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Full Research Article

Chestnut Bur Tannins: A Novel Approach to Eimeria tenella Control

N
Na Zhao1
https://orcid.org/0009-0002-3144-4766
L
Li-Zhong Wang2
https://orcid.org/0009-0008-8469-1078
H
Hua-Wei Li1
https://orcid.org/0000-0002-8024-9871
L
Li-Yun Chang1
https://orcid.org/0000-0001-9104-9464
C
Cheng-Hui Li1
https://orcid.org/0000-0002-7704-3610
S
Shuang Li3,*
https://orcid.org./0009-0002-5752-7308
1Faculty of Life Science, Tangshan Normal University, 156 Jianshe Road, Tangshan City, Hebei Province, P.R. China.
2Ocean College of Tangshan Normal University, 156 Jianshe Road, Tangshan City, Hebei Province, P.R. China.
3School of Public Health, North China University of Science and Technology, 21 Bohai Road, Tangshan City, Hebei Province, P.R. China.

ABSTRACT

Background: Coccidiosis, caused by Eimeria species, significantly impacts the poultry industry. Resistance to residues of conventional anticoccidials have emerged. Novel efforts are needed to address the limitations of current treatment. The aim of this study was to evaluate the anticoccidial activity of chestnut bur tannic acid extracts (CTE) in Eimeria tenella-infected chickens.

Methods: 105 one-day-old Lifeng broiler chickens were randomly categorized into 5 groups (n = 21) and each group included 3 replicates (n = 7 per replicate). The groups were as follows: CON (uninfected-unmedicated), ET (infected-unmedicated), DIC (infected-medicated with 1 mg/L diclazuril), CTE-5 (infected-medicated with 5 mg/L CTE), CTE-10 (infected-medicated with 10 mg/L CTE). Clinical signs, survival rate, oocyst output, body weight gain and lesion scores were documented. The anticoccidial activity of CTE was assessed by anticoccidial index (ACI), antioxidant parameters and histopathologic examination of the cecum.

Result: The results demonstrated that 5mg/L CTE addition in drinking water of E. tenella-infected broiler chickens improved growth and survival rate, decreased oocyst production and cecal lesion scores, with the corresponding ACI of 163.70. Additionally, the use of CTE alleviated the pathological changes in the cecum. Moreover, CTE counteracted the E. tenella-induced loss of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) and controlled the increase of malondialdehyde (MDA), exhibiting the antioxidant function. Based on our results, CTE may be a promising candidate as anticoccidial agent.

INTRODUCTION

Eimeria tenella (E. tenella), as the most pathogenic agent of chicken coccidiosis, can result in decreased growth rate, reduced food intake, diarrhea, whitish droppings, inflammation and poor mental state (Jia et al., 2023; Fu et al., 2021). E. tenella causes an estimated annual loss of approximately ≤10.4 billion annually worldwide and poses a significant challenge in poultry industry (Blake et al., 2020). The application of anticoccidial chemicals and live oocyst vaccines is the major way to prevent and control coccidiosis. However, the limitations of these approaches hinder their wide application. Most of the anticoccidiosis drugs in the market cause some subsequent troubles, such as antibiotic resistance and drug residues (Sobral et al., 2020). Moreover, the application of live oocyst vaccines has the risk of virulence reversion and pathogen dissemination, further raises safety concerns (Xiang et al., 2024; Liu et al., 2013). Therefore, exploring new antiparasitic strategies is essential.
       
Feed additives, such as plant-based products, have beneficial properties, such as antioxidant, antimicrobial, antifungal, immunomodulatory and anticoccidial effects (Desalegn and Ahmed, 2020). Numerous studies have reported that phytochemicals have anticoccidial properties with lesser or no side effects (Alem, 2024). Chestnut (Castanea mollissima blume), is widely cultivated and the output had reached more than 1.9 million tons annually by 2020 in China (Peng et al., 2023). Chestnut burs, as one of by-products, are usually discarded or burned as industrial waste upon harvesting. The reuse of chestnut burs can reduce the impact of agro-industrial processes on the environment and economy, further obtaining new functional products. It has been reported that chestnut burs have abundant bioactive compounds, such as polyphenols, tannins and flavonoids, which have been known as its antioxidant, antimicrobial and anti-inflammatory potentials (Rodrigues et al., 2023; Vella et al., 2018). Tannins, as the polyphenolic compounds, show beneficial pharmacological effects including antioxidant activity, anti-cancer, antimicrobial, anti-nematodes, cardio-protective properties, the regulation of metabolic disorders and the prevention of several oxidative stress-related diseases (Smeriglio et al., 2017; Atiba et al., 2021). Recent studies suggest that tannic acids have antiparasitic, especially anticoccidial potential (Zargar et al., 2017; Molnar et al., 2024; Muthamilselvan et al., 2016). Tonda et al., (2018) found that supplementation of diets with gallnut tannic acid extract alleviated the adverse effects of Eimeria. However, the effect of the chestnut bur tannins in Eimeria-infected chickens has not been elucidated. Our research was schemed to assess the anticoccidial effect of the CTE in broiler chickens.

MATERIALS AND METHODS

E. tenella oocyst
 
E. tenella wild strain was isolated from Changchun in China and was donated by Key Laboratory of Zoonosis Research of Jilin University. The oocysts were passaged in broiler chickens, isolated from fresh faecal samples, sporulated at 28°C with ventilation for 48 h and preserved in 2.5% potassium dichromate.
 
Animals and experimental design
 
This experiment was carried out at Laboratory Animal Center, North China University of Science and Technology and Tangshan Normal University. The research was conducted from April to November 2023. One hundred and five one-day-old Lifeng broiler unsexed chickens obtained from Shandong Yisheng Livestock and Poultry breeding company (Tangshan, China) were used in this study. During the study, chickens were reared under free access to water and a commercial diet without coccidiostat additives. The anticoccidial vaccines were not allowed. The recommended temperature was maintained with the help of electric radiators and ventilation fans. The animal experiments had passed scrutiny of the Ethics Committee of North China University of Science and Technology (No. 2022-SY-055).
       
A preliminary pilot project was conducted to evaluate the appropriate dose of CTE. Based on the results of pilot project, the dose ≤10 mg/L in drinking water which could reduce the oocyst number was standardised. The 105 seven-day-old chickens were randomly allocated into 5 groups (n = 21) as the following: CON: uninfected-unmedicated; ET: infected-unmedicated; DIC: infected-medicated with 1 mg/L diclazuril supplementation; CTE-5: infected-medicated with 5 mg/L CTE supplementation; CTE-10: infected-medicated with 10 mg/L CTE supplementation. Each group was subdivided into 3 replications (n = 7). CTE were extracted using 20% absolute ethanol at a solid to solvent ratio of 1:40 (w/v), subsequently ultrasound (35 kHz) for 30 min and a 90°C water bath for 3 h. A centrifugation at 5000 rpm for 10 min was used to obtain the supernatant. The extracted supernatant was collected and then evaporated under vacuum at 50°C. After the concentration, the extract was lyophilized at -40°C and finally stored at room temperature for further research. The content of total tannic acid in the dry extract was 34.39%, which was determined using the Folin-Denis spectrophotometric method (Shahzad et al., 2022).

At 7th day of age, diclazuril and CTE were administered in drinking water for 14 consecutive days. At 14th day of age, all chickens excluding CON group were orally inoculated with 1 mL suspension of 50000 sporulated oocysts of E. tenella. Chickens in CON group received 1 mL PBS alone. Before infection, feces from all groups were examined to confirm the coccidia-free status of the broiler birds used in the study. All chickens were weighed and their weights were recorded. At 21st day of age, after weighing, blood samples were aspired from heart. All chickens were sacrificed by neck dislocation. Cecum tissues were quickly collected for hematoxylin and eosin (H and E) staining.
 
Anticoccidial test
 
The cecum samples were opened and its contents were collected. Levels 0 to 4 based on the degree of intestinal wall hemorrhage and thickening and bloody cecal contents were used to record cecal lesions as previously described (Johnson and Reid, 1970). Lesion score = the average lesion score in each group × 10.
       
The McMaster method was used to calculate the number of oocysts per gram (OPG) of cecal contents as previously described (Hodgson, 1970).

 
Oocyst value was determined by oocyst ratio. Oocyst value was defined as 0, 5, 10, 20 and 40 according to an oocyst ratio of 0-1%, 1-25%, 26-50%, 51-75% and 76-100%, respectively.
       
The following equations were used to calculate survival rate, body weight gains (BWG) rate and relative body weight gain (rBWG).





 
Anticoccidial index (ACI) was applied to evaluate the anticoccidial ability of CTE.
ACI = (survival rate + relative rate of weight gain) - (lesion score + oocyst value).
       
An ACI ≥180 was considered to be highly effective; 160 ≤ ACI < 180 was considered moderately effective; 120 ≤ ACI <160 was considered limited effective; ACI <120 was considered ineffective (Yong et al., 2020).

Histopathological examination
 
The cecums were fixed in 4% buffered formaldehyde solution for 24 h, then dehydrated in ascending ethanol concentrations (70%, 80%, 90% and 100%), cleared by xylene and embedded by paraffin at 60°C. The thickness of tissue section was 4 μm approximately. The histopathological changes were observed by staining tissue sections with H and E.
 
Antioxidant biomarkers
 
Serum was collected by incubating the blood at room temperature for 30 min, followed by a centrifugation at 2500 rpm for 15 min. The levels of antioxidant biomarkers (T-SOD, GSH-Px, MDA) in serum were detected according to the instructions in test kits (Nanjing Jiancheng Biological Engineering Research Institute).
 
Statistical analysis
 
Data was analyzed by One-way ANOVA and significant differences between various groups were determined by Tukey multiple comparisons using the SPSS software (version 19, USA). p<0.05 was considered a statistically significant difference.

RESULTS AND DISCUSSION

Anticoccidial activity of CTE
 
No deaths occurred except for ET group. In CON group, no bloody droppings were observed and all members were healthy. Severe clinical features of coccidiosis including bloody droppings, fatigue, decreased appetite, emaciation and fluffy feathers were displayed in ET group after 5 days of infection. In contrast, no obvious abnormalities were observed in CTE-5 and DIC groups during the experimental period. In CTE-10 group signs of weakness, inappetence and weight loss, rough feathers and bloody droppings in were observed which were slightly lesser than the signs observed in the ET group birds.
       
As shown in Table 1, the BWG in ET and CTE-10 groups was significantly lower than those the other groups (p<0.05). Chickens in CTE-5 and DIC groups showed a significant increase in BWG when compared with ET group (p<0.05), with the best BWG and rBWG in DIC group. Cecal lesions of all infected groups were observed and the degree of damage in CTE-5 and DIC groups was lower than other infected groups. When compared to the ET group with an ACI of 77.31, a reduction in oocyst production in CTE-5 and DIC groups was observed and the ACI was 163.70 and 176.57, respectively. The ACI was the overall parameter in the evaluation of the anticoccidial potential. The 5 mg/L CTE supplementation with an ACI of 163.70 showed better anticoccidial potential.

Table 1: Anticoccidial effects of CTE against E. tenella (n = 21).


       
Diclazuril, as a triazine chemical compound, is one of the widely used assays in coccidiosis controlling. Diclazuril affects different stages of Eimeria spp., especially the first-generation schizont, second-generation schizont and gamont stages and its mechanism of action is quite complex (Ahmadi et al., 2022). Diclazuril may hinder the synthesis of amylopectin abundantly existing in the cell walls of the schizonts of E. tenella. Therefore, the second-generation schizogony was severely affected and the merozoite formation was impeded, thereby preventing the development of E. tenella (Verheyen et al., 1988; Maes et al., 1988). Research reported diclazuril could downregulate the expression of enolase and microneme genes which played crucial roles in parasite invasion, immunoregulation and energy metabolism in second-generation merozoites of E. tenella (Zhou et al., 2020; Zhou et al., 2010). In this study, diclazuril was used as a criterion to evaluate the anticoccidial efficacy of CTE. According to the ACI, the anticoccidial effects of 5 mg/L CTE (ACI = 163.70) was slightly lower than that of diclazuril (ACI = 176.57), that may indicate differences in the mechanism of action.
       
The precise anticoccidial mechanism of tannins remains poorly understood. The tannins may penetrate the oocyst wall and inhibit endogenous enzymes that play important role in oocyst sporulation (Molan et al., 2009). Choi et al., (2022) reported that an appropriate dose of tannic acid improved gut barrier integrity, activated the immune system and exhibited defensive effects against E. maxima infection in broilers. Another study also indicated tannins strengthened the humoral immune responses and provided protection against coccidiosis (Kaleem et al., 2014). Further studies for exploring the anticoccidial mechanism of CTE are warranted. Taking into account the advantages of CTE (e.g., minimum side effects, low cost, wide source and a relatively good protection), the potential synergistic effects of CTE use in association with diclazuril to control avian coccidiosis should be concerned.
       
The ACI in CTE-10 group was 117.72, which indicated no therapeutic effect of 10 mg/L CTE on chicken coccidiosis. Choi et al., (2022) revealed anti-nutritional effects and cytotoxicity of high concentrations of TA (5000 mg/kg TA) with reduced growth, increased of gut permeability and decreased nutrient digestibility in E. maxima-infected broilers. In currrent study, high dosage of CTE might have precipitated protein and inhibit digestive enzymes, interfering with the growth, reducing the anticoccidial potential, consistent with the results of previous study (Choi et al., 2022). Hence, it is essential to investigate the safety of CTE for long-term use in chickens.
 
Histopathological observations
 
Histopathologically, the structure of the cecums in CON group appeared intact, showing no oocyst and no obvious lesions in mucosal epithelial cells and intestinal crypts. (Fig 1a). In ET group, chickens suffered heavy infestation with different coccidial stages (developing schizonts and macrogametes) in the enterocytes and intestinal crypts. The structure of mucosa was observed to be severely damaged. The mucosa and submucosa showed hemorrhages and infiltration with inflammatory cells (Fig 1b). These findings concurred with those of Zhou et al., (2010) and Cheng et al., (2023). In DIC group, no obvious damage was seen, only limited numbers of developing schizonts were observed in the enterocytes and intestinal crypts (Fig 1c). Similarly, in CTE-5 group, the cecum possessed normal morphology and coccidia were markedly reduced in the intestinal crypts (Fig 1d). In CTE-10 group, there was an increased number of different coccidial stages (developing schizonts, macrogametes and oocyst with oocyst wall) and inflammatory cells infiltration in the mucosa and submucosa was observed (Fig 1e). By comparing the histopathological changes, we concluded that CTE can reduce the number of oocysts in cecal mucosa. The reduction in parasite load decreased the inflammatory response alleviating the E. tenella-induced cecal injury. Goblet cells, the products of stem cells differentiation in the intestinal crypts, played a vital role in defense against pathogens (Zhao et al., 2024; Knoop and Newberry, 2018). The increased proportion of goblet cells in DIC, CTE-5 and CTE-10 groups was observed in this study. The capacity of parasites to invade the epithelial cells could be affected by the change of goblet cells (Alajmi et al., 2023). The increase of goblet cells suggest that CTE may have a significant role in barrier maintenance, which is beneficial for prevention of coccidiosis.

Fig 1: Histopathological lesions in chicken cecum.


 
Antioxidant parameters
 
The T-SOD and GSH-Px activities in ET group were significantly reduced, while the content of MDA significantly increased (p<0.05) than the CON group. The antioxidant biomarkers in CTE-5 and DIC groups showed significantly improvement when compared with ET group (p<0.05). The CTE-10 group, which received 10 mg/L CTE showed no significant difference in biomarker levels compared to ET group (p>0.05) (Table 2). According to the previous research, tannic acid had a certain antioxidant effect (Amarowicz and Pegg, 2024). The coccidial infection can destroy the balance between the body’s antioxidant defense system and its release of free radicals, further induced tissue damage caused by oxidative stress and lipid peroxidation (Murshed et al., 2023; El-Shall et al., 2022). The levels of antioxidant enzymes (T-SOD and GSH-Px) reflect the ability of eliminating free radicals. Lipid oxidation produces MDA, which reflect the severity of the body’s attack by oxygen radicals (Cheng et al., 2023; Alsulami et al., 2023). Increased T-SOD and GSH-Px and decreased MDA indicated reduction in E. tenella infection induced oxidative stress. Reduced T-SOD and GSH-Px and increased MDA indicated more free radicals production. The DIC and CTE-5 groups showed increased activities of T-SOD and GSH-Px and reduced MDA contents, indicating improved antioxidant potential. However, CTE at high dosage of 10 mg/L may cause damage to antioxidant system and couldn’t alleviate oxidative stress effectively, which was consistent with the previous report (Choi et al., 2022). The results indicated that CTE supplementation in drinking water may alleviate the damage caused by free radicals, restore their antioxidant capacity and resist the E. tenella infection.

Table 2: Antioxidant effects of CTE in E. tenella-infected broiler chickens (n = 21).


       
This research demonstrated the anticoccidial potential of CTE. The data provide a basis for CTE application in the prevention and control of coccidiosis. The limitations, such as a relatively small sample size, the lack of information on the chemical composition of CTE, should be dealt with in future studies. Guo et al., (2023) reported that hydrolyzed gallotannin strengthened the intestinal physical barrier in broilers by upregulating the gene expression of tight junction protein. We speculate that the improved intestinal barrier can resist the invasion of coccidia. More molecular biology research should focus on elaborating the anticoccidial mechanism of CTE, which will be beneficial for controlling coccidiosis precisely.

CONCLUSION

The present study confirmed the anticoccidial effect of CTE in E. tenella infected broiler chickens. The application of CTE, as a supplementary or alternative therapy to control avian coccidiosis, could potentially reduce reliance on chemical anticoccidials and contribute to a more sustainable poultry production system. More studies are needed to elucidate the anticoccidial mechanisms of CTE, evaluate its optimal concentration and clinical security and explore its efficacy against other Eimeria species.

ACKNOWLEDGEMENT

This work was supported by the Talent Funding Project of Tangshan, China (Grant No. C202303028), Science Research Project of Tangshan Normal University, China (Grant Nos. 2024B05 and 20266129063) and Hebei Provincial Department of Science and Technology Centrally Guided Local Development Fund Project, China (Grant No. 236Z7705G).
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by North China University of Science and Technology of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Chestnut Bur Tannins: A Novel Approach to Eimeria tenella Control

    N
    Na Zhao1
    https://orcid.org/0009-0002-3144-4766
    L
    Li-Zhong Wang2
    https://orcid.org/0009-0008-8469-1078
    H
    Hua-Wei Li1
    https://orcid.org/0000-0002-8024-9871
    L
    Li-Yun Chang1
    https://orcid.org/0000-0001-9104-9464
    C
    Cheng-Hui Li1
    https://orcid.org/0000-0002-7704-3610
    S
    Shuang Li3,*
    https://orcid.org./0009-0002-5752-7308
    1Faculty of Life Science, Tangshan Normal University, 156 Jianshe Road, Tangshan City, Hebei Province, P.R. China.
    2Ocean College of Tangshan Normal University, 156 Jianshe Road, Tangshan City, Hebei Province, P.R. China.
    3School of Public Health, North China University of Science and Technology, 21 Bohai Road, Tangshan City, Hebei Province, P.R. China.

    ABSTRACT

    Background: Coccidiosis, caused by Eimeria species, significantly impacts the poultry industry. Resistance to residues of conventional anticoccidials have emerged. Novel efforts are needed to address the limitations of current treatment. The aim of this study was to evaluate the anticoccidial activity of chestnut bur tannic acid extracts (CTE) in Eimeria tenella-infected chickens.

    Methods: 105 one-day-old Lifeng broiler chickens were randomly categorized into 5 groups (n = 21) and each group included 3 replicates (n = 7 per replicate). The groups were as follows: CON (uninfected-unmedicated), ET (infected-unmedicated), DIC (infected-medicated with 1 mg/L diclazuril), CTE-5 (infected-medicated with 5 mg/L CTE), CTE-10 (infected-medicated with 10 mg/L CTE). Clinical signs, survival rate, oocyst output, body weight gain and lesion scores were documented. The anticoccidial activity of CTE was assessed by anticoccidial index (ACI), antioxidant parameters and histopathologic examination of the cecum.

    Result: The results demonstrated that 5mg/L CTE addition in drinking water of E. tenella-infected broiler chickens improved growth and survival rate, decreased oocyst production and cecal lesion scores, with the corresponding ACI of 163.70. Additionally, the use of CTE alleviated the pathological changes in the cecum. Moreover, CTE counteracted the E. tenella-induced loss of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) and controlled the increase of malondialdehyde (MDA), exhibiting the antioxidant function. Based on our results, CTE may be a promising candidate as anticoccidial agent.

    INTRODUCTION

    Eimeria tenella (E. tenella), as the most pathogenic agent of chicken coccidiosis, can result in decreased growth rate, reduced food intake, diarrhea, whitish droppings, inflammation and poor mental state (Jia et al., 2023; Fu et al., 2021). E. tenella causes an estimated annual loss of approximately ≤10.4 billion annually worldwide and poses a significant challenge in poultry industry (Blake et al., 2020). The application of anticoccidial chemicals and live oocyst vaccines is the major way to prevent and control coccidiosis. However, the limitations of these approaches hinder their wide application. Most of the anticoccidiosis drugs in the market cause some subsequent troubles, such as antibiotic resistance and drug residues (Sobral et al., 2020). Moreover, the application of live oocyst vaccines has the risk of virulence reversion and pathogen dissemination, further raises safety concerns (Xiang et al., 2024; Liu et al., 2013). Therefore, exploring new antiparasitic strategies is essential.
           
    Feed additives, such as plant-based products, have beneficial properties, such as antioxidant, antimicrobial, antifungal, immunomodulatory and anticoccidial effects (Desalegn and Ahmed, 2020). Numerous studies have reported that phytochemicals have anticoccidial properties with lesser or no side effects (Alem, 2024). Chestnut (Castanea mollissima blume), is widely cultivated and the output had reached more than 1.9 million tons annually by 2020 in China (Peng et al., 2023). Chestnut burs, as one of by-products, are usually discarded or burned as industrial waste upon harvesting. The reuse of chestnut burs can reduce the impact of agro-industrial processes on the environment and economy, further obtaining new functional products. It has been reported that chestnut burs have abundant bioactive compounds, such as polyphenols, tannins and flavonoids, which have been known as its antioxidant, antimicrobial and anti-inflammatory potentials (Rodrigues et al., 2023; Vella et al., 2018). Tannins, as the polyphenolic compounds, show beneficial pharmacological effects including antioxidant activity, anti-cancer, antimicrobial, anti-nematodes, cardio-protective properties, the regulation of metabolic disorders and the prevention of several oxidative stress-related diseases (Smeriglio et al., 2017; Atiba et al., 2021). Recent studies suggest that tannic acids have antiparasitic, especially anticoccidial potential (Zargar et al., 2017; Molnar et al., 2024; Muthamilselvan et al., 2016). Tonda et al., (2018) found that supplementation of diets with gallnut tannic acid extract alleviated the adverse effects of Eimeria. However, the effect of the chestnut bur tannins in Eimeria-infected chickens has not been elucidated. Our research was schemed to assess the anticoccidial effect of the CTE in broiler chickens.

    MATERIALS AND METHODS

    E. tenella oocyst
     
    E. tenella wild strain was isolated from Changchun in China and was donated by Key Laboratory of Zoonosis Research of Jilin University. The oocysts were passaged in broiler chickens, isolated from fresh faecal samples, sporulated at 28°C with ventilation for 48 h and preserved in 2.5% potassium dichromate.
     
    Animals and experimental design
     
    This experiment was carried out at Laboratory Animal Center, North China University of Science and Technology and Tangshan Normal University. The research was conducted from April to November 2023. One hundred and five one-day-old Lifeng broiler unsexed chickens obtained from Shandong Yisheng Livestock and Poultry breeding company (Tangshan, China) were used in this study. During the study, chickens were reared under free access to water and a commercial diet without coccidiostat additives. The anticoccidial vaccines were not allowed. The recommended temperature was maintained with the help of electric radiators and ventilation fans. The animal experiments had passed scrutiny of the Ethics Committee of North China University of Science and Technology (No. 2022-SY-055).
           
    A preliminary pilot project was conducted to evaluate the appropriate dose of CTE. Based on the results of pilot project, the dose ≤10 mg/L in drinking water which could reduce the oocyst number was standardised. The 105 seven-day-old chickens were randomly allocated into 5 groups (n = 21) as the following: CON: uninfected-unmedicated; ET: infected-unmedicated; DIC: infected-medicated with 1 mg/L diclazuril supplementation; CTE-5: infected-medicated with 5 mg/L CTE supplementation; CTE-10: infected-medicated with 10 mg/L CTE supplementation. Each group was subdivided into 3 replications (n = 7). CTE were extracted using 20% absolute ethanol at a solid to solvent ratio of 1:40 (w/v), subsequently ultrasound (35 kHz) for 30 min and a 90°C water bath for 3 h. A centrifugation at 5000 rpm for 10 min was used to obtain the supernatant. The extracted supernatant was collected and then evaporated under vacuum at 50°C. After the concentration, the extract was lyophilized at -40°C and finally stored at room temperature for further research. The content of total tannic acid in the dry extract was 34.39%, which was determined using the Folin-Denis spectrophotometric method (Shahzad et al., 2022).

    At 7th day of age, diclazuril and CTE were administered in drinking water for 14 consecutive days. At 14th day of age, all chickens excluding CON group were orally inoculated with 1 mL suspension of 50000 sporulated oocysts of E. tenella. Chickens in CON group received 1 mL PBS alone. Before infection, feces from all groups were examined to confirm the coccidia-free status of the broiler birds used in the study. All chickens were weighed and their weights were recorded. At 21st day of age, after weighing, blood samples were aspired from heart. All chickens were sacrificed by neck dislocation. Cecum tissues were quickly collected for hematoxylin and eosin (H and E) staining.
     
    Anticoccidial test
     
    The cecum samples were opened and its contents were collected. Levels 0 to 4 based on the degree of intestinal wall hemorrhage and thickening and bloody cecal contents were used to record cecal lesions as previously described (Johnson and Reid, 1970). Lesion score = the average lesion score in each group × 10.
           
    The McMaster method was used to calculate the number of oocysts per gram (OPG) of cecal contents as previously described (Hodgson, 1970).

     
    Oocyst value was determined by oocyst ratio. Oocyst value was defined as 0, 5, 10, 20 and 40 according to an oocyst ratio of 0-1%, 1-25%, 26-50%, 51-75% and 76-100%, respectively.
           
    The following equations were used to calculate survival rate, body weight gains (BWG) rate and relative body weight gain (rBWG).





     
    Anticoccidial index (ACI) was applied to evaluate the anticoccidial ability of CTE.
    ACI = (survival rate + relative rate of weight gain) - (lesion score + oocyst value).
           
    An ACI ≥180 was considered to be highly effective; 160 ≤ ACI < 180 was considered moderately effective; 120 ≤ ACI <160 was considered limited effective; ACI <120 was considered ineffective (Yong et al., 2020).

    Histopathological examination
     
    The cecums were fixed in 4% buffered formaldehyde solution for 24 h, then dehydrated in ascending ethanol concentrations (70%, 80%, 90% and 100%), cleared by xylene and embedded by paraffin at 60°C. The thickness of tissue section was 4 μm approximately. The histopathological changes were observed by staining tissue sections with H and E.
     
    Antioxidant biomarkers
     
    Serum was collected by incubating the blood at room temperature for 30 min, followed by a centrifugation at 2500 rpm for 15 min. The levels of antioxidant biomarkers (T-SOD, GSH-Px, MDA) in serum were detected according to the instructions in test kits (Nanjing Jiancheng Biological Engineering Research Institute).
     
    Statistical analysis
     
    Data was analyzed by One-way ANOVA and significant differences between various groups were determined by Tukey multiple comparisons using the SPSS software (version 19, USA). p<0.05 was considered a statistically significant difference.

    RESULTS AND DISCUSSION

    Anticoccidial activity of CTE
     
    No deaths occurred except for ET group. In CON group, no bloody droppings were observed and all members were healthy. Severe clinical features of coccidiosis including bloody droppings, fatigue, decreased appetite, emaciation and fluffy feathers were displayed in ET group after 5 days of infection. In contrast, no obvious abnormalities were observed in CTE-5 and DIC groups during the experimental period. In CTE-10 group signs of weakness, inappetence and weight loss, rough feathers and bloody droppings in were observed which were slightly lesser than the signs observed in the ET group birds.
           
    As shown in Table 1, the BWG in ET and CTE-10 groups was significantly lower than those the other groups (p<0.05). Chickens in CTE-5 and DIC groups showed a significant increase in BWG when compared with ET group (p<0.05), with the best BWG and rBWG in DIC group. Cecal lesions of all infected groups were observed and the degree of damage in CTE-5 and DIC groups was lower than other infected groups. When compared to the ET group with an ACI of 77.31, a reduction in oocyst production in CTE-5 and DIC groups was observed and the ACI was 163.70 and 176.57, respectively. The ACI was the overall parameter in the evaluation of the anticoccidial potential. The 5 mg/L CTE supplementation with an ACI of 163.70 showed better anticoccidial potential.

    Table 1: Anticoccidial effects of CTE against E. tenella (n = 21).


           
    Diclazuril, as a triazine chemical compound, is one of the widely used assays in coccidiosis controlling. Diclazuril affects different stages of Eimeria spp., especially the first-generation schizont, second-generation schizont and gamont stages and its mechanism of action is quite complex (Ahmadi et al., 2022). Diclazuril may hinder the synthesis of amylopectin abundantly existing in the cell walls of the schizonts of E. tenella. Therefore, the second-generation schizogony was severely affected and the merozoite formation was impeded, thereby preventing the development of E. tenella (Verheyen et al., 1988; Maes et al., 1988). Research reported diclazuril could downregulate the expression of enolase and microneme genes which played crucial roles in parasite invasion, immunoregulation and energy metabolism in second-generation merozoites of E. tenella (Zhou et al., 2020; Zhou et al., 2010). In this study, diclazuril was used as a criterion to evaluate the anticoccidial efficacy of CTE. According to the ACI, the anticoccidial effects of 5 mg/L CTE (ACI = 163.70) was slightly lower than that of diclazuril (ACI = 176.57), that may indicate differences in the mechanism of action.
           
    The precise anticoccidial mechanism of tannins remains poorly understood. The tannins may penetrate the oocyst wall and inhibit endogenous enzymes that play important role in oocyst sporulation (Molan et al., 2009). Choi et al., (2022) reported that an appropriate dose of tannic acid improved gut barrier integrity, activated the immune system and exhibited defensive effects against E. maxima infection in broilers. Another study also indicated tannins strengthened the humoral immune responses and provided protection against coccidiosis (Kaleem et al., 2014). Further studies for exploring the anticoccidial mechanism of CTE are warranted. Taking into account the advantages of CTE (e.g., minimum side effects, low cost, wide source and a relatively good protection), the potential synergistic effects of CTE use in association with diclazuril to control avian coccidiosis should be concerned.
           
    The ACI in CTE-10 group was 117.72, which indicated no therapeutic effect of 10 mg/L CTE on chicken coccidiosis. Choi et al., (2022) revealed anti-nutritional effects and cytotoxicity of high concentrations of TA (5000 mg/kg TA) with reduced growth, increased of gut permeability and decreased nutrient digestibility in E. maxima-infected broilers. In currrent study, high dosage of CTE might have precipitated protein and inhibit digestive enzymes, interfering with the growth, reducing the anticoccidial potential, consistent with the results of previous study (Choi et al., 2022). Hence, it is essential to investigate the safety of CTE for long-term use in chickens.
     
    Histopathological observations
     
    Histopathologically, the structure of the cecums in CON group appeared intact, showing no oocyst and no obvious lesions in mucosal epithelial cells and intestinal crypts. (Fig 1a). In ET group, chickens suffered heavy infestation with different coccidial stages (developing schizonts and macrogametes) in the enterocytes and intestinal crypts. The structure of mucosa was observed to be severely damaged. The mucosa and submucosa showed hemorrhages and infiltration with inflammatory cells (Fig 1b). These findings concurred with those of Zhou et al., (2010) and Cheng et al., (2023). In DIC group, no obvious damage was seen, only limited numbers of developing schizonts were observed in the enterocytes and intestinal crypts (Fig 1c). Similarly, in CTE-5 group, the cecum possessed normal morphology and coccidia were markedly reduced in the intestinal crypts (Fig 1d). In CTE-10 group, there was an increased number of different coccidial stages (developing schizonts, macrogametes and oocyst with oocyst wall) and inflammatory cells infiltration in the mucosa and submucosa was observed (Fig 1e). By comparing the histopathological changes, we concluded that CTE can reduce the number of oocysts in cecal mucosa. The reduction in parasite load decreased the inflammatory response alleviating the E. tenella-induced cecal injury. Goblet cells, the products of stem cells differentiation in the intestinal crypts, played a vital role in defense against pathogens (Zhao et al., 2024; Knoop and Newberry, 2018). The increased proportion of goblet cells in DIC, CTE-5 and CTE-10 groups was observed in this study. The capacity of parasites to invade the epithelial cells could be affected by the change of goblet cells (Alajmi et al., 2023). The increase of goblet cells suggest that CTE may have a significant role in barrier maintenance, which is beneficial for prevention of coccidiosis.

    Fig 1: Histopathological lesions in chicken cecum.


     
    Antioxidant parameters
     
    The T-SOD and GSH-Px activities in ET group were significantly reduced, while the content of MDA significantly increased (p<0.05) than the CON group. The antioxidant biomarkers in CTE-5 and DIC groups showed significantly improvement when compared with ET group (p<0.05). The CTE-10 group, which received 10 mg/L CTE showed no significant difference in biomarker levels compared to ET group (p>0.05) (Table 2). According to the previous research, tannic acid had a certain antioxidant effect (Amarowicz and Pegg, 2024). The coccidial infection can destroy the balance between the body’s antioxidant defense system and its release of free radicals, further induced tissue damage caused by oxidative stress and lipid peroxidation (Murshed et al., 2023; El-Shall et al., 2022). The levels of antioxidant enzymes (T-SOD and GSH-Px) reflect the ability of eliminating free radicals. Lipid oxidation produces MDA, which reflect the severity of the body’s attack by oxygen radicals (Cheng et al., 2023; Alsulami et al., 2023). Increased T-SOD and GSH-Px and decreased MDA indicated reduction in E. tenella infection induced oxidative stress. Reduced T-SOD and GSH-Px and increased MDA indicated more free radicals production. The DIC and CTE-5 groups showed increased activities of T-SOD and GSH-Px and reduced MDA contents, indicating improved antioxidant potential. However, CTE at high dosage of 10 mg/L may cause damage to antioxidant system and couldn’t alleviate oxidative stress effectively, which was consistent with the previous report (Choi et al., 2022). The results indicated that CTE supplementation in drinking water may alleviate the damage caused by free radicals, restore their antioxidant capacity and resist the E. tenella infection.

    Table 2: Antioxidant effects of CTE in E. tenella-infected broiler chickens (n = 21).


           
    This research demonstrated the anticoccidial potential of CTE. The data provide a basis for CTE application in the prevention and control of coccidiosis. The limitations, such as a relatively small sample size, the lack of information on the chemical composition of CTE, should be dealt with in future studies. Guo et al., (2023) reported that hydrolyzed gallotannin strengthened the intestinal physical barrier in broilers by upregulating the gene expression of tight junction protein. We speculate that the improved intestinal barrier can resist the invasion of coccidia. More molecular biology research should focus on elaborating the anticoccidial mechanism of CTE, which will be beneficial for controlling coccidiosis precisely.

    CONCLUSION

    The present study confirmed the anticoccidial effect of CTE in E. tenella infected broiler chickens. The application of CTE, as a supplementary or alternative therapy to control avian coccidiosis, could potentially reduce reliance on chemical anticoccidials and contribute to a more sustainable poultry production system. More studies are needed to elucidate the anticoccidial mechanisms of CTE, evaluate its optimal concentration and clinical security and explore its efficacy against other Eimeria species.

    ACKNOWLEDGEMENT

    This work was supported by the Talent Funding Project of Tangshan, China (Grant No. C202303028), Science Research Project of Tangshan Normal University, China (Grant Nos. 2024B05 and 20266129063) and Hebei Provincial Department of Science and Technology Centrally Guided Local Development Fund Project, China (Grant No. 236Z7705G).
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by North China University of Science and Technology of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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