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. 2025 Apr 23;104(7):105205. doi: 10.1016/j.psj.2025.105205

Transcriptomic and untargeted metabolomic studies on the anticoccidial activity of eugenol in broilers☆

Tiantian Geng a, Hongjun Yang b, Jing Liu a, Junlong Zhao a, Yanqin Zhou a,⁎
PMCID: PMC12138420  PMID: 40344707

Abstract

Coccidiosis, a protozoan disease caused by Eimeria parasites, significantly impacts the poultry industry. Traditional control methods involve the addition of anticoccidial drugs to feed, which has led to concerns over drug residues. Thus, the search for alternative treatments has become a research priority, with plant essential oils emerging as a promising option. In the study, we evaluated the anticoccidial effects of seven plant-derived products on Eimeria tenella using a broiler cage trial and calculated the anticoccidial index (ACI) to assess their efficacy. The results revealed that eucalyptus oil had the highest ACI (157.79), followed closely by eugenol (155.41), both nearing the 160.00 threshold. Eugenol demonstrated a lower oocyst output compared to eucalyptus oil, leading us to focus on the mechanism of eugenol's anticoccidial activity using transcriptomic and untargeted metabolomic analyses. Transcriptomic analysis of cecal tissue revealed 749 upregulated and 1057 downregulated differentially expressed genes (DEGs). The top three enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were the extracellular matrix (ECM)-receptor interaction, cell adhesion molecules, and cytokine-cytokine receptor interaction, with the latter pathway showing significant expression differences in 40 genes. This suggests that eugenol modulates the immune response in broilers by regulating the expression of various cytokines. Metabolomic analysis identified 103 upregulated and 22 downregulated differential metabolites, with a high enrichment of the gut IgA production-related immune network pathway. Notably, vitamin A acid, a key metabolite in this pathway, was significantly upregulated. ELISA assays confirmed the upregulation of SIgA, a terminal product of this metabolic pathway. Additionally, several anti-inflammatory metabolites and prebiotics, such as fumaric acid, quinolinic acid, succinic acid, and d-raffinose, were significantly upregulated. These results indicate that eugenol modulates the intestinal immune network and levels of various anti-inflammatory metabolites and prebiotics, suggesting its role in anticoccidial activity through the regulation of DEGs and differential metabolites. This study demonstrates that eugenol has the potential to serve as a safe alternative or adjunct to anticoccidial drugs in poultry and deepens our understanding of its anticoccidial activity.

Keywords: Coccidiosis, Poultry industry, Anticoccidial drugs, Transcriptomics, Untargeted metabolomics

Introduction

Coccidiosis in chickens is a significant protozoal disease that severely impacts the poultry industry, resulting in substantial annual expenditures for its prevention and control. Currently, the primary method for controlling coccidiosis is the administration of anticoccidial drugs through feed. However, the increasing prevalence of drug resistance in coccidia has become a major concern (Chapman and Jeffers, 2014; Akram et al., 2023). Therefore, the search for alternative anticoccidial agents has become imperative. Plant-derived natural products have emerged as a promising option. Numerous studies have demonstrated the potential anticoccidial properties of various plant extracts and compounds. For instance, extracts from Fructus Meliae toosendan, maslinic acid, Ulmus macrocarpa have all shown potential efficacy against coccidian (Youn and Noh, 2001; De Pablos et al., 2010; Yong et al., 2020).

The main components of plant-derived natural products are complex mixtures of secondary plant metabolites (Sharifi-Rad et al., 2015). These natural products not only exhibit antibacterial properties, but also show significant antiparasitic effects (Ma and Zhong, 2015). Firstly, plant-derived natural products have excellent antigerm effects, it has been reported that eugenol can cause 100 % damage to Leishmaniasis amazonensis within 60 min of treatment (Ueda-Nakamura et al., 2006). Secondly, some plant-derived natural products also have a pronounced anthelmintic effect, in an in vivo study using a mouse model, Schistosoma mansoni showed a significant reduction (19.2 %) when used in combination with conventional drugs (El-Kady et al., 2019). Plant-derived natural products are also characterized by high safety. For instance, eugenol does not exhibit cytotoxicity to healthy cells even at high doses (Lev-Ari et al., 2007; Park et al., 2008). Eugenol has been recognized by the U.S. Food and Drug Administration (FDA) as a non-mutagenic and non-carcinogenic agent (Jaganathan and Supriyanto, 2012). Additionally, certain plant-derived natural products possess anti-inflammatory and antitumor activities, effectively enhancing the host's immunity and promoting growth.

Botanical driven compounds and essential oils have shown promising effects on improving immunity, health and production parameters of birds as proven by recent studies (Abbas and Alkheraije, 2023; Al-Hoshani et al., 2023; Hussain et al., 2023a; Mairizal et al., 2023; Ullah et al., 2023; Hussain et al., 2024). Natural materials and phytochemicals have diverse therapeutic effects when used as as crude materials or after reformulating them in alternate form like as essential oils (Ahmad et al., 2023; Batool et al., 2023; Khan et al., 2023; Issa, 2024; Rashid et al., 2024). Furthermore, the development in novel products has promising values and significant potential as proven by in vitro and in vivo trials (Batool et al., 2023; Rehman et al., 2023; Hailat et al., 2024; Hayajneh et al., 2024). Therefore, plant-derived natural products are potential alternatives to parasiticidic drugs. There is limited research on the effects of plant-derived natural products on Apicomplexa parasites.

Huang et al. (2021) showed Pelargonium x asperum oil inhibits Toxoplasma gondii by reducing invasion. Pundir et al. (2023) found plant-derived Alpha-cyperone and Humulene oxide inhibit Plasmodium falciparum, potentially improving malaria treatment. As a member of Apicomplexa, Eimeria tenella, which causes coccidiosis in chickens, has been the subject of studies on the anticoccidial activity of plant-derived natural products. In recent years, numerous reports have emerged on the anticoccidial potential of these natural products. For instance, artemisinin, eugenol, and garlic essential oil have all demonstrated good anticoccidial effects (Chang et al., 2021; Wang et al., 2021; Geng et al., 2024c). These studies highlight the potential of plant-derived natural products as alternatives to synthetic antiparasitic drugs, with a particular focus on their safety and efficacy against parasites causing significant diseases in veterinary health. Building on these studies, the present research employs transcriptomics and metabolomics approaches to elucidate the anticoccidial potential of plant-based products.

Advanced technologies such as transcriptomics and untargeted metabolomics are playing increasingly vital roles in the research of novel anticoccidial drugs and alternative anticoccidial products. Studies have shown that treatment with Bacillus subtilis can enhance the expression levels of multiple proteins in broiler cecal tissue cells related to immune response, intestinal barrier, homeostasis, and metabolism (Memon et al., 2022). Zhao and colleagues, by integrating untargeted metabolomics and transcriptomics analysis, revealed the differential effects of maduramicin on energy balance and amino acid metabolism regulation between drug-resistant and sensitive strains of chicken coccidia (Zhao et al., 2024). Li and other scientists utilized metabolomics and gut microbiome techniques to study the effects of semduramicin and sulfachlorpyridazine on the cecal microbiota and metabolites of broilers, finding that changes in specific metabolites such as N-carboxymethylglutamic acid were positively correlated with the anticoccidial effects of semduramicin (Li et al., 2022). This study providing a scientific foundation for the development of safer and more effective anticoccidial strategies. Previous studies on the mechanisms of plant-derived anticoccidial agents have been relatively limited. This study represents the first attempt to integrate both transcriptomics and metabolomics to elucidate the anticoccidial effects of eugenol.

Currently, there is a lack of systematic research on the inhibitory effects of common plant-derived natural products against Eimeria tenella. Therefore, the purpose of this study is to evaluate the anticoccidial effects of seven plant-derived natural products through the calculation of the anticoccidial index, thereby screening potential plant-derived natural product that could replace anticoccidial drugs for chickens. This study also makes an initial attempt to explore the mechanism of action of eugenol's anticoccidial activity using transcriptomics and untargeted metabolomics techniques, thereby laying the foundation for the development of plant-derived natural products with anticoccidial effects.

Materials and methods

Drugs and essential oils

Cinnamic aldehyde (20231115, Wuhan Shan Chuan Biotechnology Co., Ltd, China), eugenol (20230710), origanum vulgare oil (20230710), carvacrol (20231130), thymol (20231115), and eucalyptus oil (20231025) were sourced from Wuhan Shan Chuan Biotechnology Co., Ltd. Apigenin (D220833, Shanghai Yuan Ye Biotechnology Co., Ltd, China) was obtained from Shanghai Yuan Ye Biotechnology Co., Ltd. Artemisinin (20210803) was also procured from Shanghai Yuan Ye Biotechnology Co., Ltd. Garlic Essential Oil (w14930v, Kool Biological Engineering Co., Ltd, China) was derived from Kool Biological Engineering Co., Ltd. Decoquinate solution (20210826, Shandong Luxi Animal Pharmaceutical Co., Ltd, China) was provided by Shandong Luxi Animal Pharmaceutical Co., Ltd.

Ethics statement

All animal experiments were conducted in strict accordance with the National Institutes of Health (NIH) "Guide for the Care and Use of Laboratory Animals". Adhering to the principles of animal welfare and ethics, this study optimized the experimental design and strictly planned the number of animals required. The research was approved by the Ethics Committee of Huazhong Agricultural University (Approval Number: HZAUCH-2019-019 and HZAUCH-2024-0052).

Study design

Eimeria tenella Oocysts (Xiantao Strain)

The Eimeria tenella Xiantao strain used in this study is preserved in the parasitology laboratory at Huazhong Agricultural University. Prior to conducting animal experiments, the strain was passaged to ensure the vitality and virulence of the parasite (Geng et al., 2024a).

Broilers

A total of 128 one-day-old Arbor Acres broilers were procured from Zhengkang Livestock and Poultry Co., Ltd (Jingzhou city, China). All chickens were raised in a coccidia-free environment within cages measuring 0.7 × 0.7 × 0.4 meters, and were provided with ad libitum access to feed and water under conditions of 25 ± 2°C temperature and 55 ± 15 % humidity.

Anticoccidial test

One hundred and eight 14-day-old broiler chickens were randomly divided into nine groups, ensuring that the initial cage weight was almost identical in each group, with 12 chickens per group. Except for the healthy control group, all groups were orally administered 5 × 104 oocysts at 14 days of age. Cinnamic aldehyde group (CIN) was applied by adding 0.10 g of cinnamic aldehyde per 1 kg of feed for 9 days, eugenol group (EUG) by adding 0.20 g of eugenol per 1 kg of feed for 9 days, origanum vulgare oil group (ORE) by adding 0.10 g of origanum vulgare oil per 1 kg of feed for 9 days, carvacrol group (CAR) by adding 0.20 g of carvacrol per 1 kg of feed for 9 days, thymol group (THY) by adding 0.20 g of thymol per 1 kg of feed for 9 days, and eucalyptus oil group (EUC) by adding 0.25 g of eucalyptus oil per 1 kg of feed for 9 days. Healthy control group (HCG) not challenged with parasites and without treatment. Decoquinate solution group (DSG) was treated with 1.00 mL of decoquinate solution added to 1 L of water for 7 days, and infected control group (ICG) was only infected with parasites without treatment (Table 1). Clinical symptoms of the chickens in each group were observed and recorded, and the chickens were individually weighed at 22 days of age to calculate the relative body weight gain (rBWG).

Table 1.

The usage and dosage of the seven plant-derived natural products.

Groups Drugs or Essential Oils Method of administration Dosage of oral oocysts
CIN Cinnamic aldehyde Feed with 0.10 g/kg of cinnamic aldehyde for 9 d 5 × 104
EUG Eugenol Feed with 0.20 g/kg of eugenol for 9 d 5 × 104
ORE Oregano vulgare Feed with 0.10 g/kg of oregano vulgare for 9 d 5 × 104
CAR Carvacrol Feed with 0.20g/kg of carvacrol for 9 d 5 × 104
THY Thymol Feed with 0.20/kg of thymol for 9 d 5 × 104
EUC Eucalyptus oil Feed with 0.25/kg of Eucalyptus oil for 9 d 5 × 104
DSG Decoquinate solution Feed with 1.00 ml/L of decoquinate solution for 7 d 5 × 104
HCG Healthy control group / /
ICG Infection control group / 5 × 104
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Note: ICG, Infection control group, HCG, Healthy control group, DSG, Decoquinate solution group, CIN, Cinnamaldehyde group, ORE, Oregano vulgare group, CAR, Carvacrol group, THY, Thymol group, API, Apigenin group, EUC, Eucalyptus oil group, EUG, Eugenol group.

Therelativebodyweightgain=(Thetotalweightofallthebroilersat22daysofageofthetreatmentgroups,includingHCG,drugs,andessentialoils−Thetotalweightofallthebroilersat13daysofageofthetreatmentgroups,includingHCG,drugs,andessentialoils)÷(Thetotalweightofallthebroilersat22daysofageoftheHCG−Thetotalweightofallthebroilersat13daysofageoftheHCG)×100%

After euthanizing the chickens on the 8th day after infection, ceca from each group were collected for the statistical scoring of cecal lesions (Johnson and Reid, 1970). Feces were collected on the 6th, 7th, and 8th days post-infection. The McMaster method was used to calculate the oocyst per gram (OPG) value of the feces (Geng et al., 2024a). The formula for calculating the oocyst ratio is as follows:

Theoocystratio=(Theoocystoutputofthetreatmentgroups,includingHCG,drugs,andessentialoils)÷(TheoocystoutputofICG)×100░% (Chang et al., 2021).

The oocyst count conversion standard refers to the method introduced by Chang et al. (2021).

The ACI is calculated based on rBWG, survival rate, lesion index, and oocyst value. The specific formulas for calculating the lesion index, survival rate, and ACI are as follows:

Lesionindex=Lesionscore×10

Survivalrate=(survivalnumberofchickensingroups÷totalnumberofchickensingroups)×100░%

ACI=(rBWG+survivalrate)×100−−(lesionindex+oocystvalue) (Chang et al., 2021).

Anticoccidial test of eugenol

Twenty 14-day-old broiler chickens were randomly divided into four groups, ensuring that the initial cage weight was nearly identical for each group, with five chickens per group. All chickens in the Eugenol-treated infected group (EUI) and the infected control (IC) groups were orally administered 5 × 104 oocysts. The EUI group was treated with 0.20 g/kg of eugenol for 9 days, while the EU group was also treated with 0.20 g/kg of eugenol for 9 days. The HC group served as the healthy control group, not challenged with parasites and without treatment, and the IC group was only infected with parasites without treatment (Table 2).

Table 2.

Usage and dosage of eugenol.

Groups Method of administration Dosage of oral oocysts
EUI Feed with 0.20 g/kg of eugenol essential oil for 9 d 5 × 104
EU Feed with 0.20 g/kg of eugenol essential oil for 9 d /
HC / /
IC / 5 × 104
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Note: HC: Healthy control group, EU: Eugenol essential oil-treated non-infected group, EUI: Eugenol essential oil-treated infected group, IC: Infected control group.

Transcriptome sequencing

At 22 days of age, all broilers were euthanized, and approximately 1 cm segments of the cecum were harvested from each. The cecal segments were longitudinally dissected and rinsed three times with cold PBS. Following this, the segments were placed into cryovial tubes, snap-frozen in liquid nitrogen for 15 min, labeled, and stored at −80°C until shipment on dry ice to novogene corporation in beijing for transcriptome sequencing. Initially, samples were inspected, and total RNA was extracted from the cecal tissues using a standard extraction protocol. mRNA with polyA tails was enriched using oligo(dT) magnetic beads. Subsequently, the isolated mRNA was randomly fragmented in fragmentation buffer containing divalent cations, and a sequencing library was constructed following the standard new england biolabs (NEB) protocol. After library construction, the libraries were quantified using a qubit2.0 fluorometer, diluted to a concentration of 1.5 ng/μL, and the insert size of the libraries was assessed using an Agilent 2100 bioanalyzer. Once the insert size was verified to meet expectations, quantitative real-time PCR (qRT-PCR) was employed for accurate quantification of the library's effective concentration to ensure library quality. Qualified libraries were pooled based on their effective concentration and the desired amount of data output, and then subjected to illumina sequencing. Bioinformatics analysis was conducted as follows: raw data were processed using the fastp software to remove reads containing adapters, poly-N sequences, and low-quality reads, yielding clean reads. The clean data were also analyzed for Q20 and GC content. The reference genome index was constructed using HISAT2 v2.0.5, and paired-end clean reads were aligned with the reference genome. Differential expression analysis between comparative groups was performed using DESeq2 software (version 1.20.0, European Molecular Biology Laboratory). The resulting p-values were adjusted using the method of Benjamini and Hochberg to control the false discovery rate. Genes with adjusted p-values of ≤ 0.05 were considered differentially expressed. Finally, KEGG pathway analysis of the differentially expressed genes was conducted using the ClusterProfiler software (version 3.8.1).

Untargeted metabolomics sequencing

In this study, 250 mg of cecal content was collected from 22-day-old broiler chickens. The samples were immediately snap-frozen in liquid nitrogen for 15 min, labeled, and stored at −80°C until further analysis. Subsequently, the samples were shipped with ample dry ice to novogene corporation in beijing. Metabolite extraction was initiated by taking 100 mg of the frozen-ground cecal content and placing it into an eppendorf tube. To this, 500 μL of 80 % methanol in water was added, followed by vortex mixing and a 5-minute incubation on ice. The samples were then centrifuged at 15,000 × g for 20 min at 4°C, and the supernatant was collected. The supernatant was diluted with mass spectrometry-grade water to a methanol concentration of 53 %. Finally, the samples were centrifuged again at 15,000 × g for 20 min at 4°C, and the supernatant was collected for LC-MS analysis. Following the LC-MS analysis, the raw files obtained from the mass spectrometry were imported into compound discoverer 3.3 (CD3.3) software (Thermo Fisher Scientific, USA) for spectral processing and database searching, yielding the qualitative and quantitative results of the metabolites. Data quality control was performed to ensure the accuracy and reliability of the results. Subsequently, the metabolites were subjected to principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), hierarchical cluster analysis (HCA), and correlation analysis to elucidate the metabolic profiles.

Detection of secretory IgA in cecal tissue lavage fluid

The cecal tissues collected from each group were longitudinally incised and placed into preprepared 6-well plates containing cold PBS with PMSF (100 mM, 070522220902, Shanghai Beyotime Biotech Inc., China). After rinsing three times, the lavage fluid was frozen at −80°C for subsequent analysis. In this study, a double-antibody sandwich ELISA method was employed to detect the samples, using a chicken SIgA detection kit (YJ002778, Shanghai Enzyme-Linked Biological Technology Co., Ltd., China). The study strictly adhered to the procedures recommended in the kit's instructions for use.

Statistical analysis

In this study, GraphPad Prism 8.0 (Software Inc., La Jolla, CA, USA) was utilized to perform one-way ANOVA and Duncan's multiple range test, in order to analyze the statistical differences among the various groups.

Results

Screening of plant-derived products

In this study, we conducted a cage-raising trial with seven types of plant-derived natural products on broiler chickens. The results regarding relative weight gain rate indicated that eucalyptus oil (91.12 %), eugenol (87.91 %), and apigenin (88.73 %) had higher relative weight gain rates (Fig. 1A). Meanwhile, the oocyst output in the eucalyptus oil and eugenol groups was significantly reduced compared to the infected control group (Fig. 1B). Additionally, the cecal tissue lesion scores revealed that the lesion scores in the eucalyptus oil and eugenol groups were significantly lower compared to the infected control group (Fig. 1C). By integrating four evaluation indicators (rBWG, S%, LI, and OI), we derived the anticoccidial index (ACI). The ACI results demonstrated that eucalyptus oil and eugenol had relatively good anticoccidial effects, with ACI values of 157.79 and 155.41, respectively. Both exhibited a moderate level of efficacy against coccidiosis (Fig. 1D).

Fig. 1.

Fig 1

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Anticoccidial effect of six plant essential oils and apigenin. A: Relative weight gain rate, B: Oocyst output, C: Lesion score, D: ACI value, ICG, Infection control group, HCG, Healthy control group, DSG, Decoquinate solution group, CIN, Cinnamaldehyde group, ORE, Oregano vulgare group, CAR, Carvacrol group, THY, Thymol group, API, Apigenin group, EUC, Eucalyptus oil group, EUG, Eugenol group.

Cecal tonsil transcriptome analysis

Given the promising anticoccidial effects of eugenol, this study will focus on eugenol as the subject of further research. Initially, we employed transcriptomic technology to sequence the cecal tissues from eugenol-treated groups. The differential gene analysis revealed that there were 749 upregulated and 1057 downregulated DEGs in the eugenol-treated group compared to the infected control (IC) group (Fig. 2A). Utilizing the ClusterProfiler software for GO functional enrichment analysis of the differential gene set between the eugenol-treated (EUI) and infected control (IC) groups, the results indicated that the top three Gene Ontology (GO) Terms enriched according to biological processes were G-protein-coupled receptor signaling pathway, immune response, and cell adhesion. In terms of cellular components, the top three GO Terms were extracellular region, actin cytoskeleton, and plasma membrane. Regarding molecular functions, the top three GO Terms were extracellular matrix structural constituent, G-protein-coupled receptor signaling pathway, and signal transducer activity (Fig. 2B). The KEGG enrichment analysis showed high enrichment levels in pathways such as ECM-receptor interaction, cell adhesion molecules, and cytokine-cytokine receptor interaction (Fig. 2C). A detailed analysis of the cytokine-cytokine receptor interaction pathway revealed 40 differentially expressed genes between the EUI and IC groups, with significant upregulation of IL1RL2, IL1R2, IL2RB, IL2RG, IL7R, IL16, IL18R1, IL20RA, IL21R, IL31RA, IFNG, TNFRSF1B, CSF2RA, and CSF2RB, and significant downregulation of IL13RA1 and CNTFR (Fig. 2D). These results suggest that the EUI group modulates the immune response in broiler chickens by regulating the expression of these various cytokines.

Fig. 2.

Fig 2

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Transcriptome Analysis of the Eugenol Group and Infected Control Group (A) Differential gene volcano map compared with EUI group and IC group, (B) EUI and IC group differential gene GO analysis (C) KEGG enrichment analysis scatter plot, (D) Cytokine-cytokine receptor interaction differential gene clusters regulation heat map. EUI: Eugenol essential oil-treated infected group, IC: Infected control group.

Untargeted metabolomics

In this study, we employed non-targeted metabolomics technology to analyze the effects of eugenol treatment. Our results revealed that the volcano plot of differential metabolites displayed 103 upregulated and 22 downregulated metabolites (Fig. 3A). The results of the pearson correlation coefficient among all metabolites indicated that psychosine exhibited significantly negative correlations with DL-norvaline, quinic acid, d-raffinose, and methyl acetoacetate (p < 0.05) (Fig. 3B). To assess the relative abundance of metabolites on the same level, we utilized the Z-score analysis method for the EUI and IC groups. The results indicated that the Z-score values of quinic acid in the EUI group were consistently higher than those in the IC group (Fig. 3C) (note that the Z-score values of the remaining three samples, which were significantly greater than 4, are not displayed in the Fig. 3C, the other three values were 5.09, 4.90, and 4.13). The box plots of differential metabolites in the cecal contents of the EUI and IC groups showed that positive ion scanning identified differential metabolites such as psychosine, 2-(3,4-dihydroxyphenyl) acetamide, and N-(5-aminopentyl) acetamide (Fig. 3D-F). Negative ion scanning revealed differential metabolites including DL-norvaline, fumaric acid, quinic acid, methyl acetoacetate, succinic acid, and d-raffinose (Fig. 3G-L). These results suggest that eugenol modulates the levels of various anti-inflammatory metabolites and prebiotic contents.

Fig. 3.

Fig 3

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Eugenol essential oil treated non-targeted metabolomics analysis, (A) Volcano plot of differential metabolites between EUI and IC groups, (B) Differential metabolite correlation plot, C: Z-score plot, D: Psychosine box plot, E: 2-(3,4-dihydroxyphenyl) acetamide box plot, F: N-(5-Aminopentyl) acetamide box plot, G: DL-Norvaline box plot, H: Fumaric Acid box plot, I: Quinic acid box plot, J: Methyl acetoacetate box plot, K: Succinic acid box plot, L: d-Raffinose box plot, EUI: Eugenol essential oil-treated infected group, IC: Infected control group.

In this study, we conducted an in vivo metabolite analysis of the EUI and IC groups and plotted a KEGG classification diagram based on the differential metabolites between the two groups (Fig. 4A). The results revealed that there was 1 metabolite associated with the immune system, 3 metabolites related to lipid metabolism, 9 metabolites involved in carbohydrate metabolism, and 19 metabolites linked to amino acid metabolism. Utilizing the KEGG pathways as units, we employed the hypergeometric test to identify enriched pathways among the differential metabolites. The enrichment results were then visualized in a bubble chart of KEGG pathways (Fig. 4B). The findings indicated that there was 1 metabolite associated with the intestinal immune network for IgA production pathway, which was retinoic acid, with an x-coordinate value of 1 signifying a high degree of enrichment of differential metabolites in this pathway.

Fig. 4.

Fig 4

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KEGG analysis of differential metabolites compared to EUI and IC groups, (A) KEGG classification analysis map, (B) KEGG enrichment bubble map, EUI: Eugenol essential oil-treated infected group, IC: Infected control group.

The non-targeted metabolomics KEGG enrichment analysis in this study revealed a high enrichment of the immune network pathway related to intestinal IgA production. Consequently, we conducted a joint analysis with transcriptomic results. As shown in Fig. 5A, compared to the IC group, the expression of genes BLB2, ICOS, TNFSF13B, and CD28 (not shown in the Fig. 5A) was significantly upregulated in the EUI group (Fig. 5A). Compared to the IC group, the metabolite retinoic acid was significantly upregulated in the EUI group (p = 0.044578), while in the HC group compared to the IC group, retinoic acid was upregulated but without statistical significance (Fig. 5B). These results suggest that there may be differences in the final product of this metabolic pathway, secretory IgA (SIgA). Therefore, we used a double-antibody sandwich ELISA method to detect the content of SIgA in the cecal lavage fluid of the EUI and IC groups. The results showed that, compared to the IC group, SIgA was slightly upregulated in the EUI group, but the difference was not significant (Fig. 5C). These results suggest that eugenol modulates the intestinal immune network pathway in the cecum.

Fig. 5.

Fig 5

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Analysis of KEGG metabolic and transcriptional pathways intestinal immune IgA, (A) Heatmap of differentially expressed genes in the EUI group compared to the IC group, (B) Differential metabolite retinoic acid level, (C) Levels of SIgA in cecum lavage fluid, HC: Healthy control group, EU: Eugenol essential oil-treated non-infected group, EUI: Eugenol essential oil-treated infected group, IC: Infected control group.

Discussion

This study focused on plant essential oils, which are natural compounds extracted from aromatic plants, and have garnered significant attention due to their multifaceted pharmacological effects, including antipyretic, analgesic, anti-inflammatory, antioxidant, and anthelmintic properties (Yang et al., 2021; Wang et al., 2019; Bouabdallah et al., 2022). The primary constituents of plant essential oils encompass monoterpenes, flavonoids, and alkaloids (Pintatum et al., 2020), which exert positive impacts on poultry health, such as anti-inflammatory activities (Movahedi et al., 2024).

In this study, seven plant-derived natural products were investigated: eucalyptus oil, eugenol, apigenin, cinnamaldehyde, origanum vulgare oil, thymol, and carvacrol. Previous studies have demonstrated their potential benefits in broiler chickens. For instance, Colombian oregano oil (100 mg/kg) has been shown to effectively improve body weight gain and intestinal microbiota structure in broiler chickens infected with coccidiosis (Betancourt et al., 2019). Similarly, eucalyptus oil also exhibits good anticoccidial effects (Geng et al., 2024b). Eugenol (200 mg/kg) has an anticoccidial index of 167.37 (Geng et al., 2024c), while cinnamaldehyde (100 mg/kg) and thymol (200 mg/kg) enhance relative weight gain (Jamroz et al., 2005; Bolukbas et al., 2008). Carvacrol (200 mg/kg) promotes growth and improves the intestinal environment (Liu et al., 2018). The dosage of apigenin (250 mg/kg) was selected based on its significant antitumor effects in nude mice, considering the body weight of broiler chickens (Hu et al., 2015). Although these studies have different focuses, they collectively provide a reference for the dosage selection in the preliminary screening experiments of this study. In this study, we evaluated the anticoccidial efficacy of seven plant-derived natural products, and the results showed that eugenol exhibited a good anticoccidial effect with an ACI value close to 160.00. Previously, in our laboratory, we conducted a drug resistance profile study on the Eimeria tenella Xiantao isolate using six commonly available commercial anticoccidial drugs (Geng et al., 2024a). The results indicated that the isolate was completely resistant to diclazuril premix, sulfachloropyrazine sodium, toltrazuril, halofuginone, and monensin, while it was susceptible to decoquinate solution. Notably, the ACI values of these commercial drugs were all lower than that of eugenol. Therefore, these findings suggest that, under the same isolate and rearing conditions, eugenol demonstrates superior anticoccidial efficacy compared to the tested commercial drugs.

In this study, the anti-coccidial index of eugenol was found to be 155.41, and it exhibited a lower oocyst output compared to eucalyptus oil group, leading to the selection of eugenol for subsequent mechanistic studies. We attempted to preliminarily explore the anti-coccidial mechanism of eugenol using techniques such as cecal tissue transcriptomics and non-targeted metabolomics. The results of the cecal transcriptome analysis revealed that compared to the IC group, there were 749 upregulated and 1057 downregulated DEGs in the EUI group. The KEGG enrichment analysis of differential genes showed that the top 3 significantly enriched pathways were ECM-receptor interaction, cell adhesion molecules, and cytokine-cytokine receptor interaction, which is similar to the findings of Memon et al. (2022). Specifically, cell adhesion molecules are cell surface proteins that primarily facilitate connections between cells or between cells and the extracellular matrix. The extracellular matrix (ECM) is a complex network composed of various macromolecules, providing not only physical support and structural framework for cells but also regulating cellular behaviors, including adhesion, proliferation, migration, and differentiation, through interactions with cell surface receptors. When specific ECM components, such as methyl acetoacetate, are significantly upregulated, it may indicate that cells are responding to certain physiological changes. For instance, it could be associated with increased cellular energy metabolism and the activation of fatty acid synthesis pathways. The significant upregulation of the differential metabolite methyl acetoacetate in this study also supports this notion. Additionally, we specifically analyzed the differential gene groups in the cytokine-cytokine receptor interaction pathway. Compared to the IC group, the EUI group showed significantly upregulated expression of genes such as IL1RL2, IL1R2, IL2RB, IL2RG, IL7R, IL16, IL18R1, IL20RA, IL21R, IL31RA, IFNG, TNFSR1B, CSF2RA, and CSF2RB, and significantly downregulated expression of genes such as IL13RA1 and CNTFR. These results suggest that the EUI group modulates the immune response in broiler chickens by regulating the expression of various cytokines.

In this study, untargeted metabolomics analysis revealed a significant downregulation of the metabolite psychosine in the EUI group compared to the IC group. Reports have indicated that psychosine can disrupt mitochondrial potential, activate caspase 3 and caspase 9, release cytochrome C, and promote inflammatory responses in glial cells (Gowrishankar et al., 2020). It has also been reported that eugenol controls the progression of inflammation by downregulating caspase 3, which supports the results of this study (Patlevič et al., 2016). Compared to the IC group, the EUI group showed a significant upregulation of metabolites such as 2-(3,4-dihydroxyphenyl)acetamide, N-(5-aminopentyl)acetamide, DL-norvaline, fumaric acid, quinic acid, methyl acetoacetate, succinic acid, and d-raffinose. Specifically, fumaric acid can inhibit the expression of cell adhesion molecules involved in the inflammatory process, such as the inhibition of TNF-α-induced ICAM-1, E-selectin, and VCAM-1 in endothelial cells. Derivatives of fumaric acid, such as dimethyl fumarate, have been shown to modulate immune responses by affecting the activity of T cells and monocytes, reducing the production of inflammatory cytokines (Moharregh-Khiabani et al., 2009). Quinic acid and its derivatives may be converted into tryptophan and niacinamide through the gut microbiota, ultimately inhibiting the activity of NF-κB and enhancing DNA repair (Pero et al., 2009). Succinic acid may regulate intestinal inflammatory responses and systemic immune function, affecting the expression and secretion of inflammatory cytokines. It has been reported that under inflammatory conditions, succinic acid may exert anti-inflammatory effects by activating specific receptors, such as SUCNR1 (Keiran et al., 2019). d-raffinose, a prebiotic that promotes the proliferation of bifidobacteria, contributes to intestinal health (Kanwal et al., 2023). Additionally, KEGG enrichment analysis of differential metabolites revealed a significant upregulation of retinoic acid in the immune network related to intestinal IgA production, which is supported by the significant enrichment of immune response in the transcriptomics GO analysis. These results suggest differences in the final product of this metabolic pathway, secretory IgA (SIgA). Therefore, this study utilized a double-antibody sandwich ELISA method to detect the content of SIgA in the cecal lavage fluid of the EUI and IC groups, finding a slight upregulation in the EUI group. It is indeed necessary to conduct a large number of related studies on detection sensitivity or time points in the future in an orderly manner. Additionally, we have attempted to investigate the inhibitory effect of eugenol on chicken coccidia from the pathogen perspective. Although a mature in vitro culture model for chicken coccidia is currently lacking, we are actively engaged in this research.

Currently, there are limited reports on the application of multi-omics technologies in elucidating the anticoccidial mechanisms of plant-derived natural products. Zhao et al. employed transcriptomics and non-targeted metabolomics to sequence and analyze Eimeria tenella strains that were either sensitive or resistant to maduramicin. Their results revealed that, compared with the sensitive strains, the drug-resistant strains exhibited differences in the regulation of energy homeostasis and amino acid metabolism (Zhao et al., 2024). When compared with our study, the findings of Zhao et al. differ significantly. Although both studies utilized multi-omics approaches, the subjects of investigation were distinct. Our study focused on cecal tissues and cecal contents, whereas their study targeted Eimeria strains. Li et al. conducted analyses using gut microbiota sequencing and metabolomics technologies and found that the variation trends of certain metabolites, such as N-carbamylglutamate, were consistent with the anticoccidial effects of pyrimethamine (Li et al., 2022). Although both studies focused on cecal tissues and cecal contents, the mechanisms underlying the anticoccidial effects of anticoccidial drugs and plant-derived natural products may vary. Therefore, the results of their study also differ from ours.

In summary, this study fills the research gap in elucidating the underlying mechanisms of the anticoccidial activity of eugenol using transcriptomics and non-targeted metabolomics.

Conclusions

In this study, we conducted a preliminary screening of the anticoccidial effects of plant-derived natural products, and found that eucalyptus oil (ACI = 157.79) and eugenol (ACI = 155.41) exhibited promising anticoccidial effects. Transcriptomic analysis of the cecal tissue in the eugenol treatment group revealed that pathways with high KEGG enrichment after eugenol treatment included extracellular matrix-receptor interaction, cell adhesion molecules, and cytokine-cytokine receptor interaction. Non-targeted metabolomics results indicated that the immune network pathway for intestinal IgA production was highly enriched, with retinoic acid being a key differential metabolite. Eugenol was found to exert its anticoccidial effects by modulating differential gene expression and metabolite profiles. These results have enhanced our understanding of the mechanisms underlying the anticoccidial effects of eugenol.

Data availability

The raw data of transcriptome sequencing was submitted to the Sequence Read Archive database and received the data certification number: PRJNA1200123 and PRJNA1200559.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Footnotes

☆

Health and Disease

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2025.105205.

Appendix. Supplementary materials

mmc1.pdf (224.5KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.pdf (224.5KB, pdf)

Data Availability Statement

The raw data of transcriptome sequencing was submitted to the Sequence Read Archive database and received the data certification number: PRJNA1200123 and PRJNA1200559.

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