Protective Effect of Lactobacillus plantarum P8 on Growth Performance, Intestinal Health, and Microbiota in Eimeria-Infected Broilers

Coccidiosis is one of the major parasitic diseases in the commercial broiler industry. Probiotics can protect poultry against Eimeria infection. However, the mechanisms are not fully known. Therefore, Lactobacillus plantarum P8 (P8) was used to investigate its anti-coccidial property and mechanism. Five hundred broilers were allocated to five treatments: control diet (NC), control diet + Eimeria infection (IC), control diet containing 1 × 107 cfu/g P8 + Eimeria infection (P8L), control diet containing 1 × 108 cfu/g P8 + Eimeria infection (P8H), and control diet + Eimeria infection + Diclazuril (DIC). At day 14, all treatments except NC were inoculated with sporulated oocysts. Results indicated that Eimeria infection increased the mortality and oocysts shedding, and declined the growth performance as well as the intestinal barrier in Eimeria-treated broilers. On the contrary, dietary supplementation of low level P8, high level P8 and DIC decreased the mortality and oocysts shedding, but improved the growth performance and intestinal barrier. The impaired intestinal morphology in the IC group was also improved by P8H and DIC treatments. Besides, the elevated oxidative stress and pro-inflammation in Eimeria-infected broilers were reduced by P8L, P8H, and DIC treatments. Metagenomic analysis indicated P8 altered the structure of the gut microbiota, and the alteration was more obvious at day 21 than day 42. Notably, IC also increased the abundances of Eimeriidae, Eimeria and Eimeria tenella at day 21, while P8L and DIC decreased the abundances. Correlation analysis revealed that bacteria in Eimeria-treated broilers positively correlated with the intestinal permeability, oxidative stress and inflammation, while bacteria in broilers receiving P8L and DIC negatively correlated with the aforementioned pathological indices. Functional prediction demonstrated that the metagenomes of Eimeria-infected broilers were involved in several diseases. But the metagenomes of P8L-treated broilers were involved in energy metabolism and replication repair. In conclusion, dietary P8 supplementation inhibited oocyst shedding and improved the growth performance as well as the intestinal health of broilers infected with Eimeria, which was closely related to the regulation of gut microbiota. Moreover, the effects of P8 may be more effective in the early infection of coccidia.

The culture and preparation of P8 was prepared by the Department of Animal Nutrition, Qingdao Agricultural University, China. P8 was cultured on Man Rogosa Sharpe media, kept at 37 ℃ for 24 h. Pure bacterial cells were collected after centrifugation at 5000 × g for 10 min at 4 ℃. Then, these cells were washed twice with sterile 0.85 % sodium chloride solution. Ultimately, the culture purity and identi cation were constantly checked by the spreading plate method [18].

Measurement of fecal oocyst enumeration and growth performance
Excreta samples of individual birds were collected on day 19 (5 days after challenge infection), and the number of fecal oocysts was determined according to the method described by Molan et al. [20]. The amounts of provided and refused feed were measured daily on a replicate basis to calculate the average daily feed intake (ADFI). Body weight (BW) was measured at day 0, 14 and 42 to calculate average daily gain (ADG) and the feed:gain ratio (F:G) on a replicate basis. Mortality was recorded daily.

Sample collection
At day 21 and 42, blood samples from 1 broiler of each replicate were randomly collected by cardiac puncture into vacuum tubes containing coagulant and centrifuged for 10 min (3000 × g) at 4 ℃. Pure serum samples were collected and stored in 1.5 mL Eppendorf tubes at -20 ℃. The segments of duodenum, jejunum and ileum from 1 broiler of each replicate were collected and xed in 10 % buffered formaldehyde for 24 h. Mucosa was scraped from 10 cm of the jejunum using a glass slide (5 cm proximal to the Meckel's diverticulum) from 1 broiler of each replicate. At day 21, the cecal contents of 6 broilers per treatment were collected for Illumina Sequencing. At day 42, the cecal contents of 8 broilers per treatment were collected for Illumina Sequencing. Cecal contents and all intestinal samples except the intestine segments in buffered formaldehyde were placed immediately in liquid nitrogen and then held at -80 °C.

Analysis of biochemical indices
The level of malonaldehyde (MDA) and the activities of superoxidase (SOD), catalase (CAT), glutathione peroxidase (GPX) and total antioxidant capacity (T-AOC) in the jejunal mucosa were determined spectrophotometrically using commercial kits (Suzhou Grace Biotechnology Co., Ltd) according to manufacturer's protocol. The levels of sIgA, interleukin 6 (IL-6), IL-10 and tumor necrosis factor (TNF-α) in the jejunal mucosa, and the levels of diamine oxidase and D-lactate in the serum were determined using ELISA kits (Shanghai Enzyme-linked Biotechnology Co., Ltd) according to manufacturer's protocol.

Intestinal morphology
The intestinal segments were embedded in para n, and the section of each sample was placed on a glass slide and stained with hematoxylin-eosin (HE). The villus was observed under an OLYMPUS microscope (OLYMPUS, Japan) using the HMIAS-2000 image analysis system. Villus height was measured from the top of the villus to the villus crypt junction, and crypt depth was measured as the depth of the invagination between adjacent villus [21].

Analysis of tight junction proteins
A Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Biotechnology, China) was used to extract the protein from the mucosa of jejunum according to manufacturer's protocol. Equal amounts of proteins from each sample were subjected to SDS-PAGE, then proteins on the gel were transferred to nitrocellulose membrane. Membranes were blocked by 5 % skimmed milk and then incubated with the primary antibodies (anti-β-actin, anti-Claudin-1, and anti-Occludin) overnight at 4 °C. After washing with Tris Buffered Saline Tween, membranes were incubated with secondary antibody adjusted with Horseradish Peroxidase (Beyotime Biotechnology, China) [22]. The blots were then developed with an electrochemiluminescence detection system according to the manufacturer's instruction. Densitometric quanti cation of band intensities was determined using Image J software (National Institutes of Health, USA).

DNA extraction and metagenomic analysis
Bacterial DNA was extracted using a TIANamp stool DNA kit (Tiangen Biotech Co. Ltd., Beijing) according to the manufacturer's instruction. DNA samples were quanti ed using a Qubit 2.0 Fluorometer (Invitrogen, USA) and DNA quality was con rmed using 0.8 % agarose gel electrophoresis. Puri ed DNA was sent to Novogene Biotech Co., Ltd. (Beijing, China) for Illumina MiSeq sequencing. A library consisting 300 bp paired-end reads were generated before sequencing. After removing sequences contaminated by N bases, adapter sequences, low quality sequences, and replicate sequences, according to the quality control pipeline recommended by Beijing Genome Institute (BGI, Shenzhen, China), the quality-ltered reads were obtained for subsequent metagenomic analysis of cecal contents from broilers [23].
Quality ltering of the raw tags was performed to generate high-quality clean tags according to QIIME (Quantitative Insights Into Microbial Ecology, version 1.2.1; http://qiime.org/). Operational taxonomic units (OTUs) were clustered at 97 % sequence similarity following the Uclust (version 1.2.22; https://drive5.com/usearch/manua l/uclust_algo.html), and representative sequences of each cluster were used to assign taxonomy through annotation against the SILVA database. The alpha diversity of the samples, Chao 1, Ace, Shannon, Invisimpson and Simpson indices were evaluated. Principal coordinate analysis (PCoA) of the OTUs in different groups was conducted using R version 3.5.1 Feather Spray (https://www.r-project.org/). The prediction of the functional genes in the gut microbiota was done using the protocol from PICRUSt. A closed-reference OTU table in a biom-format from the script pick_closed_reference_otus.py generated in QIIME was used. The taxonomy assignment was made with the reference sequences from Greengenes database v13.8 with a 97 % similarity. After that, the OTU table was normalized with the PICRUSt work ow using the Langille Lab Online Galaxy Instance (http://galaxy.morganlangille.com) to obtain the nal metagenome functional prediction from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database at hierarchy level 1 pathways. Finally, the Statistical Analysis of Metagenomic Pro les (STAMP) software v2.1.3 was used to analyze the PICRUStpredicted metagenomes in order to obtain signi cant differences in the functional genes between the groups using the Kruskal-Wallis test, and we used Storey's FDR approach for multiple test correction [24].

Statistical data analysis
One-way ANOVA was used for single factor analysis by SPSS 20.0 for windows (SPSS Inc. Chicago, IL). Spearman's correlation coe cient was calculated using SPSS Version 20.0 (SPSS Inc., Chicago, IL) and GraphPad Prism 8 (GraphPad Software, Inc.) software and used to assess bivariate relationships between variables. Results were expressed as means and the differences were considered signi cant at P < 0.05.

Results
Effects of P8 on the fecal oocyst shedding and growth performance of Eimeria-infected broilers At the 5th day post infection, Eimeria signi cantly increased the oocysts per gram of excreta (OPG) (P < 0.01). Whereas, dietary supplementation with low level P8, high level P8 and DIC signi cantly decreased the OPG compared with the IC group (P < 0.01). Notably, P8H and DIC treatments resulted in lower OPG than the P8L treatment (P < 0.01). No signi cant changes were observed for the growth performance among groups during day 0 to 14. However, Eimeria-infection signi cantly decreased the ADG (P < 0.01) and increased the F:G (P < 0.05) compared to the NC group during day 14 to 42. On the contrary, in comparison to the IC group, P8L, P8H and DIC treatments signi cantly increased the ADG (P < 0.01) and decreased the F:G (P < 0.05) during day 14 to 42. In addition, compared to the NC group, the IC treatment increased the mortality signi cantly (P < 0.05), however, P8L, P8H and DIC treatments decreased the mortality signi cantly compared to the IC group (P < 0.05) ( Table 2).

Effects of P8 on the intestinal morphology of Eimeria-infected broilers
At day 21, duodenal HE staining sections showed that the IC treatment signi cantly decreased the villus height compared to the NC group (P < 0.01), however, P8H and DIC treatments signi cantly increased the villus height compared to the IC treatment (P < 0.01). Although the crypt depth was not obviously changed among groups, the IC treatment signi cantly decreased the villus height / crypt depth (V/C) value (P < 0.05), which was signi cantly reversed by the DIC treatment (P < 0.05). As for the jejunum, the IC treatment led to the lower villus height (P < 0.01) and V/C value (P < 0.05) compared to the NC group, however, P8H and DIC treatments increased the villus height (P < 0.01) and V/C value (P < 0.05) compared to the IC treatment. There were no signi cant impacts of the different treatments on the tested morphology parameters in the ileum (Fig. 1A, Table 3). At day 42, compared to the NC group, the IC treatment signi cantly decreased the villus height and V/C value (P < 0.01) of duodenum, but compared to the IC group, broilers in P8H and DIC groups had the increased villus height (P < 0.01). Moreover, the jejunal and ileal villus heights were also decreased in the IC group compared to the NC group (P < 0.01). Nevertheless, in comparison to the IC treatment, the DIC treatment effectively increased the villus height (P < 0.01). Besides, compared to the NC group, the jejunal V/C value was also down-regulated in broilers with coccidiosis (P < 0.05), but the DIC treatment elevated V/C value signi cantly compared to that of the IC treatment (P < 0.05) (Fig. 1B, Table 3).
Effects of P8 on the expressions of jejunal tight junction proteins and the levels of D-lactate and diamine oxidase of Eimeria-infected broilers The expressions of tight junction proteins Claudin-1 and Occludin are shown in Fig. 2 and Table 4. At day 21, Claudin-1 and Occludin expressions in the IC group were signi cantly decreased compared to the NC group (P < 0.01). However, P8L and P8H treatments signi cantly increased the expressions of Claudin-1 and Occludin compared to the IC group (P < 0.01). Besides, the DIC treatment induced the highest Claudin-1 and Occludin expressions among groups (P < 0.01). At day 42, the expressions of Claudin-1 and Occludin were also signi cantly reduced in broilers with the IC treatment compared to that of the NC treatment (P < 0.01), however, P8L, P8H and DIC treatments signi cantly up-regulated the Claudin-1 and Occludin expressions compared to the IC treatment (P < 0.01). Furthermore, the serum D-lactate and diamine oxidase levels were signi cantly elevated in the IC group at day 21 and 42 (P < 0.01) compared to the NC group, however, P8L, P8H and DIC treatments effectively declined the levels of D-lactate at day 21 and 42 (P < 0.01) compared to the IC treatment. The diamine oxidase levels were also reduced by P8L, P8H and DIC treatments at day 21 (P < 0.01), but only reduced by P8H and DIC treatments at day 42 (P < 0.01) ( Table 4).

Effects of P8 on the jejunal antioxidant capacity of Eimeria-infected broilers
At day 21, different treatments had no signi cant effects on CAT activity. Compared to the NC group, the IC treatment decreased the activities of T-AOC (P < 0.05), GPX (P < 0.05) and SOD (P < 0.01). However, compared to the IC group, the P8L treatment did not alter the activities of aforementioned antioxidases, but broilers in P8H and DIC groups had increased SOD activities (P < 0.01). In addition, compared to the NC group, the IC treatment induced a higher MDA level (P < 0.01), but P8L, P8H and DIC treatments reduced the MDA level signi cantly (P < 0.01). At day 42, different treatments had no signi cant effects on CAT and GPX activities. But compared to the NC group, the IC treatment decreased T-AOC and SOD activities (P < 0.05). Compared to the IC group, the DIC supplementation signi cantly increased T-AOC and SOD activities (P < 0.05), nevertheless, P8 treatments had no signi cant effects on SOD activity, while the P8H treatment could increase the activity of T-AOC (P < 0.05). Additionally, the Eimeria infection increased the MDA level (P < 0.01), but P8L, P8H and DIC treatments had reversion effects of this change (P < 0.01) ( Table 5).
Effects of P8 on the jejunal immunity of Eimeria-infected broilers At day 21, compared to the NC group, the IC treatment signi cantly increased the IL-6 level (P < 0.01), but P8L, P8H and DIC treatments signi cantly decreased the IL-6 level (P < 0.01). Moreover, Eimeria infection signi cantly decreased the IL-10 level but increased the TNF-α secretion compared to the NC group (P < 0.01). However, broilers receiving P8L, P8H and DIC showed lowered TNF-α levels compared to that of the IC group (P < 0.01). Eimeria infection also induced a higher sIgA level compared to the NC group (P < 0.05). But neither P8 nor DIC treatment reversed the sIgA secretion to normal. At day 42, all treatments had no obvious effects on the level of IL-10. Nevertheless, the IC treatment increased the IL-6 level compared to the NC group (P < 0.01), but P8 or DIC supplementation did not alter the IL-6 level signi cantly compared to the IC group. Besides, in comparison to the NC group, Eimeria infection also induced a higher TNF-α level (P < 0.01), which was signi cantly decreased in P8L, P8H and DIC groups (P < 0.01). Moreover, similar to day 21, the up-regulated sIgA level in the IC group (P < 0.05) was not signi cantly altered by the treatments of high level P8 and DIC ( Table 6).
Effects of P8 on the α-diversity and β-diversity of gut microbiota of Eimeria-infected broilers At day 21, the IC treatment signi cantly declined the α-diversity parameters, such as Chao1 (P < 0.01), Ace (P < 0.01), Shannon (P < 0.05) and Invsimpson (P < 0.05) compared to the NC group. In addition, P8L and P8H treatments had no signi cant impacts on Chao1, Ace, Shannon and Invsimpson, but the DIC treatment markedly elevated Chao1 and Ace (P < 0.01) compared to the IC treatment. All the different treatments had no signi cant effect on the Simpson parameter. Thereafter, at day 42, Chao1, Ace and Shannon indices were not signi cantly altered by different treatments. In comparison to the IC treatment, P8L, P8H or DIC treatment played no signi cant roles in regulating the values of Invsimpson and Simpson (Table 7). Moreover, as for the β-diversity, at day 21, the UniFrac PCoA revealed a distinct clustering of the gut microbiota composition between NC and IC groups (P < 0.05). Moreover, P8L, P8H and DIC treatments also induced a distinct clustering of the gut microbiota compaired to the IC treatment (P < 0.05) (Fig. 3A). However, at day 42, no signi cant difference was observed for the clustering of the gut microbiota among groups (Fig. 3B).

Effects of P8 on the abundance of gut microbiota and coccidia of Eimeria-infected broilers
The composition of the bacteria at phylum, family, genus and species levels were analysed. At day 21, at phylum level, the abundances of Bacteroidetes and Proteobacteria and Actinobacteria were not altered signi cantly among groups. Nevertheless, compared to the NC group, broilers in the IC group had a signi cantly down-regulated Firmicutes abundance (P < 0.05) and a signi cantly up-regulated Chlamydiae abundance (P < 0.05). Then, compared to the IC group, the DIC treatment signi cantly decreased the Chlamydiae abundance (P < 0.05). Although P8L and DIC had no signi cant effects on Firmicutes and Chlamydiae abundances in comparison to the IC group, P8L and DIC could increase the Firmicutes abundance and decrease the Chlamydiae abundance to the levels that were not signi cantly different from the NC group. At family level, the abundances of the top 5 abundant bacteria (Bacteroidaceae, Ruminococcaceae, Chlamydiaceae, Clostridiaceae and Lachnospiraceae) were not signi cantly changed by different treatments. At genus level, there were no signi cant differences in the abundances of Bacteroides, Flavonifractor and Pseudo avonifractor among groups. Additionally, the IC treatment led to elevated Chlamydia abundance (P < 0.05) and decreased Clostridium abundance (P < 0.01) compared to the NC group. Nevertheless, the DIC treatment signi cantly decreased Chlamydia (P < 0.05) and increased Clostridium (P < 0.01) compared to the IC treatment. At species level, IC, P8 or DIC treatment had no signi cant impacts on the abundances of Bacteroides fragilis, Anaerotruncus colihominis and Flavonifractor sp. An306. However, the abundances of Chlamydia abortus and Chlamydia psittaci were increased by the IC treatment (P < 0.05), and the DIC treatment had reversion effects of the changes induced by the IC treatment (P < 0.05). Moreover, we also observed alterations for coccidia abundance. Speci cally, IC treatment elevated Eimeriidae, Eimeria and E.tenella abundances (P < 0.01), which were signi cantly decreased in P8L and DIC groups (P < 0.01) (Fig. 4A, Table S1).
At day 42, no signi cant differences were noticed for the abundance of the major bacteria among groups at phylum, family, genus and species levels (Fig. 4B, Table S2).

Functional prediction of the metagenomic genes in the cecal contents
Predicted KEGG pathways from metagenomic sequences were pro led via PICRUSt. Differences in functional capacity were observed at day 21 but not day 42 (Table S3, Table S4 and Fig. 6). Compared to the NC group, the metagenomes of the infected broilers showed enrichment of host genes modulating pathways involving neurodegenerative diseases, cardiovascular diseases, infectious diseases (viral) and infectious diseases (parasitic) (P < 0.05), but showed decrease of host genes modulating pathways involving energy metabolism, amino acid metabolism, translation, carbohydrate metabolism, glycan biosynthesis and metabolism, lipid metabolism and metabolism of cofactors and vitamins (P < 0.05). Moreover, compared to the IC group, the metagenomes in the P8L-treated broilers showed enrichment of host genes modulating pathways involving energy metabolism and replication repair (P < 0.05). Furthermore, the metagenomes in the DIC-treated broilers showed enrichment of host genes modulating pathways involving energy metabolism, amino acid metabolism, translation, replication and repair, carbohydrate metabolism, glycan biosynthesis and metabolism, lipid metabolism and metabolism of cofactors and vitamins (P < 0.05), but showed decrease of host genes modulating pathways involving neurodegenerative diseases, infectious diseases (viral) and infectious diseases (parasitic) (P < 0.05) in comparison to the IC group.

Discussion
In the past decades, reports have proved that Lactobacillus were effective anti-coccidiosis candidates [25][26][27]. The anti-coccidiosis mechanisms of Lactobacillus may include the enhancement of cytokine levels, the elevation of anti-Eimeria antibody and the inhibition of oocyst shedding [13,25]. It was demonstrated that P8 provided protective immune response to broilers [28] and improved human gastrointestinal health. However, the anti-coccidiosis property and mechanisms of P8 are still unknown.
Hence, in the present study, the effects of P8 on Eimeria infection in boilers were evaluated, and compared to non-infected and infected birds fed with or without the conventional anticoccidial drug Dikzuril. Eimeria infection may lead to depressed growth rate and intestinal lesions [9]. Results of this work also indicated that during day 14 to day 42, Eimeria infection decreased the ADG and increased the F:G of broilers, however, low level and high level P8-supplemented broilers in the event of Eimeria infection showed better growth performance parameters than non-supplemented ones, approaching to those of DIC-treated birds. According to previous studies, broilers with coccidiosis also showed an impaired growth rate, while probiotics treatment improved the growth performance parameters [7,11,29,30]. Moreover, Eimeria infection caused an increased mortality during the whole period, but both doses of P8 and DIC down-regulated the mortality of boilers with coccidiosis. Similar results were found in the studies of Giannenas et al. [7,9]. Moreover, P8 and DIC protected against coccidia in this study as the oocyst output at the 5th day post infection was decreased in comparison with the IC group.
The reduction in growth performance due to the Eimeria challenge were caused by the damage to the intestinal mucosa [6]. Consistently, here the impaired gut morphology, tight junctions and immune function of intestinal mucosa were observed in Eimeria-infected broilers. Diamine oxidase is an intracellular enzyme catalyzing the oxidation of diamines and exists in high concentrations in the intestinal mucosa. Most diamine oxidase activity in the blood comes from the intestine. The serum diamine oxidase activity is reportedly proportional to the amount of intestinal diamine oxidase, therefore, it is a reliable marker of intestinal mucosal integrity [31]. D-lactate is produced by some intestinal bacteria. Normally, serum levels of D-lactate are quite low. However, when the mucosa is injured and the intestinal permeability is increased, the elevated e ux of bacteria and their metabolisms, including Dlactate into the circulation occurred [32]. In the present study, the increased intestinal permeability was also increased in the IC group, as re ected by increased diamine oxidase and D-lactate levels in serum. Probiotics and their effector molecules can in uence the gut barrier and mucosal immunity by numerous methods including modulation of mucus production, reduction of bacterial adhesion, enhancement of tight junctions and cell survival, and induction of cytokines [33,34]. These effects can be accomplished by in uences on the intestinal barrier functions [35]. In the present study, high level P8 signi cantly improved the intestinal morphology at day 21 and 42, besides, both levels of P8 and DIC treatments upregulated the expression of tight junction proteins and lowered the secretions of pro-in ammatory cytokines. The enhanced immune cell activity in Eimeria-infected chickens could cause the overproduction of free radicals [36], resulting in higher oxidative stress. Our data demonstrated that the decreased antioxidant capacity and increased oxidative stress were induced by the IC treatment, while, both levels of P8 and DIC down-regulated MDA levels in the condition of infection. Similar results were found in the study of Mengistu et al., in which the antioxidant capacity of chickens with coccidiosis was effectively elevated by probiotics [37]. Besides, Dalloul et al. also found that Lactobacillus-based probiotics could regulate the local immune system of Eimeria-infected chickens [4].
Gut microbiota, the microbe population in the intestines, is one of the central defense components in the gastrointestinal tract against enteric pathogens, which works by modulating host responses to limit the colonization of pathogens [38]. Recent study also showed that the functional additives alleviated the impact of coccidiosis challenge on the microbiome of broilers [39]. By exploring gut microbial composition in chickens, we found that DIC could signi cantly increase the α-diversity in the infected broilers, as re ected by the Chao1 and Ace indices, however, P8L and P8H treatments had no signi cant effects on the α-diversity compared to the IC treatment. There is evidence suggesting that higher diversity microbiota is bene cial in chickens but the cause and effect relationships have not been elucidated [40]. However, other authors found that the overall microbial diversity is not signi cantly disturbed by feed additives [41,42]. Furthermore, β-diversity indicated that the gut microbiota structure was altered by P8 and DIC treatments at day 21. By analysing the taxa of gut microbiota, we noticed that the altered gut microbiota composition was more obvious at day 21 than that of day 42. At day 21, as for the major bacteria, broilers in the IC group had decreased Firmicutes and Clostridium abundances, and increased Chlamydiae, Chlamydia, Chlamydia abortus and Chlamydia psittaci abundances. Although the P8 treatment had a tendency on reversing these changes, no signi cant statistical differences were found. However, it is worth noting that compared to the IC group, P8L and DIC increased the Firmicutes abundance and decreased the Chlamydiae abundance to the normal levels. Our results were in accordance with previous ndings that Eimeria infection resulted in decreased Firmicutes [43], which may reduce the production of short-chain fatty acids [44], leading to decreased intestinal barrier function [45]. Studies also suggest that probiotic supplementation could up-regulate Firmicutes [46,47]. Unfortunately, no other studies have reported data on Chlamydiae in broilers with coccidiosis to serve for comparison with our results. It is also worth noting that at day 21, the Eimeriidae, Eimeria and E.tenella abundances were increased by Eimeria infection, however, low level P8 and DIC in the event of infection reversed these abundances to normal.
Positive correlation exists between gut microbial communities and bird performance [48], and gut dysbiosis in broiler chickens corresponded with gut in ammation and reduced growth and production [49]. Research indicated that coccidiosis was correlated with chicken gut pathology [50]. With correlation analysis, we found that the SOD activity and IL-10 level positively correlated with the decreased abundance of Firmicutes in the IC group. Besides, the level of IL-10 and the expression of Claudin-1 negatively correlated with, but the level of MDA positively correlated with increased abundances of bacteria (Chalmydiae, Chlamydia, Chlamydia abortus, Chlamydia psittaci) and coccidia (Eimeriidae, Eimeria, E.tenella) by Eimeria infection, indicating the challenge of Eimeria led to decreased antioxidant capacity, impaired tight junctions and lowered anti-in ammatory ability. On the contrary, the downregulated Eimeriidae, Eimeria, E.tenella in P8L and DIC groups implied decreased oxidative stress and increased tight junctions as well as anti-in ammatory ability. At day 42, although correlation also existed between gut microbiota and biochemical parameters, the abundances of the major bacteria were not signi cantly altered by different treatments.
The change of gut microbiota may induce the altered microbial metabolic function [51]. Differences in functional capacity were observed at day 21 but not day 42. Predicted function of metagenomic genes in the cecal contents showed that the pathways involved in neurodegenerative diseases, cardiovascular diseases, infectious diseases (viral) and infectious diseases (parasitic) were enriched, while, pathways involved in energy metabolism, amino acid metabolism, translation, carbohydrate metabolism, glycan biosynthesis and metabolism, lipid metabolism and metabolism of cofactors and vitamins were decreased in cecal microbiota of broilers with coccidiosis at 21 days of age, implying that Eimeria infection may lead to diseases and had adverse effects on the nutrients metabolism. Similar to the DIC treatment, in the event of Eimeria infection, P8L treatment was predicted to induce a greater capacity for energy metabolism and replication repair. The greater capacity for energy metabolism is likely due to an increase in the level of Firmicutes as Firmicutes are related to energy metabolism and promote more e cient absorption of calories and subsequent weight gain [52]. Besides, Chlamydia infection causes host DNA damage and proliferation but impairs the DNA damage response [53], thus, the enhanced replication repair capacity of gut microbiota in P8L-treated broilers may be attributed to the decrease of Chlamydia abundance.

Conclusions
The current study con rms that during the infection of Eimeria, P8 gave substantial reduction in oocysts shedding and improvement in growth performance and intestinal health in comparison to infected broilers, and these results approached to those of the DIC treatment. These bene cial effects may be partly due to the alteration of the gut microbiota as re ected by the correlation analysis between the gut microbiota, Eimeria and biochemical indices. The low level P8 had a more effective role in regulating the gut microbiota of broilers with coccidiosis than the high level P8. Functional predictions of metagenomic genes in the cecal contents suggest changes in pathways favouring diseases, and a reduction in nutrients metabolisms in Eimeria-infected broilers, and suggest a greater capacity for energy metabolism and replication repair in P8L-treated broilers. Furthermore, by comparing the data of anti-coccidial effect and gut microbiota at day 21 and 42, we found that the effects of P8 may be more effective in the early infection of coccidia.