At day-of-hatch, 240 off-sex male broiler chicks were randomized and allocated to one of two treatment groups. The treatment groups included: (1) Non-challenged (NC, n = 5 replicate pens); and (2) challenged control (PC, n = 7 replicate pens) with 20 chickens/pen. Pen weights were recorded at d0, d16, d31, d42, and d52 to determine average body weight (BW) and body weight gain (BWG). Feed intake was measured at d16, d31, d42, and d52 to calculate the feed conversion ratio. Four diet phases included a starter d0–16, grower d16–31, finisher d31–42, and withdrawal d42–52 diet. The experimental diets were formulated to approximate the nutritional requirements of broiler chickens as recommended by the NRC (7) and adjusted to the breeder’s recommendations (8). At d18, chickens were orally challenged with 200 Eimeria acervulina (EA), 3,000 Eimeria maxima (EM), and 500 Eimeria tenella (ET) sporulated oocysts/chicken. At d24 (6-day post-challenge), intestinal lesion scores based on the method described by Johnson and Reid (9) were recorded for four chickens/pen (n = 20 for NC; n = 28 for PC). The entirety of the gastrointestinal tract was scored with scores being specifically assigned to the duodenal, jejunal/ileal, or cecal sections of the intestine. Additionally, on the same day, four random chickens per pen were selected and orally gavaged with 8.32 mg/kg of body weight of fluorescein isothiocyanate-dextran (FITC-d, MW 3–5 KDa; Sigma-Aldrich Co). One hour after FITC-d administration, chickens were humanely euthanized by CO₂ inhalation. Blood samples were collected from the femoral vein and centrifuged (1,000 × g for 15 min) to separate the serum. Serum levels of FITC-d were used as a biomarker to evaluate intestinal permeability as described by Baxter et al. (10), and ileal tissue sections were collected to evaluate gene expression of tight junction proteins (n = 8/group). Ileal and cecal contents were also collected at d24 for 16S rRNA sequencing and microbiome analysis (n = 8/group). At d37 (19-day post-challenge), lesion scores were also recorded for four chickens/pen (n = 20 for NC; n = 28 for PC) but no additional samples were collected at this time. Animal handling and experimental procedures were approved by the University of Arkansas Division of Agriculture Institutional Animal Care and Use Committee (#21134).

Eimeria maxima M6 and E. tenella oocysts were donated by Dr. John. R. Barta, University of Guelph, Canada, and wild-type E. acervulina oocysts were used for the challenge. Single oocyst-derived stocks of E. maxima M6, a strain recovered from a broiler flock in Florida, United States, in the mid-1990s (11) and was propagated in vivo in Eimeria-free chickens and sporulated in vitro to obtain a challenge stock. The E. acervulina strain was obtained from broiler chickens in Arkansas, United States and species confirmation was based on oocyst morphology and intestinal pathology. A preliminary dose titration study was conducted to determine the challenge dose for the trial (Table 1). The multi-species challenge dose was selected based on % reduction in body weight gain during the challenge period and lesion scores 6-day post-challenge. A 25% reduction of body weight gain during the challenge period (compared to the non-challenged control) and a lesion score of ~2 is target for subclinical coccidiosis challenge models (12). Thus, based on preliminary dose titration results, chickens in the PC group were orally challenged with 200 EA, 3,000 EM, and 500 ET sporulated oocysts/chicken at d18.

Total RNA was extracted from 50 mg of ileal tissue (n = 8/treatment) collected at d24 or 6-day post-challenge. The tissue was homogenized in 1 mL Trizol following the manufacturer’s protocol (Invitrogen, Waltham, MA, United States). RNA was then resuspended in 40 μL nuclease-free water and then treated with DNase 1 (New England Biolabs, Ipswich, MA, United States). RNA was repurified using the Trizol RNA isolation protocol and resuspended in 35 μL nuclease-free water. RNA concentration and purity were determined using a Nanodrop 1000 (Nanodrop Technology, Willmington, DE, United States). To obtain template cDNA for qPCR, 1 μg of RNA was added to PrimeScript RT Master Mix (Takara Bio USA Inc., San Jose, CA, United States) per the manufacturer’s instructions. cDNA was diluted 1:10 with nuclease-free water. Power SYBR Green Master Mix (Life Technologies, Carlsbad, CA, United States) was used for real-time quantitative PCR (Applied Biosystems 7500 Real-Time PCR system). The oligonucleotide primers for adhesion, tight junction, and gap junction genes have been listed in Table 2 and were previously described by Tabler et al. (13). The qPCR conditions were as follows: 50°C for 2 m, 95°C for 10 m, 40 cycles at 95°C for 15 s, and 58–61°C for 1 m (varied by primer). Data were analyzed by the delta–delta Ct method (14) using 18S as reference.

At d24 or 6-day post-challenge, ileal and cecal contents were collected (n = 8/treatment). Samples were stored at RT in RNA/DNA shield. Total genomic DNA of ileal and cecal content samples were extracted using the DNeasy Power Lyzer PowerSoil Kit (Qiagen, Germantown, MD, United States) according to the manufacturer’s protocol. The concentration of DNA was measured using a NanoDrop One (Thermo Fisher Scientific, Madison, WI, United States). The extracted DNA was then diluted to 10 ng/μL. The V4 region of the 16S rRNA was amplified using primer sequences (forward: 5′-GTGCCAGCMGCCGCGGTAA-3′ and reverse: 5′-GGACTAC HVGGGTWTCTAAT-3′) attached with gene-specific Illumina adapters for each sample. The PCR products were determined on a 1% agarose gel and then normalized using a commercial normalization plate [SequalPrep Normalization Plate Kit (Invitrogen, Carlsbad, CA, United States)]. All purified PCR amplicons were pooled together to generate a sequencing library. After the concentration and quality of the library were confirmed by KAPA Illumina Library Quantification Kits (Roche, Indianapolis, IN, United States) and an Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, United States), the library was sequenced on a MiSeq sequencer (MiSeq Reagent Kit v2, 500 cycles; Illumina, San Diego, CA, United States). To prevent contaminations from reagents, a mock community, ZymoBIOMICS™ Microbial Community Standard (Zymo, Irvine, CA, United States) and negative of DNA extraction and PCR amplification were included in sequencing as well. The sequencing files obtained from the Illumina sequencer were pre-processed, quality filtered (Q > 30), and analyzed using the QIIME2 (2021.4 release) software (15). The Deblur algorithm was used for sequence trimming, denoising, chimera removal, and feature binning at the amplicon sequence variants (ASV) level (16). A naive Bayes classifier was employed for the assignment of all sequences into bacterial taxonomy using the Greengenes (v13_8 clustered at 99% identity) reference database. The raw data are available in the NCBI SRA database with the BioProject ID PRJNA.

All data, excluding microbiome data, were analyzed by a one-way ANOVA using the GLM procedure of SAS (17). Means were further separated using Student’s T test with significance at p < 0.05. Alpha diversity, including the Shannon Index and the number of Observed ASVs, was compared using a two-tailed Wilcoxon signed-rank test between two groups. Beta diversity based on Bray-Curtis and Jaccard distances was tested using an analysis of similarity (ANOSIM). The outputs of diversity were visualized using the “ggplot2” package in R (v4.1.2). The linear discriminant analysis (LDA) effect size (LEfSe), an analytical tool for discovering and interpreting biomarkers of high-dimensional data, was used to identify the signature bacteria associated with the growth stages and intestinal segments. LDA score>2 was used as a criterion for judging the significant effect size (18). The signature bacteria were visualized in a heat map using the “heatmap” function in R.

The results of the evaluation of body weight (BWG), feed intake (FI), and feed conversion ratio (FCR) in broiler chickens in a mixed Eimeria spp. challenge model is summarized in Table 3. Significant (p < 0.05) differences in average BWG and FCR between NC and PC groups were observed from d16-31, with the NC having markedly higher BWG and lower FCR compared to the PC. There were no significant differences in feed intake observed between the NC and PC groups during any period evaluated (Table 3). There were no significant differences in mortality between the groups (data not shown).

Table 4 shows the coccidiosis lesion scores at d24 (6-day post-challenge) and d37 (19-day post-challenge). On d24, chickens in the PC group had significantly (p < 0.05) higher lesion scores for EA, EM, and ET compared to the NC. Nevertheless, by d37, only significant lesion scores were observed for EM in the PC group compared to non-challenged chickens (Table 4).

Figure shows macroscopic and microscopic lesions observed in chickens challenged with the mixed culture of Eimeria spp. Eimeria acervulina primarily affects the duodenum and upper small intestine. Macroscopic lesions include congestion, oedema, and thickening of the intestinal mucosa (Figure 1A). Microscopic examination reveals partial destruction of the duodenal and upper jejunal villi. The damaged villi may exhibit blunting or fusion. The mucosal epithelium may show sloughing or detachment, leading to erosion of the intestinal surface (Figure 1B).

Eimeria maxima, the most noticeable macroscopic lesions, occur in the small intestine. The affected intestinal segments, particularly the jejunum, appear thickened, congested, and edematous. They may exhibit hemorrhages and appear dark red or black due to the presence of blood. The mucosa may also have a velvety or grainy appearance (Figure 1C). Microscopic examination shows severe damage to the intestinal mucosa. The villi, finger-like projections on the mucosal surface, become shortened and broadened. The epithelial cells lining the villi may be detached, leading to denuded areas. Infiltration of inflammatory cells, such as lymphocytes and heterophils, may also be observed (Figure 1D).

Eimeria tenella affects the ceca. Macroscopic lesions in infected chickens involve hemorrhages, congestion, and thickening of the cecal mucosa. The ceca may become distended and contain blood, fibrin, and a characteristic reddish mucoid exudate (Figure 1E). Microscopic Lesions cause severe damage to the cecal mucosa. The cecal epithelium is destroyed, resulting in the formation of ulcers. The base of these ulcers often contains a reddish fibrin necrotic material. Inflammatory infiltrations, including heterophils, lymphocytes, and plasma cells, are commonly observed in the affected areas (Figure 1F).

The serum FITC-d and gene expression of tight and gap junctions are summarized in Table 5. Significantly increased serum FITC-d levels were observed for the PC group 6-day post-challenge, which was attributed to the severity of intestinal lesions in the PC group as compared to the NC group. Similarly, significant differences were observed in relative mRNA expressions of cadherin 1 (CDH1), calprotectin (CALPR), connexin 45 (Cx45), and villin 1 (VIL1) between the NC and PC. CDH1, CALPR, and Cx45 were upregulated in the PC group compared to the NC, whereas VIL1 was downregulated in the PC group compared to the NC. However, no significant differences were observed for the other tight or gap junction genes evaluated between both experimental groups (Table 5).

The challenge with mixed Eimeria spp. did not affect the alpha diversity of the microbial community in the ileal lumen of the PC group compared to the NC group (Figure 2). However, the PC group had a lower alpha diversity [Shannon Index and the number of Observed Amplicon Sequence Variants (ASVs)] in the cecal lumen compared to the NC group. There were no differences in the community structure within the ileal contents of the NC or PC observed on the principal coordinate analysis (PCoA) based on Bray-Curtis and Jaccard distances [Analysis of similarities (ANOSIM): R = 0.10, p = 0.108; R = 0.17, p = 0.073; Figure 2]. In contrast, the cecal microbiome of the PC group was distinct compared to NC (ANOSIM: R = 0.33, p = 0.003; R = 0.22, p = 0.001). Moreover, higher alpha diversity in the cecal luminal contents was observed in both NC and PC groups, and distinct clusters between cecum and ileum-based Bray-Curtis and Jaccard distances were observed in NC and PC groups (ANOSIM: R = 0.99, 0.43, 0.79, 0.33, p < 0.05).

At the phylum level, Firmicutes was the dominant bacteria across all samples (Figure 3). Bacteroidetes was also a major phylum in the cecal community. In the ileum, PC had a higher abundance of Proteobacteria (2.43%) compared to NC (1.51%) and a lower abundance of Actinobacteria (0.16%) compared to NC (1.83%). A similar pattern was observed in the cecal community, with Proteobacteria, Actinobacteria, and Bacteroidetes abundance in the NC (0.67, 4.07 and 5.37%) vs. the PC (6.46, 2.16, and 10.41%). At the genus level, the dominant genera across all samples were Lactobacillus (28.00%), Clostridium (22.02%), Faecalibacterium (5.48%), and Ruminococcus (5.32%; Figure 4). Although inter-bird variation within treatment groups was observed, treatment effects on the microbiome composition were observed. The average abundance of Lactobacillus in the PC (33.58%) was lower than that in the NC (58.99%), while Clostridium was higher in the PC (47.12%) compared to the NC (26.72%). For the cecal microbiome, higher abundances of Faecalibacterium (12.79%), Ruminococcus (13.94%), and Oscillospira (5.02%) were observed in the NC, while Lactobacillus (11.86%), Clostridium (13.78%), Bacteroides (9.74%), and Streptococcus (3.96%) were higher in the PC.

Linear discriminant analysis Effect Size (LEfSe) was employed to identify bacterial biomarkers for each group at the ASV level (Figure 5). In the ileum, the signature ASVs for the NC were Lactobacillus helveticus (ASV3), Peptostreptococceceae unclassified (ASV29), Streptophyta (ASV49), and Corynebacterium stationis (ASV74), while PC had greater abundances of Clostridium sordellii (ASV10), Clostridium butyricum (ASV11), Ruminococceceae unclassified (ASV51), and Bacteroides unclassified (ASV18; Figure 5A). In the cecum, the NC was enriched with Dorea (ASV13), Coprobacillus (ASV33, ASV58), Clostridiales unclassified (ASV61, 73), Bacteroides ovatus (ASV65), Oscillospira (ASV64), Coprococcus (ASV53), and Lachnospiraceae unclassified (ASV90, ASV59), while the PC had higher abundances of Clostridium perfringens (ASV1, ASV12) and Enterobacteriaceae unclassified (ASV8; Figure 5B).