 and those in birds receiving the adjuvant alone, while the relative contribution of the specific immunity attributed to these antibodies could not be precisely determined using this experimental necrotic enteritis induction model. In addition, 16S rRNA gene amplicon sequencing showed that immunization of birds with recombinant proteins had a low impact on the chicken caecal microbiota.

Keywords: Clostridium perfringens; broiler chickens; gut microbiota; immune response; necrotic enteritis; surface-exposed antigenic proteins.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Coomassie blue stained SDS-PAGE gels showing purification of His-tagged recombinant proteins by Ni-NTA affinity chromatography. A volume of 15 µL of crude E. coli extract and fractions of (a) P153, (b) P264, (c) P509, (d) P537, and (e) P561 were loaded onto 15% SDS-polyacrylamide gels. The size of the protein of interest is 4 KDa higher than the predicted size due to the insertion of V5 and His-tag at the N-terminal of each coding sequence. Negative control sample in Figure 1a corresponds to the crude cell lysate from BL21 cells that were transformed with pET empty vector. The uncropped original gels are presented in Supplementary Figure S1.
Figure 1
Figure 1
Coomassie blue stained SDS-PAGE gels showing purification of His-tagged recombinant proteins by Ni-NTA affinity chromatography. A volume of 15 µL of crude E. coli extract and fractions of (a) P153, (b) P264, (c) P509, (d) P537, and (e) P561 were loaded onto 15% SDS-polyacrylamide gels. The size of the protein of interest is 4 KDa higher than the predicted size due to the insertion of V5 and His-tag at the N-terminal of each coding sequence. Negative control sample in Figure 1a corresponds to the crude cell lysate from BL21 cells that were transformed with pET empty vector. The uncropped original gels are presented in Supplementary Figure S1.
Figure 1
Figure 1
Coomassie blue stained SDS-PAGE gels showing purification of His-tagged recombinant proteins by Ni-NTA affinity chromatography. A volume of 15 µL of crude E. coli extract and fractions of (a) P153, (b) P264, (c) P509, (d) P537, and (e) P561 were loaded onto 15% SDS-polyacrylamide gels. The size of the protein of interest is 4 KDa higher than the predicted size due to the insertion of V5 and His-tag at the N-terminal of each coding sequence. Negative control sample in Figure 1a corresponds to the crude cell lysate from BL21 cells that were transformed with pET empty vector. The uncropped original gels are presented in Supplementary Figure S1.
Figure 2
Figure 2
Lesion scores in birds submitted to NE-experimental infection and immunized with adjuvant alone, recombinant form of the candidate proteins, and whole-cell lysate from virulent C. perfringens MLG_7820. The bacitracin group was not immunized before the challenge (* p < 0.05 and lines correspond to the average lesion score).
Figure 3
Figure 3
ELISA antibody responses in birds immunized with indicated recombinant proteins mixed with the adjuvant Quil-A. The negative control group (Quil-A) was injected with the adjuvant alone. Birds in Mix group received 50 μg of each of the four candidate proteins (P153, P264, P509, P561, for a total of 200 μg of proteins) and 50 μg of Quil-A adjuvant. Immuno plate MaxiSorp wells were coated with 0.5 μg of each recombinant protein. The sera obtained from birds from the Mix group were tested four times separately against each recombinant protein. The number of asterisks indicates the level of significance: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
Figure 3
Figure 3
ELISA antibody responses in birds immunized with indicated recombinant proteins mixed with the adjuvant Quil-A. The negative control group (Quil-A) was injected with the adjuvant alone. Birds in Mix group received 50 μg of each of the four candidate proteins (P153, P264, P509, P561, for a total of 200 μg of proteins) and 50 μg of Quil-A adjuvant. Immuno plate MaxiSorp wells were coated with 0.5 μg of each recombinant protein. The sera obtained from birds from the Mix group were tested four times separately against each recombinant protein. The number of asterisks indicates the level of significance: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001.
Figure 4
Figure 4
Relative abundance of the major bacterial genera identified in the ceca of treated birds and submitted to NE infection. Only bacterial genera representing at least 2.5% of the total reads are shown.
Figure 5
Figure 5
Statistically significant changes (p-value < 0.05) in key bacterial families of interest across experimental groups.
Figure 6
Figure 6
Non-metric multidimensional scaling plot (NMDS) illustrating the dissimilarity between sample types using Bray–Curtis index.
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