Effects of tannic acid on the immunity and intestinal health of broiler chickens with Clostridium perfringens-induced necrotic enteritis

Abstract

Background

In broiler chickens, necrotic enteritis (NE) infection can reduce production performance. Tannic acid, as a kind of plant extract, has received extensive attention. However, the appropriate dosage of tannic acid in NE of broilers and the improvement effect on intestinal health are not very clear. In this study, we aimed to investigate the effects of different doses of tannic acid on the production performance, immunity, and intestinal health of broilers by constructing an NE model with C. perfringens infection and determining the appropriate dosage of tannic acid with regard to NE.

Results

Challenged birds showed significant reduction in body weights, villus heights, and the ratio of villus height to crypt depth (V/C) (P < 0.05) and increase in the feed consumption gain ratio, intestinal lesion score, and crypt depth (P < 0.05). NE infection significantly reduced the relative Bacteroides and Ligilactobacillus abundance (P < 0.05) and increased the ratio of Firmicutes/Bacteroides and cecal content of C. perfringens (P < 0.05). Challenged birds fed diets supplemented with tannic acid showed significantly increased mRNA expression of nutrient transport carriers and intestinal barrier genes and growth performance and reduced serum zonulin and endotoxin levels (P < 0.05). Addition of tannic acid to the diet inhibited the inflammatory response by reducing the number of coccidia oocysts in feces and the content of C. perfringens in the cecum. Specifically, tannin acid reduced the serum levels of C reactive protein, myeloperoxidase, and specific IgY and ileal mucosal secretory immunoglobulin A (sIgA) levels in the ileal mucosa compared with those in the NE-infected birds. NE-infected birds fed diets supplemented with tannin acid also showed significantly increased relative Anaerocolumna, Thermoanaerobacterium, and Thermosinus abundance (P < 0.05); their microbial composition and functional predictions were similar to those of the NC group.

Conclusions

Tannic acid in the diet alleviated NE by enhancing the intestinal barrier and absorption function. The recommended dietary tannic acid additive level is 500–750 mg/kg. Our study findings would be useful in reducing related economic losses in the broiler industry.

Background

In the poultry industry, ensuring that the intestinal tracts of birds are healthy plays an important role in maximizing growth performance. Necrotic enteritis (NE) in broiler chickens, caused by Clostridium perfringens (C. perfringens), is one of the most prominent intestinal diseases in modern intensive farming. The destruction of the intestinal mucosal structure in broiler chickens with NE results in poor digestion and absorption of nutrients and reduced feed efficiency, causing huge losses for the industry [1]. In addition, C. perfringens can enter the human body through the food chain, threatening public health. Due to the limited use of antibiotic growth promoters (AGP) in the poultry industry, the incidence of NE in chickens has been increasing worldwide. Therefore, effective AGP alternatives are urgently required to prevent and control NE in broiler chickens.

Tannins are water-soluble polyphenols and secondary metabolites in plants [2]. They are classified into two types according to their chemical structures—condensed tannins (CTs) and hydrolytic tannins (HTs). HTs are phenolic acid polyesters with a D-glucose core, such as gallic acid and ellagic acid. The relative molecular weight of HTs is usually 500–3000 Da, and they are easily hydrolyzed by acids, alkalis, and enzymes. CTs are composed of flavan-3-ol units connected by carbon-carbon double bonds, also called proanthocyanidins (catechins or epicatechins). CTs are the most prevalent and typical class of plant tannins, with relative molecular masses ranging from 1,900 to 28,000 Da; they are mainly found in legumes, trees, and shrubs [3].

Tannins have antibacterial, antiparasitic, antiviral, anti-inflammatory, antioxidant, antidiarrheal, and nutrient metabolism-modulating effects [4]. Dietary supplementation of broilers with tannins has been shown to improve growth performance [5, 6] and reduce the abundance of C. perfringens, production of toxins such as alpha toxin [7], and the adverse effects of C. perfringens and Eimeria tenella in chickens [8, 9]. The European Union approved tannins as a novel feed additive for livestock and poultry in 2016. However, it has been shown that the addition of tannins can result in negative effects, including reduction in production performance [10]. The differences in the results may be related to the tannic acid type or dosage used. Therefore, this study was conducted to investigate the modulating effects of different tannic acid additive levels on the intestinal health of broiler chickens co-infected with coccidia and C. perfringens to aid the development of more tailored tannic acid application strategies.

Methods

Experimental Design, Birds, And Diets

The experiment was carried out at the poultry experiment base of China Agricultural University (Zhuozhou City, Hebei Province, China). A total of 630 1-day-old Cobb 500 male broilers were weighed and randomly divided into six treatment groups according to the principle of similar body weight. There were seven replicate cages (1.0 × 0.7 × 0.38 m, length × width × height) per treatment and 15 chickens per replicate. They were grouped as follows: (i) negative control group (no tannin acid treatment or NE infection, NC group); (ii) positive control group (NE infection + 0 mg/kg tannin acid, PC group); (iii) PTA1 group (NE infection + 250 mg/kg tannic acid); (iv) PTA2 group (NE infection + 500 mg/kg tannic acid); (v) PTA3 group (NE infection + 750 mg/kg tannic acid); and (vi) PTA4 group (NE infection + 1000 mg/kg tannic acid). Broilers were reared in a room equipped with two-tiered battery cages. All chickens were vaccinated and managed (including light and temperature management) according to routine immunization and management programs of Cobb broilers. Furthermore, all chickens were provided feed and water ad libitum. Diets were formulated according to the Chinese chicken feeding standard (NY/T 33-2004) and were fed in the form of pellets; the nutritional information is summarized in Table 1.

C. perfringens challenge

NE was induced in chickens according to a previously described method, with slight modifications [11]. On day 19, birds in the PC and PTA1-PTA4 groups were orally inoculated with 1 mL of a 25-fold dose of attenuated quadrivalent coccidia vaccine suspension (Foshan Standard Bio-Tech Co. Ltd., Foshan, China). Chickens in the NC group received 1 mL sterile PBS. Except for broilers in the NC group, chickens were orally gavaged with 1 mL C. perfringens type A CVCC52 (3 × 10⁸ CFU/mL) per day on days 22–28. Uninfected birds received 1 mL sterile fluid medium.

Growth Performance

All broilers were weighed at 19, 28, and 35 d of age in replicates after 12 h of fasting. Body weight, feed consumption, and the feed consumption to weight gain ratio (F/G) were calculated at different experimental periods.

Sample Collection

One chicken with the average body weight was selected from each replicate at 28 and 35 days of age, and blood was collected from the wing vein, followed by intravenous injection of sodium pentobarbital at a dose of 30 mg/kg body weight. After the anesthesia, the chickens were bled through the jugular vein and sacrificed. The middle region (1 cm) of the ileum was excised and immediately fixed in 4% paraformaldehyde for intestinal morphological examination. Approximately 2 g of chyme from the middle ileum was collected aseptically into sterile tubes; the mucosa of the middle ileum was then gently scraped with a slide, collected in sterile tubes, and stored at − 80°C until further analysis.

Intestinal Ne Lesion Scoring

At 28 days of age, one chicken in each replicate was anesthetized and sacrificed. The intestinal cavity was opened, and chyme was removed to observe the lesions in the intestinal wall. The scoring rules for intestinal lesions described by Dahiya et al. were adopted [12]; the specific scoring criteria were as follows: 0 = normal intestinal appearance; 0.5 = severe congestion in serous surface and mesentery of the small intestine; 1 = the intestinal wall became thin and brittle, with red silting spots (more than 5); 2 = the intestinal wall appears needle-like necrosis or ulceration, and there is a small amount of gas in the intestinal cavity; 3 = flaky necrosis or ulceration of the intestinal wall, gas-filled intestines and small blood spots, and 1–2 cm long necrotic spots; 4 = diffuse necrosis, significant bleeding, and large intestinal gas.

Fecal coccidia oocyst counts and determination of intestinal C. perfringens concentration

On days 25–28, the feces of broilers were collected repeatedly, and fecal coccidia oocysts were counted. The feces collected from each replicate were mixed; then, 2 g feces was added to 58 mL saturated salt water, mixed thoroughly with a vortex shaker for 2 min, and sieved through a 40 mesh. The upper suspension was then added to a McMaster counting plate and allowed to stand for 3 min. Oocysts were counted under a light microscope (Leica, LEICA DM750), and the number of oocysts per gram of feces was calculated according to the following formula: oocyst content per gram of feces (opg) = number of oocysts in the counting chamber × 200.

Cecal C. perfringens was counted as previously described [11]. Approximately 0.5 g of each sample was collected in 10 mL sterile plastic tubes, diluted with PBS to an initial 10^− 1 dilution, and serially diluted to prepare 10^− 1 to 10^− 5 dilutions. Each diluted sample of 100 µL was plated on tryptose-sulfite-cycloserine agar (TSC, CM 138; Beijing Land Bridge Technology Co., Ltd.). The number of C. perfringens was determined after anaerobic incubation at 37°C for 24 h. The opg and C. perfringens were then log₁₀-transformed and counted.

Intestinal Morphological Analyses And Observation

The middle ileum was circumcised to a thickness of 5 µm and stained with hematoxylin and eosin. The height of the villi and depth of the crypt of the chicken ileum were measured according to the method described by Frankel et al. [13]. Briefly, 10 straight and structurally intact intestinal villi were selected from each intestinal section and measured. The mean value of each index was calculated to determine the ratio of villus height to crypt depth (V/C).

Determination Of Serum Immunoglobulin, C-reactive Protein, Myeloperoxidase, And Ileal Mucosa Siga Levels

The levels of natural serum IgA and IgM antibodies were determined using a chicken IgA enzyme-linked immunosorbent assay (ELISA) kit (Beijing Solaibao Technology Co., Ltd., SEKCN-0018) and chicken IgM ELISA kit (Beijing Solaibao Technology Co., Ltd., SEKCN-0128), respectively. C-reactive protein (CRP) (Shanghai Enzyme Link Biotechnology Co., Ltd., YJ036965) and myeloperoxidase (MPO) kits (Shanghai Enzyme Link Biotechnology Co., Ltd., YJ966521) were used to determine the serum levels of CRP and MPO, respectively. The tissue homogenate was prepared by taking 0.1 g of ileal mucosa at a weight: saline volume ratio of 1:9, and the supernatant was retained for subsequent analysis. The sIgA and total protein contents in the ileal mucosa were determined using a chicken sIgA ELISA kit (Shanghai Yuanmu Biotechnology Co., Ltd., YM-A3724) and a total protein quantification kit (Nanjing Jiancheng Institute of Biological Engineering), respectively. Serum immunoglobulin content was measured in mg/mL, and mucosal sIgA content was measured in µg/mg protein. The above-mentioned indexes were measured in strict accordance with the manufacturer's instructions.

The antibody levels of C. perfringens in the serum were detected using an improved ELISA method. The methods referred to those described by Wu et al., with slight alterations[11]. C. perfringens was cultured to a concentration of 10⁸ CFU/mL and centrifuged at 4000 rpm for 10 min; the thalli were collected and cleaned with 0.01 M sterile PBS solution thrice. The last time, the thalli were diluted with PBS and subjected to intermittent ultrasound over 10 times in an ice bath. When the bacterial solution was turbid and consistent, 9 ml PBS solution was added and incubated for more than 2 h. The supernatant was absorbed into another sterilized centrifuge tube. Protein concentration was measured using the total protein quantitative kit (Nanjing Jianguo Institute of Bioengineering) according to the manufacturer's instructions. Approximately 100 µL bacterial lysate of C. perfringens (40 µg/mL) was added to the enzyme-labeled plate and incubated overnight at 4°C. The 96-well plates were coated with 20 µg/mL albumin from bovine serum (BSA) and incubated overnight at 4°C. After washing five times with 200 µL of 1% BSA dissolved in PBS containing 0.05% Tween (PBST), the plates were incubated with the serum (1:50 dilution) at 37°C for 1.5 h. Then, the plate was washed and 100 µL diluted horseradish peroxidase-conjugated goat anti-chicken IgG (1:10,000; A30-104P, Bethyl Laboratories Inc., Montgomery, TX, USA) was added and incubated at 37°C for 30 min. After washing five times with PBST, 100 µL 0.05% tetramethylbenzidine was added to the plates and incubated at 37°C in the dark for 30 min. The reaction was terminated with 2 mol/L sulfuric acid. Readings were taken at 450 nm using an ELISA reader (SepctraMax i3x Platform, Molecular Devices, LLC).

Serum Zonulin And Endotoxin (Et) Detection

The venous blood of chicken wings was collected using common collection vessels (5 mL), and the serum was collected after centrifugation at 3500 rpm and 4°C for 10 min. Zonulin (Shanghai Fankewei Industrial Co., Ltd., F8139-A) and Endotoxin detection kits (Shanghai Meilian Biotechnology Co., Ltd., ml0122369-J) were then used according to the manufacturer’s instructions.

Quantitative Reverse Transcription-pcr To Measure Mrna Expression In The Ileum

The ileal tissues were collected, placed in RNase-free freezing tubes, and stored at − 80°C. Tissue samples (100 mg) were placed in a 2 mL centrifuge tube, 1 mL of Trizol (Invitrogen Life Technologies, Carlsbad, USA) extract was added, and total RNA was extracted according to the manufacturer’s instructions. The purity of the extracted RNA was checked using a nucleic acid spectrophotometer (AG 22331, Eppendorf, Hamburg, Germany), and samples with OD 260/OD 280 nm greater than 1.8 were used for subsequent processing. The samples were reverse-transcribed using a cDNA kit (Takara Biotechnology Co. Ltd., Beijing, China). The Applied Biosystems 7500 Fast Real-Time PCR System and SYBR Premix Ex Taq™ kit (Takara Biotechnology Co. Ltd., Beijing, China) were used to performed quantitative reverse transcription-PCR. All procedures were performed according to the manufacturer's instructions. Genes were quantified using β-actin as an internal reference, and the results were statistically analyzed according to the method described by Fu et al. [14]. The primer sequences for genes are shown in Table 2.

Ileal Microbiological Analysis

The contents of middle ileal samples collected on d 28were used for 16S sequencing analysis, according to the method described by Zhang et al. [15]. First, fecal microbial DNA was extracted using a DNA extraction kit (QIAamp Fast DNA Stool Mini Kit, Qiagen Company, Dusseldorf, Germany), and then the purity of the DNA was tested using 1% agarose gel electrophoresis. All procedures were performed according to the manufacturer's instructions. Bacterial DNA was amplified using the following V3-V4 primers: 338 F (5ʹ-ACTCCTACGGGAGGCAGCA-3ʹ) and 806r (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ). In-machine sequencing was performed using HiSeq2500 PE250, and the sequences were analyzed by Beijing Nuohe Zhiyuan Bio-Information Technology Co. Ltd. The Qiime software (Qiime2-2019.7, Nature Biotechnology) was used to generate species abundance tables at different taxonomic levels. Analysis was then performed to identify biomarkers that were statistically different between groups and α, β diversity. Venn plots and Principal Co-ordinates Analysis (PCoA) plots were plotted using the cloud platform (Beijing Nuohe Zhiyuan Bio-Information Technology Co. Ltd.), and analysis of similarities (ANOSIM) and PICRUSt analyses were performed.

Results

Fecal Coccidia Oocyst Counts

The coccidia oocyst counts are shown in Table 6. The coccidia oocysts were not observed in the NC group (Fig. 2). Addition of tannic acid to the diet linearly reduced the number of fecal coccidia oocysts (P < 0.05), and the numbers were significantly lower in the TA2 group at d 26 (P < 0.05), TA3 group at d 26–28 (P < 0.05), and TA4 group at d 25–28 (P < 0.05) than those in the PC group. 

Immune And Intestinal Barrier Function

The changes in the levels of immune indicators in the serum and intestinal mucosa are shown in Table 7. The results showed that on day 28, the levels of IgM and C. perfringens-specific antibodies IgY, CRP, and MPO in the serum and sIgA in the ileal mucosa were significantly higher in the PC group than in the NC group (P < 0.05). Contrastingly, compared with that in the PC group, the addition of tannic acid to the feed significantly reduced the sIgA content in the ileal mucosa on day 28 (P < 0.05). The levels of C. perfringens-specific antibodies IgY awas significantly lower in the PTA3 group than in the PC group on day 28 (P < 0.05), and the levels of C. perfringens-specific antibodies IgY and MPO were remarkably lower in the PTA4 group than in the PC group on day 28 (P < 0.05). On day 35, the levels of CRP and MPO in the serum of the PC group were markedly higher than those in the NC group (P < 0.05). Addition of tannic acid had no significant effect on the levels of immunoglobulins, C. perfringens-specific antibodies IgY in the serum, and sIgA in the ileal mucosa on day 35; however, the serum levels of CRP and MPO were significantly reduced (P < 0.05).

Serum zonulin and ET levels showed consistent results (Table 8). NE significantly increased serum zonulin and ET levels (P < 0.01), while the addition of tannic acid significantly reduced serum zonulin and ET levels (P < 0.05). A combined analysis of the zonulin and ET indices showed that the addition of tannic acid improved intestinal barrier function and reduced intestinal permeability. 

mRNA expression of intestinal barrier gene, MUC2, and nutrient transport carriers

The results of intestinal barrier gene and MUC2 expression on day 28 (Fig. 3a) showed that the expression of ZO-1 in the PC group was significantly reduced (P < 0.05) and the mRNA expression of MUC2 and Occludin was significantly reduced (P < 0.01) compared to that in the NC group. The addition of tannic acid to the feed significantly increased the expression of Occludin compared with that in the PC group (P < 0.05); MUC2 expression was markedly increased in the PTA1 and PTA2 groups (P < 0.05), and ZO-1 expression was significantly increased in the PTA3 group (P < 0.05). The results for intestinal barrier genes and MUC2 mRNA expression on day 35 (Fig. 3b) showed that MUC2 expression in the PC group was still significantly lower than that in the NC group (P < 0.05). The expression of MUC2 was remarkably higher in the PTA2 group than in the PC group (P < 0.05).

Regarding the ileal nutrient transport carriers on day 28 (Fig. 3c), the mRNA expression of intestinal I-FABP and PEPT1 was significantly lower in the PC group (P < 0.01) and that of SGLT1 was lower in the PC group (P < 0.05) than in the NC group. Compared with that in the PC group, the addition of tannic acid to the feed significantly increased the expression of SGLT1 in the PTA1-PTA4 groups(P < 0.05), I-FABP in the PTA1 group (P < 0.05) and PEPT1 in the PTA2 group (P < 0.05). The ileal nutrient transport carrier results on day 35 showed (Fig. 3d) that the expression of PEPT1 in the PC group was remarkably lower than that in the NC group. The relative mRNA expression of SGLT1 was significantly higher in the PTA2 group than in the PC group on day 35 (P < 0.05). Neither NE infection nor tannic acid addition significantly affected the expression of GLUT2 (P > 0.05).

We found that the expression levels of IL-1β, TLR2, and TGF-β under NE conditions were significantly increased on d 28 (Fig. 3e) (P < 0.05). After tannic acid supplementation, TLR2 mRNA expression levels in the PTA1 group were significantly increased compared with those in the PC group (P < 0.05). Tannic acid supplementation had no significant effect on cytokine expression levels. On d 35 (Fig. 3f), compared with those in the NC group, IL-1β mRNA expression levels were significantly increased in the PC group (P < 0.05), but tannic acid supplementation had no significant effect on cytokine expression levels.

Ileal Microbiological Analysis

To study the effect of tannic acid on the microbiota of the ileal midsection of broilers infected with NE, the changes in the ileal microorganisms were compared among the NC, PC, and PTA3 groups. There were 400 unique OTUs in the NC group, 288 in the NE group, and 183 in the PTA3 group (Fig. 4a). Alpha diversity was measured using the ACE, Chao1, Simpson, and Shannon indices. Figure 4b shows that NE did not significantly affect the microbial alpha diversity in mid-ileum microorganisms. The ACE and chao1 indices were significantly reduced after the addition of tannic acid (P < 0.01), but there were no significant differences in the Shannon or Simpson indices. The principal component analysis of ileal microorganisms is shown in Fig. 4c. As displayed in Table 9, the results showed that there were remarkable differences in the composition and structure of the ileal microbiota between these groups (P < 0.01). 

R values range from − 1 to 1. Differences between groups were significant for R values > 0, and differences within groups were greater than those between groups for R values < 0. P < 0.05 indicates significant differences.

At the phylum level (Fig. 5a, b), NE infection significantly reduced the relative abundance of Anaerobacteria and increased the ratio of Firmicutes/Bacteroides (P < 0.05). At the genus level (Fig. 5c, d), Ligilactobacillus abundance was significantly reduced in the PC group (P < 0.01), and addition of tannic acid significantly increased the relative abundance of Anaerocolumna, Thermoanaerobacterium, and Thermosinus (P < 0.01). The effects of challenge on the intestinal Lactobacillus-associated flora were more pronounced at the species level (Fig. 5e, f), with the abundance of Lactobacillus salivarius flora in the PC group significantly reduced (P < 0.01); addition of tannic acid significantly increased the relative abundance of Thermosinus carboxydivorans (P < 0.01).

Prediction Of Bacterial Flora Function

PICRUSt analysis was performed online using the microbial genome information from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Fig. 6a, b). This analysis allowed for a comparison of the differences in functional profiles among the groups and revealed significantly different functional clustering of the gene pathways among the NC, PC, and PTA3 groups. The results showed that eight gene pathways differed among the three groups at level 1, mainly involving metabolic and disease-related gene pathways. The gut microbes of the NC group were mainly enriched in biological systems, cellular processes, and metabolic pathways; those of the PC group were mainly enriched in disease and genetic information processing pathways; while those of the PTA3 group were mainly enriched in environmental information processing signaling pathways. The results showed 35 different gene pathways at level 2. Among them, NE infection significantly downregulated the metabolic pathways of carbohydrates, lipids, and amino acids, and the addition of tannic acid upregulated the pathways related to the metabolism of these nutrients to some extent. Furthermore, we found that the metabolic pathways related to energy, nucleotides, cell growth and death, replication and repair, cancer, disease, and disease immunity were significantly elevated in the intestinal flora after challenge and that the addition of tannic acid downregulated these metabolic pathways to a certain extent.

Indicator Correlation Analysis

To explore the relationship between the effect of tannic acid on the gut microorganisms of broiler chickens with NE and the indices for growth performance and immunity, Pearson correlation analysis was conducted based on the above-shown microbial data. Ligilactobacillus showed a highly significant negative correlation (P < 0.01, Fig. 7) with IgM levels in the serum and a significant negative correlation (P < 0.05) with CRP levels; Lactobacillus salivarius showed a highly significant positive correlation with body weight (P < 0.01) and a highly significant negative correlation with the F/G, ET, MPO, and zonulin levels (P < 0.01). The Firmicutes/Bacteroides ratio exhibited a negative correlation with body weight (P < 0.05) and a significant positive correlation with serum ET levels and C. perfringens-specific antibodies (P < 0.05); Thermoanaerobacterium showed a significant positive correlation with serum IgM levels (P < 0.05); Thermosinus carboxydivorans and Thermosinus were significantly and positively correlated with GLUT2 expression levels (P < 0.05. The number of C. perfringens in the cecum was significantly positively correlated with CRP, ET, MPO, and Zonulin levels and the F/G ratio (P < 0.01) and significantly positively correlated with IgY, sIgA, IgM, IL-1β and intestinal lesion score (P < 0.05). In addition, the number of C. perfringens was negatively correlated with body weight (P < 0.01).

Discussion

In this study, we investigated the modulating effects of different tannic acid additive levels on the intestinal health of broiler chickens co-infected with coccidia and C. perfringens. NE infection was found to significantly reduce the body weight and F/G of broilers, and their small intestines showed remarkable pathological changes, such as intestinal congestion, red bruises, thinning of the intestinal wall, and intestinal distension. The intestinal section analysis further showed damage to the villi structure in the PC group, which indicated the successful establishment of the necrotizing enteritis model and was consistent with the results of previous studies [15, 16]. Regarding the intestinal barrier genes as well as nutrient transporters, most mRNA expression in the PC group showed negative effects. Previous studies have reported that NE reduces growth performance and elevated serum ET [15], which is consistent with our findings. In addition, the serum levels of zonulin levels were elevated, further indicating the successful establishment of NE model. Tannic acid addition to the feed had beneficial effects on the broilers with NE and resulted in reduced intestinal lesion scores, increased villus height, and improved intestinal morphology.

Effects of tannic acid addition to the diet on the growth performance of broiler chickens with NE

Results showed that tannic acid in the feed could ameliorate the growth performance degradation caused by NE, which is consistent with the results of previous studies. Yang et al. observed better F/G ratios when broilers were fed 7.5 to 15 mg/kg proanthocyanidins [17]. Wang et al. investigated the effects of addition of 5–80 mg/kg grape seed extracts to the coccidia-challenged diet and found that 10–20 mg/kg significantly improved the growth performance [18]; these results were consistent with those of this study. However, some studies have shown that adding 20 g/kg tannic acid significantly reduces body weight gain and the feed conversion efficiency in broilers [10].Jamroz et al. added 0.025–0.1% sweet chestnut tannin to the diet and found that < 0.05% had no effect on growth performance, whereas 0.1% significantly reduced growth performance [19]. These findings suggest that the biological effects of tannins are highly dose-dependent, and the inconsistent effects of tannins in the literature may be due to differences in their source, dose, extraction process, encapsulation, and basal diets. In the present study, the growth performance showed a quadratic curve that increased with the addition of tannic acid; 500–750 mg/kg tannic acid was found to be the optimal concentration for improving the growth performance. The reason dietary tannic acid improves the growth performance of NE may be that it enhances the intestinal barrier, improves intestinal morphology, balances microbiota, and improves nutrient absorption capacity.

Effects Of Tannic Acid On The Intestinal Morphological Structures Of Broiler Chickens With Ne Infection

A normal villus morphological structure is a prerequisite for the intestine to perform its absorption function. An increase in villus height may lead to enhanced nutrient absorption, and a lower crypt depth indicates a decrease in the metabolic cost of intestinal epithelial renewal. Our findings suggested that NE significantly increased crypt depth, indicating that there was faster tissue renewal for the villi, as needed in response to the inflammation caused by pathogens or their toxins, which is consistent with previous findings [11, 20]. The addition of tannic acid significantly reduced the increase in the depth of intestinal crypts caused by NE and improved the V/C ratio, demonstrating the beneficial effects of tannic acid on the intestinal morphology. The V/C showed a quadratic curve change with the addition of tannic acid, which is consistent with our results regarding the growth performance, as 500–1000 mg/kg tannic acid was highly effective at ameliorating the intestinal morphology.

Effects of tannic acid on the fecal coccidia oocyst excretion in broiler chickens with NE infection

Examining coccidia oocyst shedding is an effective method to determine the level of coccidia infection [21]. The addition of tannic acid reduced the number of coccidia oocysts shed compared to that in the PC group, which is consistent with the results of previous reports [18, 22, 23]. The reduction in the number of oocysts in the feces indicated an increase in coccidia resistance in broilers. In the present study, the amount of excreted coccidia oocysts decreased linearly with an increase in tannic acid concentration. The decrease in the number of coccidia oocysts after tannic acid addition could be attributed to the modulation of intestinal microorganisms by tannic acid and its own antiparasitic biological properties [24, 25]. Taken together, tannic acid supplementation reduced coccidia proliferation, significantly reduced the number of coccidia oocysts excreted in feces, accelerated coccidia clearance, and improved intestinal health.

Effect Of Tannic Acid On The Immune Indexes In Broiler Chickens With Ne

In this study, NE infection resulted in impaired intestinal morphology and elevated lesion scores, whereas the addition of tannic acid to the feed alleviated these changes. This process was accompanied by changes in the serum levels of IgM, MPO, CRP and C. perfringens-specific antibodies in broilers. IgM is a circulating antibody secreted by B cells that first responds to initial encounters with foreign antigens; however, IgM concentrations in the blood rapidly decline due to clearance, which is consistent with our study findings, as IgM levels were found to be elevated during the first days of infection with NE and did not change significantly seven days after the infection. CRP is an acute temporal protein that activates complement and enhances phagocytosis, and MPO is mainly found in myeloid cells; both of these are markers of the inflammatory response. In this study, the increased MPO and CRP activities in the serum of the PC group indicated that NE infection could activate immune cells in the blood and promote inflammation. The addition of tannic acid to the feed reduced the inflammatory response caused by NE and the concentration of C. perfringens-specific antibodies in the serum, which may be related to the anti-inflammatory and proliferation inhibitory properties of tannic acid [4, 7]. The results of this experimental study showed that NE infection exerted long-term effects on broiler chickens, including reduced intestinal barrier function seven days after infection, continued high levels of MPO and CRP in the serum, and inferior growth performance compared to those in the NC group.

In innate immunity, the intestinal mucosa is considered the first line of defense against pathogen infection, and mucosal immunity plays an important role in NE. The results of the present study showed that NE infection significantly damaged the ileum and stimulated the expression of sIgA in the intestinal mucosa, which is consistent with the results of a previous study [11]. We found that tannic acid reduced the sIgA secretion in the ileal mucosa induced by NE, which might be because tannic acid significantly reduced the number of coccidian merozoites and the colonization of C. perfringens. In addition, we observed that TLR2 was activated and inflammatory factor expression was increased in NE, consistent with previous studies [26, 27]. We found that tannic acid addition reduced the number of C. perfringens and coccidia in the intestinal tract by stimulating TLR2 and thus activating the immune system, on the one hand, and by inhibiting the number of C. perfringens and coccidia, on the other hand, thus reducing the inflammatory response.

Effects of tannic acid addition to the feed on mRNA expression of the gut barrier genes and nutrient transport carriers in chickens with NE

The intestine plays key roles in defense against pathogens, host nutrient digestion, and absorption [28]. Numerous studies have shown that the mRNA expression of tight junctions and nutrient transport carriers decreases and nutrient digestion and absorption are diminished during intestinal inflammation [29–32]. Intestinal inflammation results in a reduction in mucin synthesis and the number of cupped cells, increasing the chances of further intestinal infection and bacterial translocation. In this study, NE led to a reduction in the mRNA expression of Occludin, ZO-1, and MUC2, causing an increase in intestinal permeability. However, the mRNA expression of intestinal tight junctions and MUC2 was increased with the addition of tannic acid compared to that in the PC group, indicating the positive effects of tannic acid in protection of chickens from NE.

Zonulin is the only known regulator of tight intercellular junctions and is involved in the regulation of intestinal barrier function. Studies have also shown that zonulin levels in the serum are remarkably elevated in patients with irritable bowel syndrome or Crohn’s disease [33]. However, the changes in the sensitivity to zonulin in poultry intestinal diseases require further investigation. In the present study, NE led to a significant increase in zonulin levels in the serum, indicating that the intestinal barrier permeability was increased. ET is a gram-negative bacterial cell wall component, and serum ET levels are elevated in broiler chickens suffering from NE [34, 35], which was also confirmed in our study. The addition of tannic acid to the feed increased the mRNA expression of Occludin, ZO-1, and MUC2 and improved the integrity of the intestinal barrier, thereby reducing zonulin and ET levels. Nevertheless, the upregulation of intestinal tight junction-related pathways by tannins requires further investigation.

I-FABP is a cytoplasmic protein located in mature cells at the top of intestinal villi, and it plays an important role in fatty acid uptake and metabolism. We observed that the mRNA expression of I-FABP was reduced in broiler chickens infected with NE, which was consistent with the results of a previous study [36]. However, it has also been shown that the mRNA expression of I-FABP does not change significantly in the dexamethasone-induced intestinal disorder model or infection with Campylobacter jejuni [37, 38], which might be due to the different disease models and severity. In addition, we observed a decrease in the mRNA expression of PEPT1 and SGLT1 in broiler chickens infected with NE, which is consistent with the results of a previous study [39]. Addition of tannic acid improved the mRNA expression of nutrient transporter carriers and reduced the negative effects of NE on nutrient absorption in chickens. A comprehensive analysis revealed that growth performance, intestinal villus height, crypt depth, V/C, and nutrient transporter expression showed relatively consistent quadratic curve changes after tannic acid was added to the diet. The reason for this is that lower doses of tannins can reduce gastrointestinal peristalsis, thereby increasing the residence time of chyme in the gastrointestinal tract and improving nutrient digestibility and intestinal health. However, high doses of tannins exert a negative impact on growth performance by binding starch and protein in the feed, reducing digestive enzyme activity, and binding to intestinal wall proteins [40, 41].

Effect Of Tannic Acid On The Ileal Microflora Of Broilers With Ne

The intestinal microbiota forms a highly complex microecosystem. To further investigate the mechanisms by which tannic acid alleviates the intestinal damage caused by NE, we analyzed the microflora structure of the ileal microorganisms using 16S rRNA sequencing. The results of this study showed that the challenge did not have a significant effect on the microbial α-diversity, which is consistent with the results of previous studies [42, 43]. We speculated that the possible reason for this result was that NE infection inhibited the proliferation of minor microorganisms in the broiler intestinal flora, which resulted in a convergence with the α-diversity of the NC group microbiota. However, NE has also been shown to result in lower or higher gut microbial α diversity [15, 44], which may be related to the collection of different parts of the chyme. In the present study, addition of tannic acid significantly reduced the α-diversity but did not affect the balance abundance of the flora, which is consistent with a previous study [45]. It has been shown that low-abundance microbes lead to the formation of simpler metabolic networks, resulting in increased concentrations of specific components used to support host energy requirements [45]. Shabat et al. reported that a reduced microbiome abundance may be closely associated with an increased feed conversion efficiency, with specific enrichment and metabolic pathways of microbes leading to better energy and carbon delivery to the animal body [46], which could be one of the reasons tannic acid improves growth performance. The significant difference in β-diversity among the three groups indicated that the addition of tannins or challenges significantly altered the microbial community structures. Bacteroidetes are an important contributor to intestinal health, and this phylum plays an important role in breaking down complex molecules into simpler compounds and producing short-chain fatty acids that are beneficial for increased growth performance [47, 48]. We found that NE led to a decrease in the abundance of Bacteroides, which is consistent with the results of a previous study [43]. The fermentation products of Bacteroidetes, such as Bacteroides fragilis, inhibit C. perfringens spore formation, and the decrease in Bacteroidetes may predispose the animal intestine to C. perfringens infection and gastroenteritis [49], which may be one of the reasons for the decrease in Bacteroidetes abundance in NE. Lately, several studies have suggested that stressed birds present a significantly increasing trend for the Firmicutes/Bacteroides ratio [50, 51]. In this study, we found that the high Firmicutes/Bacteroidetes ratio caused by NE may be one of the reasons for dysbiosis of the gut microbial community.

The results also showed that at the genus level, NE significantly reduced the abundance of the beneficial bacterium Ligilactobacillus, suggesting that NE infection inhibited the growth of beneficial intestinal bacteria. At the species level, NE significantly reduced the relative abundance of Lactobacillus salivarius, which is a probiotic in poultry that dates back more than 15 years. Furthermore, this bacterium can re-establish the proper microbial balance of bacteria by forming lactic and propionic acid, stimulate butyric acid-producing butyric acid production, and inhibit the production of pro-inflammatory cytokines [52]. In broilers, Lactobacillus_salivarius was found to improve growth performance, reduce the expression of inflammatory factors, and improve intestinal health [9, 53, 54]. In conclusion, in this study, we found that the intestinal flora of broiler chickens infected with NE was disturbed and the abundance of beneficial bacteria was reduced, leaving the intestine in an unhealthy state. The addition of tannic acid after the challenge significantly increased the relative abundance of the genus Anaerocolumna, which belongs to the Lachnospiraceae and can be metabolized by the fermentation of carbohydrates into acetate [55]. In addition, the relative abundance of Thermoanaerobacterium, which can secrete xylanase, degrade hemicellulose and even cellulose, and produce acetate and butyric acid via fermentation using xylan and others, was increased [56]. The genus Thermosinus includes gram-negative bacteria that can convert CO to carbon dioxide through a series of chemical reactions and can decompose organic substrates (glucose, sucrose, or lactose) to produce acetic acid as well as acetate, H₂, and CO₂ during glucose fermentation [57]. Thus, it regulates the intestinal pH, nourishes butyric acid-producing bacteria, protects against pathogens, and contributes to intestinal health. In conclusion, the addition of tannins increases the abundance of short-chain fatty acid-producing-related flora, which may be one of the reasons for their beneficial impacts on intestinal health.

In the PICRUSt analysis, we found that differences in the metabolic pathways were the most common in this study, especially in the PC group, where there were large metabolic differences when compared with the other two groups. The present study showed that nutrient metabolism in the intestinal flora of broilers with NE was generally downregulated, and the pathways related to intestinal flora metabolism were more enriched in the NC group, which was consistent with previous findings [43]. Bacteroidetes are the main carbohydrate-degrading bacteria in the intestine that contribute to the degradation of complex pectins. Therefore, the enrichment of the carbohydrate metabolic pathway in the NC group may be due to the high abundance of Bacteroidetes, which degrade complex polysaccharides and indigestible carbohydrates to generate short-chain fatty acids and maintain intestinal health [58]. Microbial catabolism of amino acids produces a large number of byproducts, including amines, phenols, indoles, and short-chain fatty acids [59], which can have beneficial or detrimental effects on epithelial cells, depending on the concentration of metabolic byproducts in the lumen. For example, aromatic amino acids are metabolized by colonies to produce tryptamine and indoles. Indoles play an important role in host defense by enhancing intestinal barrier function and downregulating the proinflammatory cytokines [60]. We speculated that the healthier gut in the NC group may be related to the products of carbohydrate and amino acid metabolism formed by the intestinal flora. Bacteroides, Propionibacterium, Fusobacterium, Lactobacillus, and Streptococcus play important roles in the protein hydrolysis process [61], and the downregulation of amino acid metabolic pathways in the PC group of microorganisms may be due to a decline in Bacteroides and Lactobacillus abundance. In the PC group, genes related to disease immunity, metabolic diseases, and energy metabolism were upregulated, which suggested that during the infection of chickens with C. perfringens, the intestinal flora competed with the host for energy, and more energy allocation was required for stimulation of the immune system, which may have resulted in the reduction in chicken growth performance. According to this study, the microbial communities in normal chickens were relatively healthier, and the ileal flora structure and function of the tannic acid groups were predicted to be similar to those of the NC group chickens. Therefore, we conclude that the addition of tannic acid could alleviate ileal microflora disorder in broiler chickens with NE. Taken together, the mechanism underlying the effect of tannic acid on intestinal health may involve changes in the intestinal microflora; however, relevant metabolomic analyses are still required to explore the effects of tannic acid on metabolites, which can further help us to establish the relationship among tannic acid, intestinal flora, metabolites, and intestinal health.

To investigate the correlation between intestinal flora and growth performance, immunity, and mRNA expression of intestinal barrier genes and nutrient transport carriers, simple correlation analysis was performed. Correlation analysis can indicate the correlation between indicators but cannot determine a causal relationship. The results suggested that the abundance of Lactobacillus salivarius was positively correlated with growth performance and significantly negatively correlated with MPO, CRP, ET, and zonulin, which may be because Lactobacillus salivarius can inhibit inflammation [62, 63] and improve intestinal barrier function [41]. The increase in the abundance of Thermosinus carboxydivorans and Thermosinus after the addition of tannic acid was significantly and positively correlated with the mRNA expression of GLUT2, which suggested that the regulation of intestinal flora by tannic acid may be related to nutrient translocation and absorption. However, the specific mechanisms require further investigation.

Conclusions

The present study demonstrated that tannic acid supplementation is apparently effective in reducing NE in broiler chickens. The protective effects of dietary tannic acid supplementation against NE challenge were evidenced by the suppression of coccidia and C. perfringens colonization and invasion, regulation of intestinal flora, alleviation of inflammatory reaction, and improvement in intestinal barrier function and nutrient transport. As the amount of additive tannic acid increases, the body weight, mRNA expression of nutrient transport carriers, and villus height, V/C also increase in the form of a quadratic curve, while the intestinal barrier-related indices (Zonulin and ET content in serum), coccidia oocyst counts, cecum C. perfringens concentration, and immune indices in serum decrease linearly. After comprehensive consideration of the results, we recommend tannic acid supplementation to broiler feed at a dose of 500–750 mg/kg.

Abbreviations

BW: Body weight;CRP: C-reactive protein; ELISA: Enzyme-linked immunosorbent assay; ET: Endotoxin; F/G: The ratio of feed intake and body weight gain; GLUT2: Recombinant glucose transporter 2; I-FABP: Intestinal fatty acid binding protein; IgA: Immunoglobulin A; IgM: Immunoglobulin M; MPO: Myeloperoxidase; NE: Necrotic enteritis; PEPT1: Peptide-transporters 1; sIgA: Secretory immunoglobulin A; SGLT1: Recombinant sodium/glucose cotransporter 1; V/C: The ratio of villus height to crypt depth; ZO-1: Zonula occludens-1 

Declarations

Authors’ contributions

YG designed the study; HX, JF, YL, PL, BS, and ZL performed the experiments; HX analyzed the data and wrote the manuscript; and YG revised the manuscript. All authors contributed to the data interpretation and approved the final version of the manuscript. 

Availability of data and materials

All data generated or analyzed during this study are available from the corresponding author upon request. Datasets supporting the conclusions of this study are included in this article. The 16S gene sequencing data can be obtained from the following website: https://www.ncbi.nlm.nih.gov/sra/PRJNA897828; the accession number is PRJNA897828. 

Competing interests

The authors declare that they have no competing interests. 

Consent for publication

Not applicable.

Ethics approval and consent to participate

All experiments were approved by the Institutional Animal Care and Use Committee of the China Agricultural University. The animal welfare number is AW11112022-1-4. 

Funding

This work was supported by the China Agriculture Research System program (CARS-41-G11). 

Acknowledgments

The authors are grateful to the staff of the Department of Animal Science and Technology of the China Agricultural University, especially those involved in animal nutrition, for their valuable assistance in conducting the experiments.

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