Prophylactic impact of cecal fermentation broth against gut microbiota dysbiosis of broiler chickens challenged with Escherichia coli

Background

The intestinal microorganisms make an important contribution to the development of the host immune system and resistance to pathogen infections, especially early in life. Our previous study found that early inoculation with cecal fermentation broth could change the colonization patterns of intestinal microorganisms and enhance the immune performance in broilers. However, it remains unknown whether cecal fermentation broth inoculation can be used as a novel form of treatment against pathogenic infections.

Results

In this study, a pathological model of avian pathogenic Escherichia coli (E. coli) was established to investigate the mechanism of cecal fermentation broth against pathogen infection in broilers. A total of 180 newly hatched broilers were randomly divided into three groups: (1) negative control (Con) group, inoculated with sterile normal saline from 1 to 4 days of age; (2) positive control (NS + E.coli) group, inoculated with sterile normal saline from 1 to 3 days of age, infected with E.coli at 4 days of age; (3) fermentation broth prevention (FerB + E.coli) group, inoculated with fermentation broth from 1 to 3 days of age, infected with E.coli at 4 days of age. Subsequently, 8 individuals were randomly selected from each group for weighing followed by slaughter to determine relevant indicators at 7, 10, 14 and 21 days of age. The results showed that early inoculation with fermentation broth significantly alleviates the negative effects on growth performance and intestinal permeability caused by E. coli infection. Furthermore, enhanced small intestinal health was observed, as indicated by the increase in villus height and the ratio of villus height to crypt depth (V/C). The relative abundance of Bacteroidetes was higher in the prevention group, and the number of functional genes detected was significantly greater. Additionally, the concentration of propionic acid was substantially elevated in the prevention group, while butyric acid concentrations were significantly reduced in the challenge group.

Conclusion

This study demonstrates that early intervention with cecal fermentation broth improves intestinal barrier function and immune performance in broilers by increasing the relative abundance of intestinal Bacteroides and promoting propionic acid synthesis. These findings provide a novel strategy for preventing intestinal inflammatory disorders in broilers, such as avian colibacillosis.

Supplementary Information

The online version contains supplementary material available at 10.1186/s42523-025-00514-9.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s42523-025-00514-9.

Background

There is general agreement that avian colibacillosis is a serious enteric disease in poultry production, which is mainly caused by E. coli [1, 2]. It is associated with high morbidity and mortality, which not only brings huge economic losses to poultry production, but also poses a serious threat to environmental safety and human health [3, 4]. The most frequently disease-related E. coli serogroups in poultry are serotypes O1, O2, and O78, with a prevalence of 80% for the latter two [5, 6]. Importantly, avian pathogenic E. coli shares serotypes, virulence genes, and phenotypes with human diarrheagenic E. coli [4, 7]. Gastrointestinal infections of these pathogenic bacteria in broilers consequently serve as a source that causes human gastrointestinal pathogen infections, which is harmful to both human and broiler health [8, 9]. Although antibacterial drugs can effectively prevent the occurrence of colibacillosis, many areas have strictly restricted the use of antibiotics in livestock production due to the problems of bacterial resistances [2, 10, 11]. In addition, there is currently no effective vaccine to provide protection against pathogenic infection in avian colibacillosis [12]. This urgently emphasizes the need for alternative strategies for the prevention colibacillosis in poultry flocks.

Essentially, many chronic and degenerative diseases in broilers and humans, such as necrotic enteritis and inflammatory bowel disease, are caused by dysbiosis of the gut microbiome and excessive proliferation of pathogenic bacteria [13, 14]. Therefore, how to maintain intestinal homeostatic balance and health by improving host defense and colonization resistance against pathogens is critical to prevent this disease. In recent years, a large number of studies have shown that the targeted reconstruction of the intestinal microbiota is an ideal strategy for prevention or treatment of gastrointestinal diseases [15–17]. Unlike humans or other species, newly hatched chicks almost do not acquire the maternal microbiome from the adult hens due to the characteristics of commercial poultry production [18]. Consequently, the intestinal tract of newly hatched chicks still contains a very low microbial load, and the establishment of gut microbiota occurs predominantly after hatching through rapid environmental exposure [19]. This in return makes it possible for early interventions to modulate the initial colonization and development of intestinal microbiota in broilers. The published literatures provided evidences for potential modification of poultry health using a gut microbiota preparation or fecal microbiota transplants. These applications contributed to establish chicks’ early intestinal microbiota, and successfully increased the feed intake and body weight of broilers [20–23]. Furthermore, one study showed that transplantation of the fecal microbiota from healthy adult chickens can improve the resistance of newly hatched broilers to Salmonella [24]. However, whether microbial transplantation may be used as a novel strategy against E. coli infection in broilers has not been sufficiently investigated so far.

In most reports of microbial transplantation, the commonly used transplant materials are human or animal feces, because of their convenience of collection, no intrusion into the body, and no ethical issues involved [15, 25, 26]. Of note, poultry feces and urine are not easy to separate, and the amount is relatively small, which is difficult to meet the need of experiment. Our laboratory can produce large amounts of fermentation broth using a chemostat system in which cecal contents from a donor chicken were used as the source of the inoculum. Furthermore, our previous studies have demonstrated that early inoculation with cecal fermentation broth can alter gut microbiota development and microbial-driven metabolism, improve intestinal barrier function, as well as increase growth performance of broilers [27–29]. Subsequently, we considered whether early inoculation with cecal fermentation broth could be a novel strategy to prevent avian colibacillosis, so as to reduce the colonization and proliferation of pathogenic bacteria in broilers. Thus, the present study was designed to evaluate the prophylactic impact of cecal fermentation broth against gut microbiota dysbiosis of broiler chickens challenged with E. coli serotype O78 using 16S rRNA gene sequencing and metagenomic shotgun analysis.

Materials and methods

Preparation of cecal fermentation broth and E. coli

Our previous study gave a detailed introduction of cecal fermentation broth [28]. In brief, adult free-range broilers from mountainous area without a history of gastrointestinal diseases or antibiotic exposure were selected. The cecal content of the selected donor was collected aseptically and then thoroughly mixed with sterile phosphate buffered saline (PBS) to form a 10% suspension. The suspension was immediately injected into the chemostat and fermented continuously for 11 days. Finally, the broth of fermentation for 11 days was used as the inoculum for broilers.

A chicken E. coli O78 strain (CVCC1556, China Veterinary Culture Collection Center, Beijing, China) was resurrected in Luria Bertani broth at 37 °C for 18 h. The inoculum was streaked out on MacConkey agar plate and were incubated at 37 °C for 24 h. Subsequently, overnight cultures obtaining a single colony were grown in 5 mL Luria-bacterial broth at 37 °C with shaking. On the following day, the bacterial cells were harvested by centrifugation at 2700 rpm for 15 min at 4 °C, and then resuspended in 50 mL of normal saline in successive doses until the desired optical density of 0.5 (OD600) was recorded, corresponding to 1 × 10⁸ colony forming units (CFU) per mL. Bacterial concentration was further confirmed by plating 10-fold serial dilutions of the suspension onto agar plate.

Animals and experimental treatments

The present study was performed according to the Chinese guidelines for animal welfare, and the protocol involving animals was approved by the Animal Welfare Committee of Zhejiang University (No. ZJU2013105002, Hangzhou, China). The overall experimental design is detailed in Fig. 1A. In total, 180 newly hatched broiler chicks were randomly allocated into three groups with 60 birds per group. The treatments were as follows: (1) negative control (Con) group, inoculated with 0.3 mL sterile normal saline from 1 to 4 days of age; (2) positive control (NS + E.coli) group, inoculated with 0.3 mL sterile normal saline from 1 to 3 days of age, infected with 0.3 mL 1 × 10⁸ CFU/mL E.coli at 4 days of age; (3) fermentation broth prevention (FerB + E.coli) group, inoculated with 0.3 mL fermentation broth from 1 to 3 days of age, infected with 0.3 mL 1 × 10⁸ CFU/mL E.coli at 4 days of age. The appropriate challenge dosage (0.3 mL 1 × 10⁸ CFU/mL/bird) of E. coli was obtained based on a pre-trial with the different concentration gradient. Broilers were reared in two-tiered cages with free access to water and feed and each treatment group was kept separately in an environmentally controlled animal facility in Zhejiang Academy of Agricultural Sciences. The temperature was maintained between 32℃ and 35℃ in the first week, and then decreased by 2–3℃ each week. The daily relative humidity was maintained at approximately 70% in the first week and at 65% thereafter. No antibiotics or drugs were given to the birds throughout the trial.

Fig. 1.

Effect of cecal fermentation broth supplementation or E.coli challenge on growth performance and immune organ indexes. (A) Schematic of experimental timeline, treatments, sample analysis. Con group, inoculated with 0.3 mL sterile normal saline from 1 to 4 days of age; NS + E.coli group, inoculated with 0.3 mL sterile normal saline from 1 to 3 days of age, infected with 0.3 mL 1 × 10⁸ CFU/mL E.coli at 4 days of age; FerB + E.coli group, inoculated with 0.3 mL fermentation broth from 1 to 3 days of age, infected with 0.3 mL 1 × 10⁸ CFU/mL E.coli at 4 days of age. (B) Growth performance and immune organ indexes of broilers at the 7, 10, 14 and 21 days of age. Data are presented as means ± SEM, n = 8 in each group. Means without a common letter differ significantly, P ˂ 0.05

Sample collection

Four sampling time points were set apart to observe the continuous effect. Concretely, eight birds per treatment group were randomly selected and weighed individually at 7, 10, 14 and 21 days of age. Subsequently, blood samples were collected in 1.5 mL Eppendorf tubes by puncture of the wing vein and centrifuged at 3000 rpm for 15 min at 4℃ to separate the serum for biochemical index determination. After that, the birds were euthanized by cervical dislocation. Complete organ including spleen, bursa and thymus were stripped from carcass for weighting. The organ index was calculated for each of the organs as WO/WB × 100, with WO as immune organ weight and WB as body weight of the bird [30]. Three segments (1 cm in length) of the small intestine (duodenum, jejunum and ileum) were excised, flushed with pre-cooled PBS to remove the intestinal digesta, and then fixed in 4% (v/v) paraformaldehyde for morphological examination. The ileum mucosa samples were carefully scraped off with a glass slide, frozen in liquid nitrogen rapidly, and transferred to -80℃ freezer for subsequent analysis. The contents from one cecal lobe were separated into two sterile tubes and stored at -80℃ for microbiological analysis and short-chain fatty acids (SCFAs) determination.

Gut permeability assessment

Serum diamine oxidase (DAO) activity was measured by enzymatic spectrophotometry using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The concentration of serum D-lactic acid was measured using Chicken D-Lactate ELISA Kit purchased from the above company. Serum endotoxin, that is free lipopolysaccharide (LPS) in serum, was measured by Chicken Lipopolysaccharides ELISA Kit (Shanghai Enzyme-linked Biotech Co., Ltd, Shanghai, China). These experimental procedures were strictly performed according to the manufacturer’s instructions.

Real-time quantitative PCR

Total RNA was extracted from ileum mucosa using the TRNzol Universal (Tiangen Biotech Co., Ltd. Beijing, China) according to the manufacturer’s protocol. The concentration and the purity of RNA were determined using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, USA). Subsequently, 1 µg of qualified RNA from each sample was reverse transcribed into complementary DNA (cDNA) using HiScript^® II Reverse Transcriptase (Vazyme Biotech Co., Ltd. Nanjing, China). Quantitative analysis of PCR was performed with the ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd. Nanjing, China) using the Bio-Rad CFX Connect™ Real-Time PCR Detection System (Bio-Rad, CA, USA). The primers were synthesized by Sangon Biotech (Shanghai, China), and the primer sequences were shown in Table S1. Each reaction system (20 µL) included 10 µL SYBR qPCR Master Mix, 0.4 µL forward primer (10 µM), 0.4 µL reverse primer (10 µM), 2 µL cDNA and 7.2 µL ddH₂O. All samples were analyzed in triplicates with the same reaction protocol (95℃ for 30 s, 40 cycles of 95℃ for 10 s and 60℃ for 35 s). The GAPDH gene was used as housekeeping gene to normalize the expression of the other target genes. The 2^−ΔΔCt method was used to calculate the relative expression levels of the target genes [31].

Intestinal morphological examination

Following fixation in 4% paraformaldehyde for 24 h, the intestinal segments were dehydrated with graded ethanol, cleared in xylene, and then embedded in paraffin. The embedded samples were sectioned at 5 μm thickness and mounted on slides. Hematoxylin and eosin staining was performed using standard protocols. The images were captured using an Eclipse 80i microscope (Nikon Corp., Tokyo, Japan). The morphometric analysis was performed on at least 6 randomly-selected, well-oriented villi and crypts per slide. A computerized microscope-based image analyzer (Olympus DotSlide Virtual Slide System, Tokyo, Japan) was used to determine the height of villus (from the villus tip to the villus-crypt junction) and crypt depth (from the villus-crypt junction to the base of the crypt). Then, the V/C was calculated according to the means of villus height and crypt depth.

16S rRNA gene sequencing, data collection, and analysis

Total DNA was extracted from the cecal contents of broiler chickens using the QIAamp^® Fast DNA Stool Mini Kits (Qiagen Ltd., Valencia, USA) according to the manufacturer’s instructions. The V4-V5 hypervariable regions of the 16S rRNA gene were amplified by polymerase chain reaction (PCR) with universal primers 515 F (GTGCCAGCMGCCGCGG) and 907R (CCGTCAATTCMTTTRAGTTT) [32]. The cycling conditions of PCR reactions were 95℃ for 5 min, 35 cycles of 95℃ for 30 s, 55℃ for 30 s, and 72℃ for 45 s, with a final extension step at 72℃ for 10 min. After purification and quantification, the PCR products were pooled into equal molar amounts and sequenced on the Illumina MiSeq platform to generate paired-end reads of 300 bp.

Raw sequences were quality-filtered and demultiplexed using analyzed using Quantitative Insights into Microbial Ecology platform (http://qiime.org/index.html). The high-quality sequences were assigned into operational taxonomic units (OTUs) by the USEARCH (http://drive5.com/uparse/) according to a 97% similarity threshold. UCLUST software was then employed to perform taxonomic analysis of OTUs representative sequences with reference to Silva database (http://www.arb-silva.de). Alpha and beta diversities were calculated using MicrobiomeAnalyst (https://www.microbiomeanalyst.ca/MicrobiomeAnalyst/faces/home.xhtml) [33]. The alpha diversity was evaluated using Chao1 and Shannon indexes. Principal coordinate analysis (PCoA) was conducted to evaluate the beta diversity, which was assessed using permutational multivariate analysis of variance (PERMANOVA) based on the Bray-Curtis. Differentially abundant features among groups at genus level were identified using the linear discriminant analysis (LDA) effect size analysis (LEfSe).

Metagenomic shotgun sequencing, processing and analysis

The cecal contents of 14-day-old broiler chickens were subjected to metagenomic sequencing. Sequence libraries were generated using NEBNext^® Ultra™ DNA Library Prep Kit for Illumina (NEB, USA). The DNA extracted at 16S rRNA sequencing was utilized for metagenomic sequencing after quality inspection. Sequencing was performed on the Illumina Hiseq 2500 platform with 150 bp paired-end reads at the Mingke Biotechnology Co., Ltd. (Hangzhou, China). Metagenome assemblies were generated with trimmed but un-merged reads for each sample using MEGAHIT (https://github.com/voutcn/megahit). The genes were predicated using the METAProdigal (http://prodigal.ornl.gov/). Non-redundant gene catalog was constructed by CD-HIT software (http://www.bioinformatics.org/cd-hit/) with the parameter of 95% identity and 90% coverage [34]. The reads were aligned using the Salmon program (https://github.com/COMBINE-lab/salmon) for sequence abundance estimation. The taxonomy functional assignments, including the Kyoto Encyclopedia of Genes and Genomes (KEGG) and carbohydrate-active enzymes database (CAZy), were performed using the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with parameter setting the expected value e-value as 1e-5.

SCFAs concentration determination

Based on the characteristics of microorganisms among different treatment groups, the metabolome profiles were further explored by gas chromatography. The samples of cecal content collected at each sampling time point were used to measure the SCFAs concentration. Approximately 100 mg of cecal content was resuspended in 1 mL sterile PBS with vortex for 5 min at 8000 rpm. 500 µL of supernatant was diluted with 100 µL of 25% (w/v) metaphosphoric acid solution. After 24 h of incubation at -20℃, the mixture was centrifuged at 12,000 rpm for 20 min at 4℃. Finally, the collected supernatant was filtered through a 0.22 μm syringe filter and injected into a Shimadzu GC-2010 ATF instrument for the determination of SCFAs. The specific parameters were set according to the published paper [35].

Statistical analysis

Statistical analyses were carried out with SPSS (Version 20.0, SPSS Inc., Chicago, USA). The Kolmogorov-Smirnov program was used to verify the normality of the data. Data conforming to the normal distribution were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Non-normally distributed data were analyzed using nonparametric ANOVA (Kruskal-Wallis test) to detect significant differences among the different groups. The P-value below 0.05 was considered statistically different. The results were presented as the mean ± SEM.

Results

Growth performance and immune organ indexes

The experimental design is shown in Fig. 1A. Cecal fermentation broth supplementation or E.coli challenge had no effect on the body weight of 7- and 10-day-old broilers (Fig. 1B). However, E.coli challenge significantly reduced the body weight of broilers at 14 and 21 days of age (P ˂ 0.05). The weight loss induced by E.coli at 14 and 21 days old can be successfully alleviated by the fermentation broth. As compared with the Con group, E.coli challenge led to a decrease in the bursal index and an increase in spleen index in broilers at 10, 14 and 21 days of age (P ˂ 0.05), which was abolished by early intervention with cecal fermentation broth. In addition, the thymus index of broilers in the NS + E.coli group was slightly lower than that in groups Con and FerB + E.coli, but the difference was not statistically significant. These findings support the potential of cecal fermentation broth as an effective preventive strategy to promote growth and immune health in broilers faced with pathogenic challenges.

Serum biochemical indicators

The results from the serum biochemical indicators of broilers aged 7, 14 and 21 days are shown in Table 1. There was no significant difference among the three groups for the endotoxin index, except at 7 days of age (P ˂ 0.05). Compared with the Con group, serum diamine oxidase activity in the NS + E.coli group was significantly increased at 7, 14 and 21 days of age (P ˂ 0.05). D-lactic acid was not detected in the serum of 7-day-old broilers in each group. As the age increases, serum D-lactic acid concentration gradually increases, and the NS + E.coli group was significantly higher than the Con group at 14 and 21 days of age (P ˂ 0.05). In terms of the 3 biochemical indicators tested, there was no significant difference between the FerB + E.coli group and the Con group. The results indicate that E. coli infection significantly increases serum diamine oxidase activity and D-lactic acid concentration, reflecting compromised gut integrity. However, the early intervention with cecal fermentation broth effectively mitigated these effects, as the biochemical markers in the prevention group were comparable to those in the control group, highlighting its potential role in maintaining intestinal health and reducing infection-induced damage.

Table 1.

Effect of cecal fermentation broth supplementation or E.coli challenge on serum biochemical indicators of broilers at 7, 14 and 21 days of age

Days	Parameters	Con	NS + E.coli	FerB + E.coli	SEM	P value
7	D-lactic acid, nmol/ml	ND	ND	ND	/	/
	Diamine oxidase, U/L	23.35b	55.91a	35.70ab	5.92	0.046
	Endotoxin, EU/ml	17.15ab	19.52a	15.95b	0.63	0.033
14	D-lactic acid, nmol/ml	0.89b	2.11a	0.90b	0.24	0.025
	Diamine oxidase, U/L	13.50b	29.59a	15.19ab	2.94	0.022
	Endotoxin, EU/ml	9.63	10.70	10.53	0.53	0.741
21	D-lactic acid, nmol/ml	1.50b	2.81a	2.59ab	0.25	0.040
	Diamine oxidase, U/L	15.08b	24.50a	18.93ab	1.66	0.048
	Endotoxin, EU/ml	11.56	11.34	12.46	1.23	0.943

ND, not detectable indicated the value was below the minimum detection limit. Data are presented as means ± SEM, n = 8 in each group. Means within a row without a common letter differ significantly, P ˂ 0.05.

Morphological characteristics of small intestine

Representative morphologic observations of the intestinal tissue in the duodenum, jejunum and ileum from the Con, NS + E.coli and FerB + E.coli groups are shown in Fig. 2. Morphology examination revealed that obvious villi damage or shedding were observed in broilers from the NS + E.coli group than the Con and FerB + E.coli groups. Statistically, there were no significant differences among the three groups for villus height, crypt depth, the ratio of villus height to crypt depth (V/C) of duodenum at 7, 14 and 21 days of age. However, E.coli challenge significantly reduced the jejunum villus height of 21-day-old broilers (P ˂ 0.05), and the lowest villus height was observed in the NS + E.coli group. Meanwhile, compared with the Con group, E.coli challenge resulted in a significant reduction in jejunum V/C ratio of 14- and 21-day-old broilers (P ˂ 0.05), whereas no significant difference was observed between the FerB + E.coli group and the Con group. Notably, cecal fermentation broth supplementation or E.coli challenge exerted profound influence on the ileum of broilers. E.coli challenge reduced villus height and V/C ratio of ileum at 7, 14 and 21 days of age (P ˂ 0.05), and increased crypt depth of ileum at 14 and 21 days of age (P ˂ 0.05), which could be neutralized by early intervention with cecal fermentation broth.

Fig. 2.

Effect of cecal fermentation broth supplementation or E.coli challenge on duodenum (A), jejunum (B) and ileum (C) of 7-, 14- and 21-day-old broilers. Statistics for each intestinal segment included villus height, crypt depth, and the ratio of villus height to crypt depth (V/C). n = 8 each in the Con, NS + E.coli and FerB + E.coli groups

Tight junction protein gene expression

The relative expression of genes associated with tight junction in the ileum of broilers were summarized in Fig. 3, including claudin family (CLDN), occludin (OCLN) and zona occludens family (ZO). E.coli challenge reduced the relative expression of CLDN3 in the ileum of 14- and 21-day-old broilers (P ˂ 0.05), as compared with the Con group. Whereas, the effect of E.coli on CLDN3 could be eliminated by early intervention with cecal fermentation broth (Fig. 3B). At the age of 21 days, the expression levels of ZO1 and ZO2 in the ileum of broilers were significantly lower in the NS + E.coli group than those in the Con group (P ˂ 0.05). However, there was no significant difference between the FerB + E.coli group and the Con group (Fig. 3E and F). In addition, cecal fermentation broth supplementation or E.coli challenge had no effect on the expression of CLDN1, CLDN5 and OCLN at any ages.

Fig. 3.

Effect of cecal fermentation broth supplementation or E.coli challenge on the relative mRNA expression of tight junction protein in the ileum of broilers at the 7, 14 and 21 days of age. Data are presented as means ± SEM, n = 8 in each group. Means without a common letter differ significantly, P ˂ 0.05

Cecal microbiome composition of broilers using 16S rRNA sequencing

After quality control and chimera filtering, a total of 4,377,903 sequencing reads with a mean of 45,603 (standard deviation SD, ± 8,421) sequences per sample were obtained for the 96 samples of cecal content (n = 8, 3 groups: Con, NS + E.coli, FerB + E.coli; 4 sampling time points: 7, 10, 14, 21 days old). Rarefaction curves generated from the OTUs approached a plateau with the increase of the number of reads sampled (Fig. S1), indicating the sequencing depth was sufficient for the investigation of the cecal microbiota. Venn diagram showed that 401, 426, 548 and 571 OTUs were identified based on 97% sequence similarity at 7, 10, 14 and 21 days of age, respectively (Fig. S2). The number of OUT in the NS + E.coli group was lower than that in the Con and FerB + E.coli groups at 4 sampling time points.

To evaluate alterations in the microbiota community structure among the three groups, alpha diversity analysis was conducted according to the result of 16S rRNA sequencing (Fig. 4). Community richness (Chao1) was significantly different among the groups at 7 and 21 days of age (P ˂ 0.05), and the lowest Chao1 value was observed in the NS + E.coli group (Fig. 4A). Shannon index applied to measure species diversity was lower in the NS + E.coli group than that in the Con and FerB + E.coli groups for 7- and 14-day-old broilers (P ˂ 0.05) (Fig. 4B). On the other hand, beta diversity of cecal microbiota in broilers at 7, 10, 14 and 21 days of age was investigated, as illustrated by the PCoA plots in Fig. 4C. It can be observed that the microbiome composition profiles among the Con, NS + E.coli and FerB + E.coli groups were separated obviously. The microbial community structures were dramatically different among the three groups based on the results of PERMANOVA analysis of the Bray-Curtis dissimilarity at each day of age (P ˂ 0.001).

Fig. 4.

The alpha (A, B) and beta-diversity (C) of the community richness and species diversity of the cecal microbiota of the Con, NS + E.coli and FerB + E.coli groups. n = 8 in each group. Significant differences are indicated by P values at the top of a single figure

Microbial compositions in cecum of 7-, 10-, 14- and 21-day-old broilers at both the phylum and genus levels are shown in Fig. 5. The figure manifested the bacteria with the top 5 abundance at the phylum level and the top 15 at the genus level, and merged remaining species into “Other”. Firmicutes and Bacteroidota were the most dominant phyla in the cecal microbiota, accounting for about 90% the relative abundance in all samples at each day of age. However, the relative proportions of these two phyla were different among the three groups. Compared with the Con and NS + E.coli groups, the relative abundance of Bacteroidota in the FerB + E.coli group increased, while Firmicutes decreased. Proteobacteria, as the third largest phyla, had higher relative abundance in the NS + E.coli group than that in the Con and FerB + E.coli groups (Fig. 5A). At the genus level, Bacteroides was predominant bacteria in the cecum of broilers for the three groups, among which the abundance of Bacteroides was the highest in the FerB + E.coli group. In contrast, broilers in the FerB + E.coli group, although infected with E.coli, had low level of Escherichia-Shigella, which was significantly lower than that in the NS + E.coli group (Fig. 5B). In addition, a particular finding was that broilers in the FerB + E.coli group had a more uniform cecal microbiota regardless of the taxonomic level of phylum and genus.

Fig. 5.

Relative abundance of cecal microbiota composition of 7-, 10-, 14- and 21-day-old broilers at the levels of phylum (A), genus (B) and the discriminant genera in the cecum of 7-, 10-, 14- and 21-day-old broilers (C). The sample numbers of the Con group were C7-1 to C7-8 at 7 days of age, and 7 was replaced by 10, 14 or 21 at other points in time, and NE and FE were substituted for C in the NS + E.coli and FerB + E.coli groups, respectively. n = 8 in each group

To identify whether specific bacterial taxa at the genus level varied in abundance by groups, we applied the LEfSe method to further analysis the cecal microbiota of broilers (Fig. 5C). 29, 21, 29 and 24 genera were respectively identified from 7-, 10-, 14- and 21-day-old broilers based on the criteria of LDA score for discriminative features ˃ 4.0 and adjust the p-value of the false discovery rate ˂ 0.1. More bacterial genera were significantly enriched in the Con group compared with the NS + E.coli and FerB + E.coli groups. Moreover, Incertae-Sedis was significantly higher in the Con group at all days of age. Conversely, broilers in the NS + E.coli group had higher proportions of Escherichia-Shigella regardless of their age. Three genera, including Bacteroides, Parabacteroides and Megasphaera, were obviously greater in the FerB + E.coli group at all days of age. These bacterial genera can be used as biomarkers for each group, which showed consistent enrichment characteristics in the corresponding group based on the results of 4 sampling time points.

Functional characteristics of cecal microbiota based on metagenomic sequencing

To investigate the functional profile of the cecal microbiota in the Con, NS + E.coli and FerB + E.coli groups, we selected the microbial DNA extracted from 14-old-day broilers for metagenomic sequencing. A total of 1,502,810 functional genes were predicated from the three groups (Fig. 6A). The microbial community in the NS + E.coli group had the lowest number of detected functional genes (1,084,445), which was significantly lower than that in the Con group (P ˂ 0.05). However, there was no significant difference in the number of genes between the Con and FerB + E.coli groups (Fig. 6B). As expected, principal components analysis (PCA) clustered all functional genes into three groups, with a more pronounced tendency to separate FerB + E.coli group from Con and NS + E.coli groups (Fig. 6C).

Fig. 6.

Microbial gene function differed among the Con, NS + E.coli and FerB + E.coli groups. Venn diagram analysis of the functional genes in the three groups (A). Comparison of the microbial gene count in the three groups (B). PCA analysis of bacterial functional genes in the three groups (C). Heatmap showing the abundance of 10 most significantly altered CAZy family in the three groups (D). Heatmap showing the composition and abundance of six KEGG functional genes in the three groups (E)

The CAZy database focuses on carbohydrate active enzymes that can degrade, modify, or generate Glycosidic bonds. These carbohydrate active enzymes are divided into six major protein functional modules: glycoside hydrolases (GH), glycosyl transferases (GT), polysaccharide lyases (PL), esterase, auxiliary activities (AA), carbohydrate-binding modules (CBM). As shown in Fig. 6D, the abundances of GT and CBM in the Con group were higher than that in the NS + E.coli and FerB + E.coli groups, indicating E.coli infection resulted in a negative effect on these enzymes. Correspondingly, cecal fermentation broth greatly activated the GH, whose relative abundance increased in the FerB + E.coli group. The metabolic pathway analysis database is the core of the KEGG database. By comparing the protein sequences with the KEGG metabolic pathway database, the genes predicted by the macrogenomic were annotated and classified according to the metabolic pathways. Genes were assigned to 40 Level 2 KEGG items, which were summarized in six KEGG categories: cellular processes, environmental information processing, genetic information processing, human diseases, metabolism, and organismal systems. Among these, metabolism was the most disturbed pathway among the three groups, followed by human diseases, organismal systems, cellular processes, genetic information processing and environmental information processing. Of note, the functional genes involved metabolism pathway had a higher proportion in the FerB + E.coli group. Based on this result, we speculated that cecal fermentation broth may improve metabolite production or metabolic performance of broilers to some extent.

SCFAs concentrations in cecal content

In order to confirm the functional differences among the Con, NS + E.coli and FerB + E.coli groups, the SCFAs profile in the cecum of broilers at all days of age was evaluated (Fig. 7). The dominant SCFAs in broiler cecum were acetate and propionic acid, followed by butyric acid, isobutyrate, valerate and isovalerate. Cecal contents from the NS + E.coli group had significantly reduced concentrations of isobutyrate, valerate and isovalerate when compared to the Con group (P ˂ 0.05). However, early intervention with cecal fermentation broth can effectively alleviate the decrease in the concentration of SCFAs. Notably, the concentrations of propionic acid were significantly enhanced in the FerB + E.coli group at 7, 10, 14 and 21 days of age (P ˂ 0.05). There was no significant difference in the levels of acetate and butyrate among the three groups. These findings suggest that cecal fermentation broth supplementation plays a protective role in maintaining the SCFA profile in the cecum of E. coli-challenged broilers. By stabilizing the concentrations of key SCFAs such as isobutyrate, valerate, and particularly propionic acid. Fermentation broth intervention may support gut health and functionality, enhancing the intestinal environment even under pathogenic stress.

Fig. 7.

Effects of cecal fermentation broth supplementation or E.coli challenge on short-chain fatty acids (SCFAs) concentration in the cecum of broilers at 7, 10, 14 and 21 days of age. Data are presented as means ± SEM, n = 8 in each group. Means without a common letter differ significantly, P ˂ 0.05

Discussion

Previous research has identified early microbial intervention could alter the colonization patterns of intestinal microbiota and enhance the intestinal barrier function in broilers. However, there is limited research on the use of microbial intervention as a preventative measure against intestinal inflammation in broilers. In this study, a pathological model of E.coli infection in broiler chickens was established to evaluate the preventive efficacy of cecal fermentation broth intervention on inflammatory bowel disease.

Growth performance constitutes a pivotal factor in assessing the economic benefits of broiler production. In the current study, the body weight of broiler chickens in the challenge group was significantly lower than that in the control group, whereas the body weight of broilers in the prevention group did not differ significantly from the control group. This result indicated that fermentation broth supplementation could mitigate the detrimental effects of E. coli infection on the growth performance of broiler chickens. Our findings align with those of Ateya et al. [36], who reported that E. coli infection negatively impacts broiler performance parameters, including body weight gain, feed intake, and feed conversion efficiency. However, supplementation with probiotics, acidifiers, symbiotics, and antibiotics has been shown to enhance the performance of infected broilers. The thymus, spleen and bursal are considered the primary immune organs in broilers, playing a critical role in establishing an effective immune response against a wide range of pathogens. Typically, E. coli infection adversely affects immune organ development in broilers, while this study found that prophylactic fermentation broth treatment could attenuate the detrimental effects of E. coli on immune organs. The observed improvement in broiler growth parameters may be attributed to enhanced digestibility, increased beneficial intestinal microbiota, and strengthened intestinal barrier function [37]. Therefore, it is necessary to further evaluate the impact of fermentation broth intervention on the intestinal barrier function in broilers infected with E. coli.

The intestinal barrier constitutes a sophisticated defense system encompassing mechanical, chemical, immune, and microbial components, which act together to maintain intestinal homeostasis by inhibiting the translocation of pathogens and toxins from the lumen into the circulation [38]. Generally, disruption of the intestinal barrier results in the translocation of pathogens and toxins into the bloodstream, which subsequently triggers systemic inflammation and a variety of diseases. In related studies, serum DAO activity, D-lactate levels and endotoxin concentration serve as key indicators of intestinal mucosal permeability and damage to the intestinal barrier [39]. The DAO is an enzyme predominantly located in the intestinal epithelial villi, whereas the last two indicators originate from intestinal bacteria. Disruption of the intestinal barrier function is typically associated with elevated levels of these markers in peripheral blood [40]. In this study, we observed that E. coli infection significantly increased serum D-lactate, DAO, and endotoxin levels. However, prophylactic administration of fermentation broth effectively modulated these elevated markers, suggesting its potential in mitigating the impact of E. coli on intestinal permeability. This finding indicates that E. coli infection results in increased intestinal permeability, while prophylactic administration of fermentation broth can assist in preserving intestinal integrity in chickens exposed to pathogens.

There is a close relationship between the histological morphology of the intestine and intestinal function and growth performance. Damage to the intestinal barrier and absorptive function can be directly reflected through changes in intestinal microstructure. Our study demonstrated that broilers infected with E. coli exhibited reduced villus height and a lower villus-to-crypt ratio in the jejunum and ileum, suggesting a diminished surface area for nutrient uptake and a negative impact on intestinal health. Nevertheless, fermentation broth treatment effectively counteracted these adverse effects, underscoring its beneficial role in promoting intestinal integrity and gut health in broilers.

Tight junctions are essential intercellular structures located at the apical region of the intestinal mucosa, where they function as a semi-permeable barrier to restrict the entry of harmful substances [41]. Intestinal permeability is largely regulated by tight junction proteins positioned between adjacent enterocytes, including occludin, members of the claudin family, and ZO family proteins [42, 43]. Previous studies indicate that E. coli infection in mice can alter the expression of these tight junction proteins, resulting in increased intestinal permeability [44]. In this study, we observed significant alterations in the mRNA expression levels of key tight junction proteins, such as ZO1, ZO2, and CLDN3, in broilers challenged with E. coli. Specifically, CLDN3 expression in the ileum was markedly reduced in the challenge group at 14 and 21 days of age. Similarly, ZO1 and ZO2 levels were significantly decreased in the challenge group at 21 days. However, the preventive administration of cecal fermentation broth notably mitigated the E. coli-induced disruptions in CLDN3, ZO1, and ZO2 expression, preserving intestinal integrity and reducing permeability alterations. The findings of the present study collectively demonstrate that supplementation of fermentation broth restored intestinal barrier integrity in broilers challenged with E. coli, as evidenced by complete intestinal morphology, reduced serum D-lactate, DAO, and endotoxin levels, as well as increased relative mRNA expression of tight junction proteins.

Epithelial barrier damage and immune-mediated diseases are frequently associated with disruptions to the host’s intestinal microbial composition during pathogenic bacterial infections. Intestinal microbiota is a crucial component of the mucosal barrier, playing an essential role in maintaining intestinal health and defending against pathogen invasion. Furthermore, these microorganisms significantly contribute to various physiological processes through their metabolic byproducts, such as short-chain fatty acids, amino acids, and vitamins, which serve as essential energy sources for the host. Under typical physiological conditions, the diversity of the intestinal microbiota is beneficial for maintaining the stability of the intestinal microenvironment and enhancing resistance against pathogenic microorganisms [45]. In our research, the E. coli infection significantly decreased alpha-diversity as indicated by reduced Chao1and Shannon indexes, which increased susceptibility to pathogen infection and intestinal inflammation. In contrast, the adverse effects of E. coli on alpha-diversity were diminished by fermentation broth prevention, suggesting that fermentation broth could stabilize the cecal microbiota. The beta-diversity reflects variations of community composition between samples. The distinct clustering observed in the PCoA plot indicates significant differentiation in the microbial composition of broilers across the three groups. Regarding the microbial composition, it was observed that broilers infected with E. coli exhibited significantly higher relative abundances of Proteobacteria and Escherichia-Shigella. The phylum Proteobacteria, a major component of the chicken cecal microbiota, includes various pathogenic genera such as Escherichia, Salmonella, and Vibrio [46]. Escherichia-Shigella, recognized as a pathogenic bacterium, is positively correlated with intestinal inflammation owing to its capability to produce a diverse array of exotoxins [47]. In contrast, prophylactic treatment with fermentation broth reduced the relative abundance of Proteobacteria and Escherichia-Shigella in the cecum of E. coli-infected broilers, while concurrently increasing the relative abundance of Bacteroides, which is associated with the biosynthesis of short-chain fatty acids, particularly propionic acid [48]. Consequently, the concentration of propionic acid in the cecum of broilers in the fermentation broth prevention group was markedly elevated. This is consistent with the study on the role of Bacteroides thetaiotaomicron in mice, which demonstrated that this bacterium exhibited anti-inflammatory activity by producing propionic acid [49]. Furthermore, metagenomic sequencing enabled a comprehensive evaluation of functional genes within the microbial community. Our findings revealed that E. coli infection diminished the abundance of functional genes and altered the gene composition. Functional gene annotation based on the CAZy database indicated a reduction in carbohydrate-active enzyme activity following E. coli infection. Within the six primary KEGG categories, metabolic function was impaired in the challenge group, whereas the fermentation broth prevention group exhibited a higher abundance of functional genes associated with metabolic pathways. This enhancement in metabolic function likely accounts for the observed improvements in growth performance and the increased production of short-chain fatty acid in broilers following the administration of fermentation broth.

Conclusion

In summary, prophylactic administration with fermentation broth can mitigate E. coli-induced inflammatory responses and aid in preserving intestinal functions, potentially by modulating the composition of intestinal microbial. Additionally, intervention with fermentation broth enhances the metabolic synthesis capacity of SCFAs in the cecum of broilers, thereby promoting overall intestinal health. These findings offer a novel control strategy for the prevention of pathogen infection in poultry.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AA

Auxiliary activities

CAZy

Carbohydrate-active enzymes database

CBM

Carbohydrate-binding modules

CFU

Colony forming units

CLDN

Claudin

DAO

Diamine oxidase

E. coli

Escherichia coli

GH

Glycoside hydrolases

GT

Glycosyl transferases

LDA

Linear discriminant analysis

LEfSe

Linear discriminant analysis effect size

LPS

Lipopolysaccharide

OCLN

Occludin

OTUs

Operational taxonomic units

PBS

Phosphate buffered saline

PCA

Principal components analysis

PCoA

Principal coordinate analysis

PERMANOVA

Permutational multivariate analysis of variance

PL

Polysaccharide lyases

SCFAs

Short-chain fatty acids

V/C

The ratio of villus height to crypt depth

WB

Body weight

WO

Organ weight

ZO

Zonula occludens

Author contributions

Y.G. and S.L. conceptualized the study and wrote the manuscript. S.S. and Z.C. performed the experimental process. S.Z and R.J. analyzed the data and were involved in the study design. X.Z., X.K. and Y.T. critically revised the manuscript for important intellectual content. All authors read and approved the final version of the manuscript.

Funding

This study was supported by the Special Fund for Henan Agriculture Research System (HARS-22-18-S), the Natural Science Foundation of Henan Province (252300423587), the Science and Technology Innovation Fund of Henan Agricultural University (2024CXZX009) and the Key Research and Development Special Projects of Henan Province (241111113600).

Data availability

The metagenome datasets are available at NCBI project PRJNA1224300. The raw reads of 16S rRNA sequencing are available at NCBI project PRJNA1225000.

Ethics approval and consent to participate

The present study was performed according to the Chinese guidelines for animal welfare, and the protocol involving animals was approved by the Animal Welfare Committee of Zhejiang University (No. ZJU2013105002, Hangzhou, China).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.