I. Introduction
Necrotic enteritis (NE), caused by Clostridium perfringens (CP) and exacerbated when birds are co-infected with Eimeria spp., is one of the most severe and common diseases resulting from intestinal mucosal damage (Bae et al., 2021). The stimbiotic (STB) concept has been recently introduced as a non-digestible and fermentable additive that stimulates the development of a microbiome comprising bacterial species that are principally involved in fibre degradation (Cho et al., 2020). It was reported that a low concentration of STB resulted in a better fermentation activity in colonic samples compared with 10-20 times higher doses of commercially available prebiotics. To successfully test the proposed mode of action, it is critical to develop an experimental infection model that will mimic a commercial outbreak of NE. Therefore, for successful NE induction in Experiment 1, a pre-screening study was performed to determine the optimal dosage of vaccine (coccidia and infectious bursal disease) and CP for inducing damage of the intestinal mucosa and instigating an NE infection model. Then in Experiment 2, the effects of STB on performance and intestinal barrier function in broilers challenged with the NE infection model were evaluated.
II. Methods
The experimental protocol for this study was reviewed and approved by the Institutional Animal Care and Use Committee of Chungbuk National University, Cheongju, Korea (CBNUA-1477-20-02).
1. Experiment 1
1.1. Oral administration of Vaccine and C. perfringens
Coccidia vaccine (Hipra Evalon®, Laboratorios Hipra, Girona, Spain) and infectious bursal disease (IBD) vaccine (IBD Blen®, Boehringer Ingelheim Animal Health USA Inc., Georgia, USA) were used in this experiment. These vaccines were diluted in sterile water and, when broiler chickens reached 14 days of age, 10 times or 20 times the recommended dose was administered orally through a sterile pipette. Clostridium perfringens (CP) type A NCTC 8798 (NCTC, National Collection of Type Cultures, London, UK) was used in this experiment. Four days after vaccination, a total of 3 ml of CP at 2.2 × 107 colony forming units (CFU)/mL was orally administered twice daily for 3 consecutive days (09:00 and 17:00; on days 18, 19, and 20).
1.2. Experimental animals and treatment
A total of 120 Arbor Acres (AA) broilers (45.0 ± 0.2g) were randomly assigned to 6 treatments in a 3×2 factorial arrangement (5 replicate pen with 4 broiler per pen). The duration of experiment was from d14 and d28. Coccidial and IBD vaccine treatments included nonchallenge (0), 10 times the recommended dose (×10) or 20 times the recommended dose (×20) by the manufacturer. Clostridium perfringens (CP) treatments were non-challenge (NE-) or 3 ml of 2.2 × 107 CFU/mL CP challenge (NE+).
2. Experiment 2
2.1. Induction of necrotic enteritis (NE) disease
NE was induced according to the optimal conditions determined in Experiment 1. Briefly, birds in the challenged groups were orally gavaged 20 times the recommended dose of coccidia vaccine and IBD vaccine on 14 days of age followed by oral gavage with a 3 ml of CP type A NCTC 8798 at 2.2 × 107 CFU/mL twice daily for three consecutive days (09:00 and 17:00; on day 18, 19, and 20).
2.2. Experimental animals and treatment
A total of 72 AA broilers (40.17 ± 0.03g) were randomly assigned to 6 treatments in a 3×2 factorial arrangement (4 replicate pens with 3 broiler per pen). The duration of experiment was from d1 and d 30. Birds were individually tagged and placed in floor pens from d1 to d21 and then moved in cages at d22 (3 birds/cage). Dietary treatments included non-additive (CON), 100 mg/kg STB (STB) and 100 mg/kg STB with other commercial feed additives such as essential oil, probiotics, and yeast cell wall (CB). Challenge treatments included non-NE challenge (NE-) or NE challenge (NE+).
Statistical analysis
Parametric data (growth performance, ileal morphology, blood profile, caecal microbiome and SCFA) were analysed by ANOVA using the fit model using JMP Pro 15.1 (SAS Institute Inc., Cary, NC, USA). The statistical model included the effect of the additives (CON, STB, CB), the effect of the challenge (NE- vs NE+) and the interaction between additives and challenge, and initial body weight was also included as a covariate. Treatment means were separated using Student’s t-test with significance accepted at p≤0.05. Nonparametric data (footpad dermatitis scoring, intestine lesion scores) were analyzed using contingency analysis to test the relationship between categorical variables and a Chi-square test to check if the variables are dependent or not with significance accepted at p≤0.05.
III. Results
1. Experiment 1
1.1. Growth performance
There was no interaction between vaccine overdose and CP challenge. CP challenge decreased (P < 0.01) body weight, body weight gain and feed intake on d24 compared with the non-CP challenged group. Moreover, the injected vaccines groups had (P < 0.05) lower BW, BWG and FI than non-injected group in d24, but the performance between ×10 and ×20 vaccines overdose was not significant. However, no mortalities occurred following NE challenge.
1.2. Incidence of diarrhoea and intestinal lesion
The incidence of diarrhoea (score 4) was significantly higher in the broilers challenged with ×10 and ×20 vaccine with CP. The percentage of score 3 and 4 lesions, equivalent to acute lesions, increased in the broilers challenged with ×20 vaccine with CP compared to the broilers challenged with ×10 vaccine with CP.
2. Experiment 2
2.1. Growth performance
There was no interaction between diet and NE challenge. NE challenge decreased BW, BWG and FI (P< 0.05), and increased FCR (P< 0.001) compared with the non-NE challenged groups until the end of the experiment. Compared with the CON group, the supplementation of STB and CB improved (P< 0.05) BWG, FCR and bodyweight-corrected FCR at d1-30, but there were no differences among STB and CB. However, the mortality by NE challenge was not observed.
2.2. Incidence of footpad and intestinal lesion
The percentage of score 3 and score 2 (equivalent to acute and moderate lesions in jejunum and ileum, respectively) increased when broilers were challenged with NE compared to the broilers without NE challenge. The supplementation of STB and CB reduced the percentage of the birds with the highest score.
2.3. Ileal morphology
There was no interaction between NE challenge and diets. The NE challenge decreased VH (P< 0.01) and increased CD (P< 0.01), hence reduced the VH:CD ratio (P< 0.01). The supplementation of STB and CB maintained higher VH () and VH:CD ratio (P < 0.01) than CON group, but there was no significant difference between STB and CB.
2.4. Blood profile
There was a significant interaction between NE-challenge and additives supplementation for endotoxin and TNF-α content (Figure 1; P< 0.01). The NE-challenge significantly increased serum endotoxin (P< 0.01) and TNF-α contents (P< 0.01) but in higher proportion for the control treatment. STB and CB decreased the serum TNF-α and endotoxin content only in the challenged birds.
2.5. Caecal bacteria culture
The NE challenge significantly reduced Lactobacillus counts (P < 0.01) in the caecal content and increased C. perfringens (Figure 2; P < 0.01). Supplementation of STB and CB increased Lactobacillus count (P< 0.01), while they significantly decreased E. coli (P < 0.01). An interaction between additives and challenge conditions was observed only for C. perfringens counts (P < 0.01), wherein the challenged conditions with no additives CP counts were significantly higher than for birds fed with STB or CB. The effect of STB and CB was also higher in challenged than in non-challenged conditions.
Figure 1. Effects of NE challenge and dietary treatments on TNF-α in serum and C.perfringens count in caecal content in broilers challenged with a necrotic enteritis infection model
IV. Discussion
The results were in agreement with the works of Craig et al. (2020) where the addition of XOS increased BWG in broilers. These improvements were partly explained by the enhanced gut health and functionality. In this study, NE challenge decreased growth of broilers, gut health and SCFA production, while increasing the lesion score and penetration of endotoxin into the blood. However, STB alleviated these negative effects induced by the NE challenge. XOS supplementation significantly improved the relative abundances of beneficial bacteria in addition to increased luminal concentrations of SCFAs, which are considered advantageous for intestinal health (Azad et al., 2018). The analysis by traditional microbiology methods of selected pathogenic bacteria such as E.coli and C. perfringens counts demonstrated their decrease, while the count of Lactobacillus, a well-known beneficial bacterial genus was increased by supplementing the diets with STB. NE challenge also increased endotoxin levels in the blood, while the addition of STB decreased endotoxin penetration. Liu et al (2012) reported that NE can promote the proliferation of several gram-negative bacteria in the ileum such as E. coli, resulting in the translocation of endotoxin to the blood and increasing the level of endotoxin in the blood. The reduction of endotoxin in this study can partly be attributed to the increase in beneficial intestinal microorganism populations that may have improved their capacity to ferment fibre and provide better gut integrity. Another interesting finding in this study was that other additives supplemented on top of the STB diet did not significantly improve the measured variables under these study conditions which were considered reasonably harsh, probably due to overlap of the mode of actions between the additives used.
Presented at the 33th Annual Australian Poultry Science Symposium 2022. For information on the next edition, click here.
