Effects of dietary Nisin on growth performance, immune function, and gut health of broilers challenged by Clostridium perfringens

Abstract

Nisin (Ni) is a polypeptide bacteriocin produced by lactic streptococci (probiotics) that can inhibit the majority of gram-positive bacteria, and improve the growth performance of broilers, and exert antioxidative and anti-inflammatory properties. The present study investigated the potential preventive effect of Nisin on necrotic enteritis induced by Clostridium perfringens (Cp) challenge. A total of 288 Arbor Acres broiler chickens of 1-d-olds were allocated using 2 × 2 factorial arrangement into four groups with six replicates (12 chickens per replicate), including: (1) control group (Con, basal diet), (2) Cp challenge group (Cp, basal diet + 1.0 × 10⁸ CFU/mL Cp), (3) Ni group (Ni, basal diet + 100 mg/kg Ni), and (4) Ni + Cp group (Ni + Cp, basal diet + 100 mg/kg Ni + 1.0 × 10⁸ CFU/mL Cp). The results showed that Cp challenge decreased the average daily gain (ADG) of days 15 to 21 (P<0.05) and increased interleukin-6 (IL-6) content in the serum (P < 0.05), as well as a significant reduction in villus height (VH) and the ratio of VH to crypt depth (VCR) (P<0.05) and a significant increase in crypt depth (CD) of jejunum (P<0.05). Furthermore, the mRNA expressions of Occludin and Claudin-1 were downregulated (P<0.05), while the mRNA expressions of Caspase3, Caspase9, Bax, and Bax/Bcl-2 were upregulated (P<0.05) in the jejunum. However, the inclusion of dietary Ni supplementation significantly improved body weight (BW) on days 21 and 28, ADG of days 15 to 21 (P<0.05), decreased CD in the jejunum, and reduced tumor necrosis factor-α (TNF-α) content in the serum (P<0.05). Ni addition upregulated the mRNA levels of Claudin-1 expression and downregulated the mRNA expression levels of Caspase9 in the jejunum (P<0.05). Moreover, Cp challenge and Ni altered the cecal microbiota composition, which manifested that Cp challenge decreased the relative abundance of phylum Fusobacteriota and increased Shannon index (P<0.05) and the trend of phylum Proteobacteria (0.05<P<0.10), while Ni addition increased the relative abundance of phylum Cyanobacteria (P<0.05). In conclusion, the results demonstrated that the addition of Ni effectively mitigated the negative impact of Cp challenge-induced the damage of growth performance and intestine in broilers via reducing inflammatory responses and intestinal apoptosis and modulating cecal microbial composition.

The addition of Ni in the diet can provide effective protection from the injury of Cp challenge in broilers, including the improvement of growth performance, inflammatory injury, intestinal morphology, and barrier function, as well as the optimization of the composition of the cecal microbiota. This study contributes to the theoretical foundation for clinical prevention and the underlying mechanisms of Ni in mitigating NE.

Introduction

Necrotic enteritis (NE) is a highly prevalent consequential digestive disorder that occurs in broilers within a 2 to 6 week period, with subclinical necrotizing enteritis being the most frequently observed form in poultry (Tang et al., 2022). Chicken bowel lesions, deterioration of the intestinal microbiome, an inflammatory response, and reduced production are the characteristic symptoms of NE infection (Wang et al., 2021a; Yu et al., 2022; Zhao et al., 2022). The annual economic cost attributed to NE in the global poultry industry has been estimated to be about $6 billion and represents a significant financial burden for the poultry sector (Wade and Keyburn, 2015). The main cause of NE is Clostridium perfringens (Cp), a gram-positive bacterium that forms spores and can be found in water, soil, chicken feces, and the intestines of humans and animals (Shimizu et al., 2002). Cp is known to produce up to 17 different toxins and can be classified into different types (A-G) based on their toxin secretion capabilities, in which the Cp type A and C strains can cause NE (Rood et al., 2018).

Over the past few decades, antibiotics have emerged as the foremost methods for preventing and managing NE infections (Abd El-Hack et al., 2022). Nevertheless, the prolonged use of antibiotics has led to the development of drug resistance in both humans and animals, prompting several nations to prohibit the use of antibiotics as feed supplements (Huemer et al., 2020; Evangelista et al., 2022). Therefore, there is an urgent need to develop non-antibiotic alternatives that are both safe and effective in addressing NE. Bacteriocins, which are antimicrobial peptides produced by bacteria, exhibit antimicrobial properties against certain gram-positive bacteria (Guryanova, 2023). Moreover, studies have found the positive impact of bacteriocins on poultry growth and their ability to enhance immune capacity by improving anti-inflammatory activity and disease resistance (Cesa et al., 2021; Guryanova, 2023). Consequently, bacteriocins are not only viewed as substitutes and supplements to conventional antibiotics but also hold promise as therapeutic agents for the prevention, control, and treatment of animal diseases (Ben Lagha et al., 2017).

Nisin (Ni) is a polypeptide produced by lactic streptococci (probiotics) and possesses a molecular weight of 3.4 K Da and 34 amino acid residues. It falls under the classification of a lantibiotic (Class Ia) bacteriocin (Ben Lagha et al., 2017). In 1983, the European Union granted approval for the utilization of Ni in food, while the United States Food and Drug Administration bestowed upon it the designation of being “Generally Recognized as Safe.” Previous studies have indicated that the inclusion of Ni in diets resulted in elevated daily weight gain and feed intake among broilers, as well as exhibiting anti-inflammatory characteristics (Kindrachuk et al., 2013; Kierończyk et al., 2016). Furthermore, Ni supplementation was found to enhance the composition of beneficial bacteria in the gut, such as Lactobacillus, while concurrently inhibiting the growth of pathogenic strains of E. coli in a mouse model of diarrhea (Field et al., 2023). Recently, Kierończyk et al. (2016) observed that Ni had inhibitory effects on gram-positive bacteria, including Staphylococcus aureus and Listeria. However, there is currently no reported research on the preventive effects of Ni in the NE of broilers caused by Cp.

In order to elucidate the protective effects of Ni against Cp-induced NE, our study aimed to investigate the impact of Ni on growth performance, immune functions, intestinal development, and intestinal microflora in broilers. This research contributes novel insights into the nutritional regulation of Ni in maintaining poultry gut health.

Materials and Methods

Preparation of Ni and Cp

The Ni (95% purity) used in this study was obtained from Liaoning Vetland Bio-Technology Co., Ltd. and Cp type C was isolated and prepared in the laboratory (Northeast Agricultural University, China). First, Cp was cultured on tryptose sulfite cycloserine agar (TSC, Perfringens agar) under anaerobic conditions at 37 °C for 18 h, and then the inoculation of the bacteria strains was put into a broth medium of heart-leaching liquor and growth at 37 °C in an anaerobic chamber for 18 h. The infection was carried out for 7 consecutive days.

Experimental design, animals, and diets

A total of 288 1-d old Arbor Acres broilers obtained from Harbin Yi Nong Corporation of China were randomly assigned into four groups based on a 2 × 2 factorial completely design (six replicates, 12 chickens per replicate), including: (1) control group (Con, basal diet), (2) Cp challenge group (Cp, basal diet + Cp infection), (3) Ni group (Ni, basal diet + 100 mg/kg Ni), and (4) Ni + Cp group (Ni + Cp, basal diet + 100 mg/kg Ni + Cp infection). For a period of 28 d, every replicate was placed in a separate cage (1.0 m × 1.0 m × 0.8 m). Starting from day 15, each broiler in the Cp group and Ni + Cp group received an inoculation of 1 mL of actively growing Cp, with a concentration of 1.0 × 10⁸ CFU/mL. The inoculations were performed at the same time every day. Infections were conducted continuously for 7 d, during which the Con group and the Ni group were given an equal amount of sterilized cooked meat medium. The ambient temperature in the room was initially set at 33 °C for the first week and subsequently decreased by 2 °C each week until reaching 24 °C. The indoor humidity is maintained at 65% to 70% within the first two weeks and then reduced to 55% to 60%. Continuous artificial lighting was provided by an incandescent lamp throughout the entire day. Adequate amounts of food and water were made available to all the birds without restriction during the experiment. Mortality, feed intake, and weight were registered throughout the entire trial. Ingredients and nutrient levels are listed in Table 1. Diets were formulated in accordance with NRC guidelines (2004). The Ni was weighed at 5 g and uniformly mixed into 2 kg of basic diet, then mixed with 48 kg of basic diet in stages with a mini blender to obtain the 100 mg/kg levels of Ni feed. Experimental procedures complied with the standards in the Northeast Agricultural University Guidelines for Animal Welfare Committee protocol (# NEAU-2013).

Table 1.

Ingredients and nutrient levels of the basal diet (air-dry basis)

Ingredients		Nutrients levels3
Corn	56.85	Metabolic energy (Mcal/kg)	3.0
Soybean meal (45%)	37.10	Crude protein (%)	21.50
Soybean oil	2.50	Methionine + Cystinol (%)	0.91
Methionine	0.10	Lysine (%)	1.20
Cystine	0.02	Calcium (%)	1.02
Calcium hydrogen phosphate	1.50	Total phosphorus (%)	0.70
Stone powder	1.10	Available phosphorus (%)	0.44
Nacl	0.36
Choline chloride (50%)	0.12
Trace mineral premix1	0.30
Vitamin premix2	0.03
Phytase	0.02
Total	100.00

¹Provided the following per kilogram of diet: Cu, 8 mg; Fe, 80 mg; Zn, 65 mg; Mn, 0.11 mg; I, 1.1 mg; Se, 0.3 mg.

²Provided the following per kilogram of diet: vitamin A, 12,000IU; vitamin D₃, 4,500IU; vitamin E, 25 mg; vitamin K₃, 2 mg; vitaminB₁, 2 mg; vitaminB2, 8 mg; vitamin B₆, 4 mg; vitamin B₁₂, 0.021 mg; D-pantothenic acid, 1 mg; folate, 1 mg; D-biotin, 0.1 mg.

³Nutrients levels were calculated values.

Growth performance and sample collection

Broiler body weight (BW) and feed consumption were recorded on days 0, 14, 21, and 28 of age to determine the average daily gain (ADG), the average daily feed intake, and the feed gain ratio (FCR). On day 22, one chick selected from each replicate of each group was weighted and euthanized. About 10 mL of blood samples from each broiler were centrifuged at 2,500 × g at 4 °C for 15 min to obtain serum, which was then stored at −20 °C for subsequent analysis. The jejunum was fixed in a 4% paraformaldehyde solution and processed for hematoxylin and eosin staining. In order to detect mRNA contents, mucosa samples were scraped from jejunum and stored at −80 °C. We harvested the cecal contents, frozen them in liquid nitrogen, and stored them at −80 °C for analysis of intestinal microbial 16S rDNA.

Inflammatory factors

Enzyme-linked immunosorbent assays (ELISA) were employed to quantify the levels of serum inflammatory factors, namely interleukin-2 (IL-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). The ELISA assays were conducted using commercially available kits obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), following the manufacturer’s instructions. Absorbance was measured at 450 nm using microplate plate readers (AMR-100, Aosheng, Hangzhou, China). A standard curve was generated using optical density values of standard concentrations to facilitate the measurement of serum inflammation.

Intestinal morphology

Following fixation with 4% paraformaldehyde, the duodenum, jejunum, and ileum tissues were embedded in paraffin. Subsequently, consecutive serial 5 µm paraffin sections were prepared for hematoxylin-eosin staining. The resulting intestinal morphology was evaluated and documented using an optical microscope equipped with a camera (Olympus BX-50, Tokyo, Japan). For each section, a minimum of three representative visual fields were captured, and three well-oriented villi and crypts of each view were selected for villus height (VH) and crypt depth (CD) measurement using Image-J software. Finally, the VH to CD ratio (VCR) was calculated.

Quantitative real-time PCR

Total RNA was extracted from jejunal mucosa (0.1 g) using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the protocol described by Bai et al. (2023). The purity and concentration of RNA were assessed using NanoDrop spectrophotometry (Jiapeng Instruments, Shanghai, China), ensuring that the RNA concentration exceeded 300 ng/μL and the A260/A280 ranged from 1.8 to 2.0. After normalizing the concentration, RNA samples (1.0 μg) were reverse-transcribed into cDNA using the ABScript III RT Master Mix (ABclonal, Wuhan, China) as per the manufacturer’s instructions. RT-qPCR was conducted using ABScript III RT Master Mix Kits (ABclonal) on a Line Gene9600 Plus qPCR instrument (Bioer, Hangzhou, China). All steps above were done on ice or at 4 °C. The primer sequences for the target genes were designed and synthesized by Shanghai Sangon Bioengineering Company (Supplementary Table S1). The β-actin housekeeping gene was utilized as an internal reference for the calculation of relative gene expression using the 2^−ΔΔCT method (Livak and Schmittgen, 2001).

16S rRNA sequencing and bioinformatic analysis

The microbial composition of broilers treated with Cp and Ni was profiled by 16S rRNA gene sequencing. DNA was extracted from cecal contents samples according to the manufacturer’s instructions (Tiangen Biotech, Beijing, China). Subsequently, the V3-V4 region of the 16S rRNA gene was amplified with universal primers (341F: 5’-CCTACGGGNG GCWGCAG-3’ and 805R: 5’-GACTACHVGGGTATCTAA TCC-3’). After 2% agarose gel electrophoresis, the PCR products were extracted, purified, and used for library preparation with the Library Quantification Kit for Illumina (Kapa Biosciences, Woburn, MA, USA), followed by sequencing on the NovaSeq PE250 platform (Hangzhou Lianchuan Biotechnology Co., Ltd, Hangzhou, China). To obtain high-quality datasets, the raw data were spliced, quality controlled, and chimera filtered. Alpha and beta diversity metrics were analyzed with QIIME2 software and figures were prepared with the R package (v3.5.2).

Statistical analysis

Data were analyzed using a two-way analysis of variance (ANOVA) with the General Linear Model procedure of SPSS 22.0 software (IBM-SPSS Inc., Chicago, IL). The analysis accounted for the effects of Cp, Ni, and their interactions. When the interaction was significant, the data were reanalyzed by one-way analysis of variance and Duncan’s multiple comparisons. The RT-qPCR data were visualized using GraphPad Prism 5.0 (La Jolla, CA, USA). The final results are presented as the mean ± standard error of the mean (SEM). A P value ≤ 0.05 is considered statistically significant, and 0.05<P<0.10 indicates a tendency.

Results

Growth performance

Table 2 shows a significant decrease in ADG (P<0.05) and a tendency towards increased FCR from day 15 to 21 during Cp challenge (0.05<P<0.10). The inclusion of Ni in the diet improved BW on days 21 and 28 (P<0.05), as well as ADG in broilers from days 15 to 21 (P<0.05). Furthermore, Ni enhanced the ADG and ADFI propensity throughout the entire trial and at a trend towards increased ADG from days 22 to 28 (0.05<P<0.10). However, there was no discernible interaction between Ni and Cp challenge on the growth performance of broilers (P>0.05). Not surprisingly, a total of two broilers died on days 23 and 25 after Cp challenge, and there were no deaths in the Ni protection group or other groups.

Table 2.

Effect of Clostridium perfringens and Nisin on the growth performance of broilers ¹

Items	Con	Cp	Ni	Ni + Cp	SEM	P-values
						Cp	Ni	Cp × Ni
BW, g
day 1	43.25	43.26	42.64	42.85	0.11	–	–	–
day 14	352.32	351.57	359.40	360.08	1.38	–	–	–
day 21	635.32	619.65	647.00	651.40	4.87	0.529	0.023	0.268
day 28	1,060.80	1,059.28	1,120.94	1,100.28	9.14	0.485	0.005	0.546
ADFI, g/d
days 1 to 14	27.29	26.82	27.31	27.50	0.18	–	–	–
days 15 to 21	58.88	59.06	59.98	59.98	0.70	0.951	0.502	0.955
days 22 to 28	94.95	97.89	100.17	99.12	1.23	0.707	0.210	0.431
days 1 to 28	53.88	54.86	56.06	55.90	0.42	0.618	0.059	0.489
ADG, g/d
days 1 to 14	22.01	21.97	22.32	22.77	0.16	–	–	–
days 15 to 21	40.61	38.39	41.52	40.95	0.40	0.045	0.015	0.218
days 22 to 28	52.98	53.19	57.60	55.53	0.99	0.641	0.091	0.568
days 1 to 28	35.76	35.60	37.59	37.03	0.41	0.652	0.054	0.801
FCR
days 1 to 14	1.24	1.24	1.23	1.21	0.01	–	–	–
days 15 to 21	1.46	1.54	1.42	1.48	0.02	0.076	0.179	0.740
days 22 to 28	1.80	1.86	1.77	1.79	0.03	0.528	0.411	0.660
days 1 to 28	1.51	1.56	1.51	1.51	0.01	0.342	0.353	0.427

¹BW, body weight; ADFI, average daily feed intake; ADG, daily weight gain; FCR, feed gain ratio. Con, basal diet; Cp, basic diet (+ 1.0 × 10⁸ CFU/mL Clostridium perfringens, days 15 to 21); Ni, basal diet + 100 mg/kg Nisin; Ni + Cp, basal diet + 100 mg/kg Nisin (+ 1.0 × 10⁸ CFU/mL Clostridium perfringens, days 15 to 21). n = 6 broilers chicken per treatment. All of the values are expressed as the means and pooled SEM. SEM, standard error of the mean.

Inflammatory factors

The levels of serum inflammatory factors are presented in Table 3. Inoculation of Cp resulted in a significant increase in IL-6 level (P<0.01), while Ni supplementation decreased the trend of IL-6 level (0.05<P<0.10) and significantly decreased TNF-α level (P<0.05). Additionally, there was an interacting effect between Ni and Cp on the IL-2 level (P<0.05). Specifically, the serum IL-2 level in broilers from the Ni group was significantly higher (P < 0.05) compared to the Ni + Cp group.

Table 3.

Effect of Clostridium perfringens and Nisin on the inflammatory factors in serum of broilers¹

Items	Con	Cp	Ni	Ni + Cp	SEM	P-values
						Cp	Ni	Cp × Ni
IL-2, ng/L	107.28a,b	118.27a,b	131.19a	75.47b	7.88	0.123	0.501	0.027
IL-6, ng/L	19.53	34.54	19.93	23.00	1.85	0.005	0.068	0.052
TNF-α, ng/L	26.09	35.30	22.15	22.60	1.96	0.173	0.025	0.214

¹IL-2, Interleukin-2; IL-6, Interleukin-6; TNF-α, Tumor necrosis factor-α. n = 6 broilers chicken per treatment. All of the values are expressed as the means and pooled SEM. SEM, standard error of the mean.

^a,bMeans within a row without a common superscript differ significantly (P < 0.05).

Intestinal morphology

The jejunal morphology of broilers is presented in Table 4 and Figure 1. Following the challenge with Cp, HE staining revealed a significant decrease in VH and VCR (P<0.01), and an increase in CD (P<0.05). Conversely, the Ni addition exhibited a decrease in CD (P<0.01) and a slight increase trend in VCR (0.05<P<0.10). In addition, the interaction between Cp challenge and Ni did not have a significant impact on the jejunal morphology of broilers (P>0.05).

Table 4.

Effect of Clostridium perfringens and Nisin on the morphology in jejunum of broilers ¹

Items	Con	Cp	Ni	Ni + Cp	SEM	P-values
						Cp	Ni	Cp × Ni
VH, μm	1,561.05	1,250.21	1,603.56	1,325.96	46.83	0.001	0.429	0.823
CD, μm	187.38	243.38	165.44	177.86	9.63	0.033	0.009	0.158
VCR	8.63	5.80	9.92	7.62	0.50	0.003	0.052	0.727

¹VH, Villus height; CD, Crypt depth; VCR, the ratio of Villus height to Crypt depth. n = 6 broilers chicken per treatment. All of the values are expressed as the means and pooled SEM. SEM, standard error of the mean.

Figure 1.

Effect of Clostridium perfringens and Nisin on the jejunal morphology of broilers. The magnifications are 40, Bar = 500 μm. n = 6 broilers chicken per treatment.

mRNA expression of tight junction-related gene in jejunal mucosa

The mRNA expressions of tight junction-related genes in the jejunal mucosa of broilers are presented in Figure 2A, B, C. The Cp challenge resulted in a decrease in the mRNA expressions of Occludin and Claudin-1 in the jejunum of broilers (P<0.05), while also causing a slight decrease (0.05<P<0.10) in the mRNA expression of zonula occluden-1 (ZO-1). However, the supplement of Ni elevated the mRNA expression of Claudin-1 (P<0.05), as well as a slight increase in the mRNA expression of ZO-1 (0.05<P<0.10). Furthermore, no interaction effect between Cp and Ni was observed in the mRNA expressions of tight junction-related genes in the jejunal mucosa of broilers (P>0.05).

Figure 2.

Effect of Clostridium perfringens and Nisin on the jejunal-related gene expression in broilers. (A, B, and C) Barrier genes of jejunum; (D, E, F, G, and H) Apoptosis genes of jejunum. Vertical bar represents standard errors. n = 6 broilers chicken per treatment.

mRNA expression of apoptosis-related gene in jejunal mucosa

Figure 2D, E, F, G, H demonstrates that the mRNA expressions of Caspase3, Caspase9, Bax, and Bax/Bcl-2 were upregulated by Cp challenge (P<0.05). However, Ni diets reversed the elevation of the mRNA expression of Caspase9 (P<0.05). Furthermore, no significant interactions were observed between Ni and Cp in the mRNA expressions of apoptosis-related genes in the jejunal mucosa (P>0.05).

Cecal microbiota analysis

The potential effects of Cp and Ni on the microbial community structure in broilers’ ceca were investigated using 16S rRNA sequencing. After splicing, filtering, and removing chimeras, a total of 1794214 valid data were obtained from 24 samples. OTU analyses were then performed at 97% similarity, resulting in the identification of 3684 OTUs. As shown in Figure 3A, four groups shared 583 common OTUs, while the Con, Cp, Ni, and Ni + Cp groups had 582, 450, 750, and 514 exclusive OTUs, respectively. The alpha diversity of cecal microbiota is shown in Table 5, which exhibits that Cp challenge significantly increased the Shannon index (P<0.05). No interacting effect was presented between dietary Ni and Cp challenge in terms of the alpha diversity.

Figure 3.

Effect of Clostridium perfringens and Nisin in microbiota diversity. (A) Venn diagram; (B and C) The relative abundance of the top 10 phylum (B) and genus (C) from groups. n = 6 broilers chicken per treatment.

Table 5.

The alpha diversity of cecal microbiota¹

Items	Con	Cp	Ni	Ni + Cp	SEM	P-values
						Cp	Ni	Cp × Ni
Chao1	552.95	569.34	586.88	505.64	24.36	0.528	0.771	0.345
Shannon	6.61	7.38	6.52	6.72	0.13	0.047	0.110	0.221
Simpson	0.97	0.98	0.97	0.97	0.00	0.306	0.222	0.679
pielou_e	0.77	0.79	0.76	0.75	0.01	0.794	0.320	0.481
Observed_otus	546.67	560.17	578.67	499.83	23.75	0.515	0.777	0.360

¹ n = 6 broilers chicken per treatment. All of the values are expressed as the means and pooled SEM. SEM, standard error of the mean.

The first 10 dominant cecal microbiota compositions at the phylum level are illustrated in Figure 3B and Supplementary Table S2. The phylum Cyanobacteria did not exhibit significant alterations following Cp challenge (P>0.05) but possessed an increased trend of phylum Proteobacteria (0.05<P<0.10); however, the addition of Ni significantly enhanced the abundance of Cyanobacteria (P<0.05). Moreover, significant interaction effects were observed between Ni and Cp in the abundance of phylum Fusobacteriota and Desulfobacterota (P<0.05). Compared with the Con group, there was no difference in the abundance of Fusobacteriota between the Cp group and the Ni group (P>0.05); however, the Ni + Cp group exhibited a significant difference (P<0.05). The Ni + Cp group possessed a lower relative abundance of Desulfobacterota compared to the Cp group (P<0.05), but no significant difference was observed when compared to the Con group and Ni group (P>0.05). Analysis of microbial distribution at the genus level, as depicted in Figure 3C and Supplementary Table S3, reveals that the Ni and Cp challenge led to an interacting effect on the relative abundance of Clostridia_vadinBB60_group_unclassified and Intestinimonas (P<0.05). There was a higher abundance of Clostridia_vadinBB60_group_unclassified in the Ni + Cp group compared with the Cp group (P<0.05), while the abundance of Clostridia_vadinBB60_group_unclassified had no difference with the Con group (P>0.05). Additionally, the abundance of Intestinimonas in the Ni + Cp group had no difference compared with the Con group and the Cp group (P>0.05).

LEfSe analysis reveals the most differentially abundant taxa in each group (LDA score = 3.0, Figure 4, Supplementary Figure S1). Compared with the Con group, o_Lachnospirales, f_Lachnospiraceae, s_Ruminococcus_torques_group_unclassified, g_Ruminococcus_torques_group, s_UC5_1_2E3_unclassified, g_UC5_1_2E3 were higher in the Cp group and g_Pseudoflavonifractor was lower (Figure 4A). Compared with the Con group, those in the Ni group had more abundance in f_Eubacterium_coprostanoligenes_group, s_Eubacterium_coprostanoligenes_group_unclassified, g_Eubacterium_coprostanoligenes_group_unclassified, s_Bacteroides_sp_SB5, f_Selenomonadaceae, s_Phocea_unclassified, g_Phocea, s_Lachnoclostridium_edouardi, s_Collinsella_tanakaei, s_Alistipes_unclassified, f_Coriobacteriaceae, g_Collinsella but less abundance in g_Aeromonas, o_Aeromonadales, f_Aeromonadaceae, s_GCA_900066575_unclassified, g_GCA_900066575, s_Peptococcus_unclassified, g_Peptococcus, s_uncultured_lnsolitispirillum_sp_, g_Insolitispirillum, g_Anaerotruncus, g_Anaeroplasma, f_Acholeplasmataceae, o_Acholeplasmatales, s_Anaeroplasma_unclassified, g_Flavonifractor, f_Rhodospirillaceae, f_Oscillospiraceae (Figure 4B). The cecal microbiota in the Ni + Cp group possessed more enriched s_Ruminococcus_torques_group_unclassified, g_Ruminococcus_torques_group, s_Lacticaseibacillus_unclassified and g_Lacticaseibacillus, while less o_Fusobacteriales, c_Fusobacteriia, p_Fusobacteriota, s_Cetobacterium_unclassified, g_Cetobacterium, f_Fusobacteriaceae, g_Colidextribacter, s_Colidextribacter_unclassified, g_Enterococcus, f_Enterococcaceae, compared with the Con group (Figure 4C).

Figure 4.

LEfSe (LDA Effect Size) analysis (LDA = 3, P < 0.05) of the cecal microbiota. (A) Cecal microbiota between the Con and the Cp group. (B) Cecal microbiota between the Con group and the Ni group. (C) Cecal microbiota between the Con group and the Ni + Cp group.

Discussion

The NE induced by Cp commonly occurs in poultry farming and is characterized by a chronic subclinical manifestation, resulting in compromised growth performance without high mortality (El-Hack et al., 2022). Previous research has indicated that Cp challenge decreased the ADG and feed conversion ratio (Wang et al., 2021a; Zhao et al., 2022). In our study, we observed a significant reduction in ADG and a tendency towards an increased FCR during infection duration. Bacteriocins, which are secreted products of probiotics, are utilized in animal feed as a preventive against diseases and to promote animal health (Joshi et al., 2023). As a polypeptide bacteriocin, Ni is from Lactococcus lactis subsp. Related studies have shown a positive impact of Ni supplementation on the growth performance of broilers, with increasing BW, feed intake, and a simultaneous decrease in FCR during the initial rearing period (days 1 to 14) (Kierończyk et al., 2020). Additionally, Pogány Simonová et al. (2019) observed an improvement of 9.4% in the ADG of broiler rabbits fed with Nisin compared to the control group. Our findings align with previous research, indicating that the dietary Ni supplement improved BW on days 21 and 28, as well as ADG from days 15 to 21. However, other studies have shown that the Ni supplement does not provide any additional benefits to the growth performance of growing lambs, possibly due to the low concentrations of Ni added (Shen et al., 2018). In summary, supplementation with Ni improved the growth performance and mitigated the damage caused by Cp to some extent.

Inflammation serves as an indicator of overall body health. The Cp infection can induce inflammation and release cytokines, which affect pathological responses at a high level (Jiang et al., 2022). TNF-α and IL-6, as key regulators of pro-inflammatory responses, have been found to be elevated and can induce intestinal inflammatory reactions in broilers (Liu et al., 2021; Tang et al., 2022). Previous studies have reported that the toxins produced by Cp, colonizing the gastrointestinal tract, can impair the gut barrier and activate the mucosal immune response, leading to the release of TNF-α and IL-6, which in turn trigger inflammation (Xu et al., 2021; Jiang et al., 2022). In our investigation, Cp challenge substantially augmented IL-6 level. Previous studies have indicated that Ni possesses anti-inflammatory properties (Huang et al., 2022). Staphylococcus aureus infection in the uterus can be protected with Ni by reducing TNF-α and IL-6 content in rat uterus studies (Jia et al., 2019). In our investigation, the addition of Ni reduced TNF-α and IL-6 levels, indicating the anti-inflammatory effects of Ni. However, the Ni group exhibited an increase in IL-2 levels. IL-2 plays a crucial role in inducing immune responses and defending against pathogens infections (Zhang and Su, 2023). The elevation of IL-2 level typically signifies the proliferation of lymphocytes, activation of natural killer cells, and clearance of intracellular pathogens (Susta et al., 2015). The increase of IL-2 in the Ni group suggests an enhancement of organic immunity (Shan and Miao, 2022). Collectively, these studies showed that Ni can stimulate the innate immune system through its responsiveness to cytokines and autoregulation, ultimately relieving intestinal inflammation induced by Cp challenge.

Preserving the integrity of the intestinal morphology is crucial for efficient nutrient absorption and serves as a vital component in safeguarding the intestinal barrier. The villus, a crucial component of the small intestine, plays an important role in the absorption of nutrients (Wang et al., 2019). Previous studies have reported that Cp challenge severely impairs the morphology of the villi in the small intestine, resulting in a reduced capacity for nutrient absorption (Daneshmand et al., 2022). In our study, the VH, CD, and VCR of jejunum were greatly disrupted by Cp infection, which likely contributed to the decline in growth performance. Previous studies have suggested that sublancin can alleviate intestinal histopathological lesions and preserve the integrity of intestinal villi in young mice infected with Staphylococcus aureus (Wang et al., 2014). Similarly, bacteriocin supplementation improved the histomorphology parameters of the intestines in Japanese quails (Ustundag and Ozdogan, 2019). This is in agreement with our experiment that the positive effect of dietary Ni supplement played in alleviating the jejunum tract damage resulting from the Cp challenge by decreasing the CD and increasing the VCR, which indicated faster recovery of intestinal cells and effective nutrient absorption. However, Ni addition does not adversely affect the intestinal morphology of broiler chickens (Kierończyk et al., 2020). From these inconsistent results, their antimicrobial activity and dose of bacteriocin could be responsible factors for the results (Józefiak et al., 2011; Ustundag and Ozdogan, 2019). It can be concluded that the Ni addition promoted intestinal development, which could explain the improved growth performance of broilers in our study.

Occludin, Claudin-1, and ZO-1, as barrier-forming proteins, are closely linked to the function of the intestinal barrier (Wu et al., 2019), whose downregulation means weakening of tight junctions (Bai et al., 2023). Previous studies have reported that Cp challenge could decrease the expression of the intestinal barrier gene along with increasing intestinal permeability (Zhang et al., 2022). Liu et al. (2012) found that Ni has an important influence on enhancing the intestinal barrier. This study revealed that Cp challenge downregulated the mRNA expression of Occludin and Claudin-1 in the jejunal of broilers. However, Ni supplementation reversed the intestinal damage caused by Cp by upregulating the level of Claudin-1. In accordance with our results, Heeney et al. (2019) found Lactobacillus plantarum bacteriocin maintained the integrity of the epithelial barrier. In conclusion, Ni addition promising prospects for improving the reduction of intestinal barrier function caused by Cp challenge.

Apoptosis is a regulated form of cell death that contributes to maintaining homeostasis in multicellular organisms, mitochondria-mediated pathways play an important role in execution of apoptosis (Kiraz et al., 2016). Abnormal procedures for apoptosis can cause organ damage and trigger a cascade of reactions. Guo et al. (2022) found a close association between the Bcl-2 and caspase families and the apoptotic process. Bax (proapoptotic) and Bcl-2 (antiapoptotic) genes play an important influence on mitochondria-dependent apoptosis, and the balance between expression levels of Bcl‐2 and Bax determines whether a cell survives or programmed cell death (Bai et al., 2023). Caspase9 serves as a crucial protein in the initiation of the intrinsic apoptosis pathway, activating Caspase3 and initiating the caspase cascade, and finally causing cellular demise (Yang et al., 2022). To explore the underlying mechanism through which Ni alleviates intestinal injury induced by Cp challenge, we examined the expression of apoptosis-related genes. Previous studies have shown that Cp challenge upregulated the proapoptotic gene expression while downregulating the antiapoptotic genes expression, thereby accelerating apoptotic activity (Prescott et al., 2016; Wu et al., 2019). Our research indicated that Cp upregulates the gene expression of Caspase3, Caspase9, Bax, and Bax/Bcl-2, while simultaneously downregulating the Bcl-2 gene expression. This suggested that Cp participates in the apoptosis of jejunum epithelial cells via the activation of the mitochondrion-mediated apoptosis pathway. In addition, Ni was considered as anticancer peptide, with studies suggesting its potential anticancer and apoptogenic effects by mitigating mitochondrial dysfunction and oxidative stress damage in cervical cancer cells (Sadri et al., 2022). Furthermore, Ahmadi et al. (2017) discovered that Ni could induce cancer cell apoptosis by regulating apoptosis gene expression. Our experiment revealed that Ni downregulated the mRNA expression of Caspase9. Furthermore, our study illustrated that Ni addition mitigated the apoptosis caused by Cp challenge, providing further insight into the mechanism by which Ni alleviates intestinal damage.

The cecal microbiota exerts an important effect on nutrient absorption, host immunity, and intestinal defenses (Xue et al., 2020; Tang et al., 2022). However, infection with enteric pathogens can lead to disturbances in the intestinal microbiota, resulting in intestinal inflammation and reduced growth performance (Xue et al., 2020; Wang et al., 2021a). Guo et al. (2021) found that Cp disrupted the richness and diversity of the cecum microbial community. Herein, Cp challenge showed an increasing tendency for phylum Proteobacteria abundance. Studies have reported that the severity of NE is associated with an increase in Proteobacteria (Xu et al., 2018). In a study by Guo et al. (2021), phylum Proteobacteria includes pathogens that colonize the intestines of chickens, such as Escherichia coli, Salmonella, and Shigella. Kelly et al. (2018) found that Fusobacteriota activated the host’s inflammatory system to prevent pathogens responsible for tumor growth. The significant decrease in the abundance of Fusobacteriota in the Ni + Cp group indicated a substantial remission of inflammation. Cyanobacteria, a potentially pathogenic bacterium capable of producing neurotoxins and negatively impacting animal health (Mohamed et al., 2022), exhibited higher abundance in the Ni + Cp group compared with the Con group. The reason for this remains unclear and needs additional investigation. The phylum Desulfobacterota richness in the Ni + Cp group was reduced. A related study demonstrated that gut microbes have the potential to ameliorate the imbalance of gut microbiota and damage to the intestinal barrier by reducing the richness of Desulfobacterota (Tang et al., 2022).

LEfSe analysis showed that the Cp group exhibited a higher species richness of Ruminococcus_torques_group and Lachnospirales in comparison to the Con group. Another study indicated that Cp challenge increased the Ruminococcus_torques_group species richness, which was associated with the severity of intestinal disease (Tang et al., 2022). The Lachnospirales abundance was relevant to inflammation and metabolism, and the increased Lachnospirales abundance could cause organismal injury (Liu et al., 2022). Collinsella_tanakaei, functioning as a probiotic, could secrete butyrate to promote gut health and enhance disease resistance (Wang et al., 2023). Alistipes plays a crucial role in cancer immunotherapy (Parker et al., 2020). Our research revealed that Ni addition increased the abundance of Collinsella_tanakaei and Alistipes, suggesting a positive impact of Ni on probiotic growth. Moreover, the increased Eubacterium_coprostanoligenes_group abundance in the Ni group may expedite the conversion of cholesterol to coprostanol, thereby impacting the host’s fat metabolism (Wang et al., 2021b). Compared with the Con group, apart from the higher level of Ruminococcus torques group, there is a high abundance of Lacticaseibacillus in the Ni + Cp group. As a well-known member of the lactic acid bacteria, common enteric disorders can be reduced and gut health improved with Lacticaseibacillus bacteria (Lee et al., 2023). Besides, Ni treatment decreased the relative abundance of harmful bacteria. The presence of Ni + Cp resulted in decreased levels of Fusobacteriota and Enterococcus. It is worth noting that Fusobacteriota has been associated with inflammation in the gastrointestinal tract (Ostrowski et al., 2021). Enterococcus is an opportunistic pathogen in animals and causes disease in poultry (Souillard et al., 2022). In summary, the introduction of Ni appeared to enhance the colonization of beneficial bacteria and suppress the growth of harmful bacteria. Consequently, this improved the composition of the intestinal microbiota, potentially elucidating one of the underlying mechanisms through which Ni enhances the growth performance and gut health of broilers.

Conclusion

In summary, this study presents the initial evidence that dietary supplementation of Ni improves growth performance and reduces inflammation in broilers subjected to Cp challenge. Additionally, Ni supplementation mitigates subclinical NE by enhancing intestinal morphology and barrier function, as well as optimizing the composition of the cecal microbiota. Consequently, this research establishes a theoretical foundation for the clinical prevention and elucidation of the mechanisms underlying the beneficial effects of Ni on NE induced by Cp challenge.

Glossary

Abbreviations

ADG

average daily gain

ADFI

average daily feed intake

BW

body weight

Con

control group

CD

crypt depth

Cp

Clostridium perfringens

ELISA

enzyme-linked immunosorbent assays

FCR

feed gain ratio

HE

hematoxylin-eosin

IL-2

interleukin-2

IL-6

interleukin-6

Ni

Nisin

NE

necrotic enteritis

RT-qPCR

quantitative real-time PCR

TNF-α

tumor necrosis factor-α

VCR

villus height to crypt depth ratio

VH

villus height

ZO-1

zonula occluden-1

Conflict of interest statement

The authors declare no conflict of interest.