Immunoprotection of Broiler Chickens Against Necrotic Enteritis by Oral Delivery of a Live Clostridium perfringens Vaccine Adjuvanted with Cholera Toxin at Hatch Open Access

Necrotic enteritis (NE) caused by Clostridium perfringens (CP) is a complex enteric disease of broiler chickens. Based on toxin gene carriage, CP has been classified into seven toxinotypes. Among them, CP type G is responsible for inducing NE in chickens (1). Overgrowth of CP in the gut produces a wide array of enzymes and exotoxins, which are responsible for necrosis and acute death in broiler chickens (2). Mortality could range from 2% to 50% (3,4). NE in broiler chickens accounts for severe production and economic losses due to poor weight gain, increased feed conversion ratio, and increased mortality. Global production losses due to NE have been estimated around US$6 billion annually (5,6). Broiler chickens between 3 and 6 wk of age are the most susceptible to NE (3), which might be due to the decay of maternal antibodies by 2–3 wk of age (2,6,7) and the underdeveloped immune system of young birds. Major predisposing factors for NE in broiler chickens include abrupt increases of feed protein or fiber content, wheat-based diets, immunosuppressive viral agents (e.g., infectious bursal disease virus), raised without antibiotics (RWA) programs, and concurrent infections (e.g., coccidiosis). Antibiotic growth promoters (AGPs) have been an effective tool to control NE; however, the withdrawal of AGPs presented an enormous challenge to control NE. Studies have been conducted to find alternatives to antibiotics against NE, such as probiotics, prebiotics, and organic acids, but their effectiveness has been limited (8,9,10).

Mucosal surfaces are the major entryways of pathogens to the host. Hence, mucosal immunization and the development of mucosal immunity have been considered significant factors in infectious disease control. It has been demonstrated that mucosal vaccinations induce both local and systemic immunity, including cytotoxic T-cell responses (11,12). However, immune tolerance is a limitation in mucosal vaccination and requires efficient antigen delivery and identification of suitable mucosal adjuvants (13). A Vibrio cholerae enterotoxin, cholera toxin B (CT), has been shown to be a potent mucosal adjuvant that activates antigen-presenting cells (APCs) such as B cells, macrophages, and dendritic cells (DCs) (14). CT is known to be a potent and highly stable oral mucosal adjuvant in mice (15). Studies in mice have shown that CT mainly induces a Th2-type response inducing the secretion of interleukin (IL)-4, IL-5, IL-6, and IL-10, which is further denoted by the production of immunoglobulin (Ig) antibodies IgA, IgG1, and IgE (16). In a mouse model, CT abrogated the pathways involved in the development of unresponsiveness or oral tolerance by altering the regulatory mechanism of gut-associated lymphoid tissue (17). CT has also been known to induce the maturation of human DCs (18). CT has a high affinity to bind to the ganglioside receptors, GM1, which are present throughout the intestine, including on epithelial cells, DCs and Peyer’s patches (18,19). Although CT has been extensively studied in humans and mice, there is only one study available in broiler chickens, which was related to the use of CT as an adjuvant along with a nonpathogenic live CP vaccine (20). Oral vaccination using a live CP isolate with necrotic enteritis B–like toxin (netB) at 8, 9, 10, 11, and 12 days of age by oral gavage or in-feed vaccination has been shown to elicit mucosal and systemic antibody responses in broiler chickens (20). Among Gel 01, CT, Escherichia coli labile toxin (LT), and mutant LT adjuvants, only CT was shown to be an effective adjuvant, while the other adjuvants were ineffective (20). Various studies have been carried out to test the effectiveness of live attenuated CP, vectored vaccines expressing CP proteins, inactivated vaccines, and protein-based toxins to prevent NE in broiler chickens and breeder hens. Some of these vaccines have been shown to reduce the severity of NE lesions and provided partial protection against NE under experimental conditions (21,22,23,24).

An extensive range of parenteral adjuvants is in use along with vaccine candidates to improve host responsiveness against antigens (25). Synthetic cytosine-phosphorothioate-guanine–oligodeoxynucleotides (CpG-ODN) can stimulate immune cells like DCs and B cells after being recognized by pathogen-associated molecular patterns (26). In ovo delivery of CpG-ODN at 18 days of incubation has been shown to induce protective immunity against bacterial infection by inducing a rapid enrichment of various immune compartments in chickens (27). CpG-ODN enhances both T helper 1 (Th1) and Th2 immune responses and has been used as a vaccine adjuvant (28). In chickens, the immunoadjuvant effect of CpG-ODN has been reported when used along with a killed E. coli vaccine against a lethal E. coli challenge. Vaccinated birds had a significantly higher titer of total IgY and survival in contrast to birds that received a vaccine with no CpG-ODN (29). In addition, intramuscular (IM) administration of a vaccine formulation composed of CpG-ODN, recombinant interferon (IF) γ, and a whole killed Salmonella antigen improved the protection of birds by inducing an enhanced Th1-type response against Salmonella enterica serovar Enteritidis (30). Conventional adjuvants such as oil-in-water/water-in-oil are known to induce a potent immune response, particularly with bacterial antigens (31). At the injection site, the emulsion is broken down into small globules that absorb the antigens and act as an active source of antigen to the immune cells for a prolonged period (31,32). A CP type A alpha-toxoid vaccine, formulated with an oil emulsion and administered by the IM route, provided passive immunity to broiler progeny against NE (23).

Therefore, the objectives of this study were to 1) induce mucosal immunity in broiler chickens against NE by oral delivery of a wild-type live CP vaccine adjuvanted with CT at hatch with or without prior in ovo administration of CpG-ODN; 2) protect broiler chickens against NE by immunizing broiler breeder parents by oral delivery of wild-type live CP-CT vaccine. Protection was evaluated against heterologous and homologous isolates of wild-type CP.

All animal experiments were approved by the Animal Research Ethics Board, University of Saskatchewan, and adhered to the Canadian Council on Animal Care guidelines for humane animal use. For all experiments, fertile broiler hatching eggs were obtained from a commercial Ross broiler breeder operation in Saskatchewan, Canada. Fertile broiler hatching eggs were incubated at the Animal Care Unit (ACU) at the Western College of Veterinary Medicine (WCVM), University of Saskatchewan, and candled at days 11 and 18 of incubation to confirm their viability. At day 18 of incubation, viable eggs were randomly divided into groups, and CpG-ODN (50 μg/egg) was delivered in ovo to the amniotic cavity as previously described (33). At hatch, broiler chicks were transferred to a level two containment room. Each group was housed in separate pens with 3–5 cm of wood shavings as litter. Continuous lighting was provided at 30–40 lux for the first 2 days posthatch, and then lux and duration decreased until 10–20 lux and 7 hr of darkness were achieved. Room temperature was 32 C for the first 7 days posthatch and then decreased by 0.5 C per day until 20 C. Water was provided ad libitum for the entire trial. A commercial RWA broiler starter ration was provided ad libitum until 19 days posthatch. A synthetic CpG-ODN²⁰⁰⁷ type B with sequence 5′-TCGTCGTTGTCGTTTTGTCGTT-3′ was used in all the experiments, as previously described (33). It was manufactured with a modified phosphorothioate backbone (Operon Biotechnologies, Inc., Huntsville, AL) and did not contain endotoxins. It was diluted according to the manufacturer’s instruction.

A well-established animal model of NE, as described previously (34), was used in this study. Briefly, broiler chickens were fed a commercial RWA broiler starter ration containing 20% protein (Farm Choice^TM RWA; Masterfeeds, Humboldt, SK, Canada) until 18 days. Feed was withdrawn at 19 days. At 20 days of age, a new feed ration was introduced containing 28% protein. The 28% RWA feed was prepared by mixing a commercially available 25% RWA turkey starter (MasterFeeds) with 38% poultry supplement (MasterFeeds) at a 10:3 ratio. A fluid thioglycolate (FTG)–grown, clinical isolate of CP with alpha (cpa), netB, beta (cpb2), and CP lethal (tpeL) toxin genes was added to the 28% protein ration and fed to chickens for 3 consecutive days (20 to 22 days of age) (34,35). The CP culture contained approximately 1 × 10⁹ colony-forming units (CFU)/ml in a 1:1 (v/w) ratio. Birds were observed for clinical signs and mortality three times per day until the termination of the experiment at 23 days of age. Mortality was recorded, and gross and histopathologic scoring of the intestine was conducted as previously described (34,35). Histopathologic intestinal lesions were scored as: 0 = no lesions/healthy mucosa; 1 = focal necrosis of intestinal villi, acute; 2 = necrosis of intestinal villi, multifocal to coalescing, acute; and 3 = diffuse necrosis of intestinal villi, acute, severe in all the birds (34,35).

To perform homologous and heterologous vaccine protection studies, two CP isolates were selected to vaccinate broiler chickens. The CP isolates were obtained from field cases of NE in broiler chickens, and genes profiles were determined by conventional PCR (34). The CP challenge isolates (with cpa, cpb2, netB, and tpeL genes) to develop NE were the same in all experiments. For homologous challenge protection studies, the same CP challenge isolate containing cpa, cpb2, netB, and tpeL genes was used to immunize birds. For heterologous challenge protection studies, a different CP isolate containing cpa, cpb2, netB, and tpeL genes was used as previously described (34,35).

To prepare the live CP-CT oral vaccine, CP isolates were streaked on 5% Columbia sheep blood agar (Thermo Fisher Scientific, Mississauga, ON) in duplicate and incubated under anaerobic conditions in an anaerobic chamber (BD Gas Pak EZ chamber with two sachets of AnaeroGen 3.5L; Thermo Scientific) at 37 C overnight. Single colonies were individually transferred to cooked meat broth media (CMM; Sigma-Aldrich, Oakville, ON) and incubated at 37 C for 24 hr under anaerobic conditions. Then, a 10-ml aliquot of cultured CMM broth was diluted with 490 ml of CMM (dilution factor 50; v/v) and incubated for 24 hr at 37 C. Subsequently, a single colony of CP grown in CMM was inoculated in FTG media (Sigma-Aldrich) and incubated anaerobically at 37 C for 14 hr. Following incubation, the CP culture contained approximately 1 × 10⁸ CFU/ml. The CFU was determined by plating CP culture on 5% Columbia blood agar in duplicate and incubating the plates anaerobically for 18- to 24 hr. Then, the CP culture was centrifuged at 3399 × g for 20 min to obtain a bacterial pellet. Next, bacterial pellet was washed twice with phosphate-buffered saline (PBS; pH 7.4, 0.01 M), followed by CP-CT vaccine preparation.

CT (C8052; Sigma-Aldrich), a mucosal adjuvant, was formulated with the live CP vaccine in respective experiments for the oral immunization. The amount of CT was calculated based on the mean body weight of birds at the time of vaccination. CT was reconstituted with sterile water according to the manufacturer’s instructions to obtain a final concentration of 10 mg/ml. CT was added at the rate of 0.5 μg/g chicken body weight with live CP (1 × 10⁸ CFU/bird), and sterile water was added to reach a final volume of 200 μl/bird.

Fertilized eggs (n = 300) were incubated at the ACU, WCVM, University of Saskatchewan, Canada. To confirm the viability, candling was performed at 11 and 18 days of incubation. At hatch, birds were assigned to six groups (n = 20) (Table 1): (A1) no vaccine, no challenge; (A2) CP challenge only; (A3) in ovo CpG-ODN + CP challenge; (A4) in ovo saline + CT adjuvant only (CT, 0.5 μg/g body weight) + CP challenge; (A5) in ovo CpG-ODN + oral CP-CT + CP boost + CP challenge; (A6) in ovo saline + oral CP-CT + CP boost + CP challenge. Groups A5 and A6 were boosted with live CP adjuvanted with CT (CP 1 × 10⁸ CFU/bird + CT, 0.5 μg/g body weight) by the oral route at 10 days of age. At 20, 21, and 22 days of age, vaccinated birds were challenged with homologous CP and observed for the development of clinical signs and mortality as described previously (34,35). Six subgroups (n = 10/group) were treated as above but were not challenged with CP in order to collect sera and mucosal scrapings to detect IgY and IgA against CP.

After candling at 18 days of incubation, fertile eggs were randomly assigned to 10 groups (n = 35 eggs/per group) and placed into different pens (Table 2): (B1) no vaccine, no challenge; (B2) CP challenge only; (B3) in ovo CpG-ODN + live CP (homologous) with CT + CP boost + CP challenge; (B4) in ovo saline + live CP (homologous) with CT + CP boost + CP challenge; (B5) in ovo CpG-ODN + live CP (heterologous) with CT + CP boost + CP challenge; (B6) in ovo saline + live CP (heterologous) with CT + CP boost + CP challenge; (B7) in ovo CpG-ODN + live CP (homologous) with CT + no CP boost + CP challenge; (B8) in ovo saline + live CP (homologous) with CT + no CP boost + CP challenge. (B9) in ovo CpG-ODN + live CP (heterologous) with CT + no CP boost + CP challenge; (B10) in ovo saline + live CP (heterologous) with CT + no CP boost + CP challenge. Oral CP vaccination was conducted at hatch with respective homologous or heterologous CP isolates (compared to the CP challenge isolate). Each bird received 1 × 10⁸ CFU adjuvanted with CT (0.5 μg/g body weight) by the oral route. Chickens in Groups B3, B4, B5, and B6 were boosted at 10 days of age with 0.04% formaldehyde inactivated 1 × 10⁸ CFU of the respective homologous or heterologous CP isolate, adjuvanted with 20% Emulsigen-D (MVP Adjuvants, Omaha, NE) by the subcutaneous (SC) route. At 20, 21, and 22 days of age, vaccinated birds were challenged with CP and observed for the development of clinical signs and mortality as described above. Ten subgroups (n = 10/group) were treated as above but were not challenged with CP at 23 days of age to collect sera and mucosal scapings to detect CP-specific IgY and IgA antibodies, respectively.

Day-old broiler breeder parents were obtained from Aviagen Inc., Huntsville, AL. At hatch, all chicks were vaccinated against Marek’s and Newcastle disease. Birds were housed at the ACU, WCVM, University of Saskatchewan. Feeding and lighting programs were operated according to Aviagen guidelines. Males (n = 8) and females (n = 36) were reared separately until 13 wk of age. The broiler breeder parents were divided (n = 22) into two groups: 1) no CP vaccination; 2) oral live CP-CT (CP-2 × 10⁸ CFU/bird + CT 0.5 μg/g body weight). At 13 wk of age, birds were vaccinated with CP-CT vaccine orally, followed by a booster vaccination at 16 wk of age with inactivated CP (2 × 10⁸ CFU/bird) vaccine adjuvanted with CpG-ODN (10 μg/bird) via the SC route. Eggs from broiler breeders were collected at 58 wk of age from all the groups (n = 20/group), and broiler progeny were divided as follows: 1) maternal antibody negative, no challenge; 2) maternal antibody negative, CP challenge; and 3) maternal antibody positive, CP challenge. Broiler progenies were challenged with CP at 20–22 days of age as described previously (34,35).

An indirect ELISA was used to detect CP-specific serum IgY and intestinal IgA antibodies as previously described (35). Briefly, to measure mucosal IgA antibody levels, sections of jejunum (approximately 8–9 cm in length) were carefully opened, and intestinal contents were removed. Jejunum mucosa was gently scraped and transferred into an Eppendorf tube containing 500 μl of 0.1% ethylenediaminetetraacetic acid (EDTA) solution on ice. Then, jejunal scrapings were vortexed and centrifuged at 447 × g, and the supernatant was stored at −20 C until testing by ELISA. Briefly, 96-well microtiter plates were coated with 100 μl of heat-inactivated CP (0.5 optical density [OD]) and kept at 4 C overnight. Coated wells were blocked by addition of 100 μl of 10% skim milk (Sigma-Aldrich) diluted in PBS (pH 7.4, 0.1 M) and then incubated for 30 min at room temperature. Blocked plates were then washed four times with distilled water (DW). Serum was tested at 1:100 dilutions, and mucosal scrapings were tested at 1:10 dilutions in duplicate. The serum and mucosal supernatant from each bird were separately diluted in 1% skim milk diluted in PBS with Tween 20 (PBST; pH 7.4). Plates were then washed 6× with DW and incubated with 100 μl of goat anti-chicken IgA (GeneTex, Inc., Irvine, CA) [1:2000] or IgY (GeneTex, Inc.) [1:100] antibody for 2 hr at room temperature. Plates were washed 6× with DW, and a 100-μl aliquot of TMB substrate (3,3',5,5' tetramethylbenzidine; Molecular Innovations, Cedarlane, Burlington, ON) was added. The plates were then incubated for 5 min. Negative control consisted of triplicate wells with all reagents except either antigen or the primary or secondary antibodies. The OD values were measured at 450 nm using a SpectraMax Plus 340 PC Microplate Reader (Molecular Devices, LLC, San Jose, CA) acquired through SoftMax Pro Data Acquisition and Analysis Software. Corrected antibody values for each bird were calculated as the average of OD₄₅₀ values from duplicate wells minus the average OD₄₅₀ values of the negative controls (20).

Flow cytometry was performed using peripheral blood mononuclear cells (PBMC) and jejunal tissue samples collected from three unchallenged subgroups of experiment B: (B1) no vaccine, no CP challenge; (B7) in ovo CpG-ODN + live CP-CT; and (B8) in ovo saline + live CP-CT. The PBMC and sections of jejunum were collected from birds vaccinated with live CP-CT by the oral route at 2 days postvaccination. The jejunum samples (n = 5/group) were collected and placed on cold PBS containing 1% penicillin-streptomycin (Thermo Fisher Scientific Inc., Waltham, MA). The jejunum was washed several times with PBS to remove mucus and fecal contents and then cut into 1-cm sections. Then, sections of jejunum were placed in a 15-ml centrifuge tube containing collagenase I (Sigma Aldrich, Oakville, Ontario, Canada [800 U/ml]) and deoxyribonuclease I (Thermo Fisher Scientific, Mississauga, Ontario, Canada [125 μg/ml]) and incubated for 30 min at 37 C for digestion. Digested jejunum was pushed through a 40-μm cell strainer using a syringe plunger and layered on 4 ml of Histopaque 1077® (Sigma Aldrich, Canada) in a 15-ml centrifuge tube. Tubes were then centrifuged at 560 × g at room temperature for 20 min, and then the buffy coat was collected and collated in Roswell Park Memorial Institute (RPMI) 1640 medium (RPMI1640, Sigma-Aldrich, Canada) supplemented with 10% of fetal bovine serum (Sigma Aldrich, Canada) and 1% penicillin-streptomycin. Cells were washed three times with RPMI by pelleting at 400 × g for 5 min at 4 C.

Heparinized blood (3 ml) was collected from each bird (n = 5/group) and kept at room temperature until processing. Afterwards, blood and PBS were pipet mixed at a ratio of 1:1 and then layered on 4 ml of Histopaque 1077 and centrifuged for 20 min at 560 × g at 20 C. The PBMC layer was collected and washed three times with RPMI medium. The number and viability of live cells were determined by trypan blue dye exclusion method by hemocytometer. In addition, the numbers of live and dead cells were further confirmed by 7-amino-actinomycin D (7-AAD; Invitrogen, Burlington, Ontario, Canada) staining. After counting, 0.5 million live cells from each sample were seeded in a 96-well plate. Cells were washed twice with fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% of fetal bovine serum) by gentle pipet mixing. Cells were stained for a T-cells panel (CD3-APC [CT-3; Southern Biotech, Birmingham, AL]; CD4-PE [CT-4; Southern Biotech]; CD8α-fluorescein isothiocyanate (FITC) [CT-8; Southern Biotech], and 7-AAD; [Invitrogen, Canada]) and for a monocyte/macrophage panel (MoMa) (Monocyte/Macrophage-PE [KUL01; Southern Biotech]; MHC-II-FITC [2G11; Southern Biotech]; and 7-AAD [Invitrogen, Canada]) for 20 min on ice in the dark. Cells were washed twice with FACS buffer and centrifuged at 400 × g for 5 min at 4 C. Cells were resuspended in 200 μl of FACS buffer. Side scatter area vs. forward scatter area followed by forward scatter height vs. forward scatter area were used to define single cells. 7-AAD was used to exclude dead cells. Fluorescence minus one control was used to determine positive and negative populations for major histocompatibility complex class II (MHC II) and MoMa. At least 100,000 events for PBMC samples and 200,000 events for jejunal samples were acquired by CytoFlex flow cytometer (Beckman Coulter, Carlsbad, CA), and results were analyzed using FlowJo Version 10.8.1 (2021; Becton, Dickinson, and Company, Ashland, OR) (Supplemental Fig. S1).

Survival/mortality and gross and histopathologic data were analyzed using GraphPad Prism V6.0 (GraphPad Software Inc., San Diego, CA) with a significance level of p < 0.05. Survival and mortality data were compared using the log-rank (Mantel-Cox) and chi-square tests. Microscopic lesions of the intestine were analyzed between groups using nonparametric one-way ANOVA and Kruskal-Wallis tests. Comparison between two groups was performed using the Mann-Whitney test (nonparametric test, p < 0.0001). Corrected duplicate OD values of antibodies were analyzed between the groups by nonparametric ordinary one-way ANOVA and Kruskal-Wallis tests (p < 0.0001). Comparison between the two groups’ OD values was analyzed using the Mann-Whitney test (nonparametric test, p < 0.0001).

Typical gross and histopathologic lesions of NE in this animal model are shown in Fig. 1. No mortality was observed in Groups A1 (no vaccine, no challenge), A5 (in ovo CpG-ODN + oral CP-CT + CP boost + CP challenge), and A6 (in ovo saline + oral CP-CT + CP boost + CP challenge). Group A2 (CP challenge only) had a total of 30% mortality; three birds were dead at 2 days, and three birds were dead at 3 days postchallenge (Fig. 2A). Group A3 (in ovo CpG-ODN + CP challenge) had three dead birds at 3 days postchallenge (15%). Group A4 (in ovo saline + CT adjuvant only) had two dead birds at 3 days postchallenge (10%). No gross or microscopic lesions were observed in group A1 (no vaccine, no challenge). Group A2 (CP challenge only) had 85% of birds with NE lesions (55% of birds with macroscopic lesions [score 3] and 10% of birds with microscopic lesions [score 2 in 10% of birds and score 1 in 20% of birds]). The significance of the histopathologic score of Groups A3 (in ovo CpG-ODN + CP challenge), A4 (in ovo saline + CT adjuvant only), A5 (in ovo CpG-ODN + oral CP-CT + CP boost + CP challenge), and A6 (in ovo saline + oral CP-CT + CP boost + CP challenge) was compared to that for group A2 (CP challenge only). Group A3 (in ovo CpG-ODN + CP challenge) had 60% of birds with NE lesions (score 1 in 25% of birds, score 2 in 5% of birds, and score 3 in 30% of birds; p = 0.0622). Group A4 (in ovo saline + CT adjuvant only) had 45% of birds with NE lesions (score 1 in 20% of birds, score 2 in 15% of birds, and score 3 in 10% of birds; p = 0.0015). Group A5 (in ovo CpG-ODN + oral CP-CT + CP boost + CP challenge) had 35% of birds with NE lesions (score 3 in 15% of birds and score 2 in 20% of birds; p = 0.0027). Group A6 (in ovo saline + oral CP-CT + CP boost + CP challenge) had 20% of birds with NE lesions (score 3 in 20% of birds; p = 0.0004; Fig. 2B). Significantly high levels of IgY and IgA antibodies were observed in both groups vaccinated with live CP adjuvanted with CT with or without prior in ovo delivery of CpG-ODN compared to group 1 (p < 0.0001) (Fig. 2C,D). Significantly high IgY antibody levels were observed in the groups vaccinated with CP-CT (in ovo CpG-ODN + oral CP-CT + CP boost [p = 0.0085];in ovo saline + oral CP-CT + CP boost [p < 0.0001]) compared to the group that received CpG-ODN alone (Fig. 2C). Similarly, significantly high IgA antibody levels were observed in the groups vaccinated with CP-CT (in ovo CpG-ODN + oral CP-CT + CP boost [p = 0.0047];in ovo saline + oral CP-CT + CP boost [p = 0.0002]) compared to the group that received CpG-ODN alone (Fig. 2D). Significantly high IgY was noted in the groups vaccinated with CP-CT (in ovo CpG-ODN + oral CP-CT + CP boost [p = 0.0005];in ovo saline + oral CP-CT + CP boost [p < 0.0001]) compared to the group that received CT alone (Fig. 2C). Significantly high IgA was noted in the group vaccinated with CP-CT (in ovo saline + oral CP-CT + CP boost; p = 0.0011) compared to the group that received CT alone (Fig. 2D).

There was no mortality in group B1 (no vaccine, no challenge) or in all the groups vaccinated with CP-CT vaccine. Group B2 (CP challenge only) had 20% mortality (Fig. 3A). There were no gross or microscopic NE lesions (0%) in group B1 (no vaccine, no challenge; Fig. 3B). Group B2 (CP challenge only) had 100% of birds with NE lesions (score 3 lesions in 90% of birds and score 2 lesions in 10% of birds; p < 0.0001). Histopathologic score of all vaccinated Groups B3 to B10 were compared to Group B2 (CP challenge only). Group B3 (in ovo CpG-ODN + live CP [homologous] with CT + CP boost + CP challenge) had 20% of birds with NE lesions (score 1 in 10% of birds and score 2 in 10% of birds; p < 0.0001). Group B4 (in ovo saline + live CP [homologous] with CT + CP boost + CP challenge) had 30% of birds with NE lesions (score 1 in 15% of birds, score 2 in 10% of birds, and score 3 in 5% of birds; p < 0.0001). Group B5 (in ovo CpG-ODN + live CP [heterologous] with CT + CP boost + CP challenge) had 5% of birds with NE lesions (score 1 in 5% of birds; p < 0.0001). Group B6 (in ovo saline + live CP [heterologous] with CT + CP boost + CP challenge) had 5% of birds with NE lesions (score 2; p < 0.0001). Group B7 (in ovo CpG-ODN + live CP [homologous] with CT + no CP boost + CP challenge) had 15% of birds with NE lesions (score 1 in 5% of birds, score 2 in 5% of birds, and score 3 in 5% of birds; p < 0.0001). Group B8 (in ovo saline + live CP [homologous] with CT + no CP boost + CP challenge) had 20% of birds with NE lesions (score 1 in 10% of birds, score 2 in 5% of birds, and score 3 in 5% of birds; p < 0.0001). Group B9 (in ovo CpG-ODN + live CP [heterologous] with CT + no CP boost + CP challenge) had 20% of birds with NE lesions (score 2 in 5% of birds and score 1 in 15% of birds; p < 0.0001). Group B10 (in ovo saline + live CP [heterologous] with CT + no CP boost + CP challenge) had 10% of birds with NE lesions (score 1; p < 0.0001). All the groups vaccinated with either homologous or heterologous CP vaccine with or without in ovo CpG-ODN had significantly higher levels of IgY (p < 0.0001) and IgA (p < 0.0001) antibodies compared to Group B1 (Fig. 3C,D).

No mortality was observed in Group 1 (no parent vaccine, maternal antibody negative, no CP challenge). There were two birds dead in Group 2 (no parent vaccine, maternal antibody negative, CP challenge) at 2 days post–CP challenge. Group 3 (parent vaccine, maternal antibody positive, CP challenge) had no mortality after challenge with CP. Group 3 (maternal antibody positive, CP challenge) birds were from broiler breeder parents vaccinated with CP-CT vaccine (Fig. 4A). No macroscopic or microscopic lesions were observed in Group 1 (no parent vaccine, maternal antibody negative, no CP challenge). Group 2 (no parent vaccine, maternal antibody negative, CP challenge) had 80% of birds with NE lesions (score 1 in 10%, score 2 in 10%, and score 3 in 60% of birds). Group 3 (parent vaccine, maternal antibody positive, CP challenge) had 35% of birds with NE lesions (score 1 in 15% of birds, score 2 in 10% of birds, and score 3 in 10% of birds). This was significantly low compared to Group 2 (no parent vaccine, maternal antibody negative, CP challenge; p < 0.0001; Fig. 4B).

A significant increase of CD8⁺ T cells (p < 0.0001; Fig. 5A) and a decrease of CD4⁺ T (p < 0.01; Fig. 5C) cells were observed in PBMC of birds vaccinated with live CP by the oral route with or without in ovo CpG-ODN administration compared to the birds that did not receive a CP vaccine. Birds vaccinated with CP-CT vaccine had a significant increase in CD8⁺ T cells in the jejunal tissue with or without prior administration of in ovo CpG-ODN compared to the group that did not receive a CP vaccine (p < 0.001; Fig. 5B). There was a significant increase (p = 0.0197; Fig. 5D) in CD4⁺ T cells in the jejunum of groups vaccinated with live CP-CT by the oral route with prior in ovo CpG-ODN administration. The difference was not significant in broilers that were vaccinated but that did not receive in ovo CpG-ODN administration. Also, a significant difference was observed in the population of monocytes in PBMCs and macrophages in the jejunum of birds vaccinated with live CP-CT by the oral route with or without prior in ovo CpG-ODN administration compared to the birds that did not receive a CP vaccine (Fig. 5E,F).

Multiple vaccine platforms have been explored to control NE in broiler chickens. The types of vaccines range from live attenuated CP to non-inactivated CP culture supernatants, crude toxoid vaccines, recombinant proteins of CP, bacterial vectored vaccine, and modified toxins (21,22,36,37,38). Several vaccine delivery routes have also been explored, including oral, IM, and SC routes. However, these vaccines provided only partial protection (21,22,36,37,38,39,40). For instance, a significant reduction in NE lesions had been demonstrated after oral vaccination of broilers with a live recombinant Salmonella enterica serovar Typhimurium expressing the C-terminal domain of the alpha toxin of CP (38). However, administration of recombinant proteins expressing perfringolysin at day 1 and day 14 did not protect broiler chickens against NE (22,41).

Developing an effective vaccination strategy against NE, preferably delivering a single dose of vaccine at hatch, without a booster vaccine is an enormous challenge. Protection of broiler chickens against NE at 3-4 wk of age, a time when maternal antibodies decline, by inducing active immunity with a single vaccine at hatch would be ideal for the broiler chicken industry. In our first experiment (Experiment A), we observed a significant reduction of pathologic lesions of NE following a homologous CP challenge in the group vaccinated with CP-CT by the oral route at hatch followed by a CP-CT booster with or without in ovo delivery of CpG-ODN. Here, we concluded that a CP-CT vaccine at hatch followed by a CP-CT booster mounted a protective immune response against CP. Since groups of birds were primed and boosted with CP-CT vaccine in Experiment A, the objective of the second experiment (Experiment B) was to measure protective immunity induced by CP-CT vaccine alone at hatch without a CP-CT booster against heterologous CP. It was confirmed in the second experiment that CP-CT vaccination at hatch with no booster vaccine was able to protect broilers against heterologous CP challenge at a significant level. Furthermore, it was confirmed that broiler chickens that received a CP-CT vaccine by the oral route at hatch did not require CpG-ODN by the in ovo route to mount protective immunity against NE.

Interestingly, we found that a control group that received CT adjuvant alone at hatch protected broiler chickens against CP and had a significant reduction in NE lesions (p < 0.01). We hypothesize that CT modulates interactions of mucosal APCs and CP in the normal flora to induce protective immunity against CP. In order to test this hypothesis, additional methodologies such as transcriptomics, metabolomics, and single-cell RNA sequence studies of the intestine will be useful to analyze crosstalk of immune cells following oral delivery of CT.

It was demonstrated previously that oral delivery of a nonvirulent CP strain adjuvanted with CT was found to induce antibody response against NE but required multiple administrations (20). Administration of CpG-ODN to embryos at day 18 of incubation followed by a live CP vaccine adjuvanted with CT delivered by the oral route at hatch protected birds significantly against NE. This might be linked to the ability of CpG-ODN to enhance maturation of DCs, which further potentiates the T cell–mediated immune response (42,43,44). In the second experiment (Experiment B), we were able to demonstrate protection of birds against NE at a significant level after oral immunization with live CP-CT vaccine against heterologous CP with or without prior in ovo delivery of CpG-ODN and without a booster CP vaccination. The protection was correlated with significant increases of IgY and IgA antibodies in the serum and the jejunum, respectively. This signifies the ability of the CP-CT vaccine to potentially induce robust broad-spectrum mucosal immunity against NE. The objectives of this study were to demonstrate a proof of concept on the utility of CT with wild-type CP isolates, but further studies are needed to explore avenues to attenuate wild-type CP strains as a potential safe vaccine candidate in combination with CT. We used a wild-type clinical isolate of live CP in the CP-CT vaccine since we had previously demonstrated that oral delivery of this wild-type clinical isolate of CP in broiler chickens did not produce any macroscopic or microscopic lesions when delivered to broilers by in-feed administration for 3 consecutive days or by oral gavage for 3 consecutive days. Macroscopic or microscopic lesions of NE only occurred when there was an abrupt increase in the in-feed protein content following feed withdrawal as described in our NE animal model development studies (34). Furthermore, direct oral administration of our wild-type clinical isolate of CP does not cause NE in birds unless the birds are immunosuppressed with variant infectious bursal diseases, and a there is a subsequent abrupt change of protein content in the feed as described previously in our animal model of NE (34). Moreover, we did not see any adverse reactions (i.e., clinical signs or poor growth) in groups vaccinated with virulent CP-CT as a vaccine compared to the nonvaccinated, non–CP challenge group throughout the duration of the experiment. Furthermore, it is essential to culture this wild-type CP twice in CMM prior to culture in FTG in order to express the genes necessary for the development of NE in broiler chickens. Culturing this wild-type CP in FTG without culturing in CMM did not develop NE in broiler chickens, even with an abrupt change in feed protein content following feed withdrawal (data not shown). These observations indicate a delicate balance of this wild-type CP within the intestinal environment of the host.

There was an influx of CD8⁺T cells in the peripheral blood and the jejunum of birds 2 days after oral delivery of CP-CT regardless of CpG-ODN administration at the embryonic stage. In contrast, a significant decline in CD4⁺ T cells in PBMC and no significant change of CD4⁺ T cells were observed in the jejunum. Lack of T-cell responses along with diminished systemic antibody response are the hallmarks of oral tolerance or systemic unresponsiveness to antigens (45). Our results provide clear evidence of the absence of induction of oral tolerance following CP-CT oral vaccination. However, detailed investigations are required to understand immune mechanisms associated with the oral delivery of CP-CT vaccine against NE in broiler chickens, including the vaccine-mediated pathways that lead to abrogation of oral tolerance.

There was a significant reduction in NE lesions in broiler progeny following CP challenge when broiler breeder parents were vaccinated against NE using the CP-CT vaccine by the oral route followed by a CP booster vaccination using CpG-ODN as an adjuvant. Similar maternal antibody–mediated protection was reported in broiler progeny obtained from broiler breeder parents that were vaccinated with a CP type A alpha-toxoid vaccine by the IM route (23). Better protection of broiler progeny was observed when broiler breeder parents were vaccinated with type A CP than with type C CP vaccine (24). To the best of our knowledge, our study is the first to demonstrate protection of broiler progeny against NE by immunizing broiler breeder parents with an oral live CP vaccine adjuvanted with CT.

In conclusion, we were able to demonstrate protection of broiler chickens against NE by a single live CP vaccine adjuvanted with CT delivered by the oral route with or without prior in ovo delivery of CpG-ODN and with no booster vaccine. This vaccination strategy was able to protect broiler chickens against homologous and heterologous isolates of CP at a statistically significant level. Our vaccine formulation and delivery strategy were able to induce the production of both systemic IgY and mucosal IgA antibodies against CP. We also demonstrated a feasible strategy of maternal antibody–mediated protection of broiler progenies against NE by vaccinating their broiler breeder parents with an oral live CP vaccine adjuvanted with CT followed by a booster vaccine containing CpG-ODN as an adjuvant. This study demonstrates the utility of CT as a potent mucosal adjuvant with live CP vaccine against NE in broilers and broiler breeders.

Supplemental data associated with this article can be found at https://doi.org/10.1637/aviandiseases-D-24-00036.s1.

We are grateful to the animal care technicians at the ACU, WCVM, University of Saskatchewan. Financial support was provided by grants from Chicken Farmers of Saskatchewan and Poultry Research Cluster III (Canadian Poultry Research Council and Agriculture and Agri-Food Canada). Special thanks go to Aviagen North America, Huntsville, AL, for donation of broiler breeders for animal experimental studies.

The animal use protocol was approved by the University of Saskatchewan's Animal Care Committee (UACC) Animal Research Ethics Board (AREB; Certificate of Approval no. 20180085) and was conducted according to the Canadian Council on Animal Care (CCAC) guidelines.

7-AAD =

7-amino-actinomycin D;

ACU =

animal care unit;

AGPs =

antibiotic growth promotors;

APCs =

antigen-presenting cells;

CMM =

cooked meat broth media;

CP =

Clostridium perfringens;

cpa =

alpha toxin gene;

cpb2 =

beta toxin gene;

CFUs =

colony-forming units;

CpG-ODN =

cytosine-phosphorothioate-guanine oligodeoxynucleotides;

DCs =

dendritic cells;

DW =

distilled water;

FACS =

fluorescence-activated cell sorting;

FITC =

fluorescein isothiocyanate;

FTG =

fluid thioglycolate media;

IF =

interferon;

Ig =

immunoglobulin;

IL =

interleukin;

IM =

intramuscular;

LT =

labile toxin;

MoMa =

monocyte/macrophage;

NE =

necrotic enteritis;

netB =

necrotic enteritis B–like toxin gene;

OD =

optical density;

PBMC =

peripheral blood mononuclear cells;

PBS =

phosphate-buffered saline;

PBST =

PBS with Tween 20;

RPMI =

Roswell Park Memorial Institute media;

RWA =

raised without antibiotics;

SC =

subcutaneous;

Th =

T helper;

TMB =

tetramethylbenzidine substrate;

tpeL

member of the large clostridial toxin (LCT) family;

WCVM =

Western College of Veterinary Medicine