Lonicera japonica Flos as a Natural Anticoccidial Agent Against Eimeria tenella: In Vivo Efficacy and Compositional Insights

Abstract

This study aimed to evaluate the potential of Lonicera japonica Flos (LJF) as an alternative agent against Eimeria tenella (E. tenella) in chickens and to conduct phytochemical analysis to obtain compositional insights. Seventy-two one-day-old chickens were allocated to six groups and fed diets supplemented with LJF powder (LJFp) at three concentrations (LJFp-L, LJFp-M, and LJFp-H) or maduramicin (MDM) or two non-supplemented control diets, namely, an infected unmedicated control (IUC) and an uninfected unmedicated control (UUC). Dietary treatments were initiated at chick arrival (Day 0) and continued for 28 days. At 21 days of age, all groups except the UUC group were orally challenged with a field isolate of E. tenella (PT-Te003; 2.0 × 10⁴ oocysts/bird). Anticoccidial efficacy was assessed using the lesion score (LS), oocysts per gram of feces (OPG), relative body weight gain (rBWG), and anticoccidial index (ACI). The results demonstrated that all LJFp treatment groups had significantly reduced cecal OPG and LS (all LJFp treatments: p < 0.05 vs. IUC), indicating the effective suppression of E. tenella replication and intestinal damage. Regarding growth performance, the rBWG values of the LJFp-L and LJFp-M groups were the highest and comparable to those of the UUC group, showing no significant differences. In contrast, the LJFp-H and MDM groups exhibited significantly lower values (p < 0.05). Based on ACI evaluation, all LJFp-treated groups exhibited moderate to partial efficacy (LJFp-L > LJFp-M > LJFp-H), while MDM showed limited effectiveness. A gas chromatography–mass spectrometry (GC-MS) analysis of the LJFp ethanol extract revealed 15 essential oils, 10 organic acids, and three other compound classes, several of which have been associated with anticoccidial activity. Overall, the in vivo results suggest that LJF may exert potential anticoccidial effects against a field isolate of E. tenella. Phytochemical analysis provided preliminary compositional insights, and further studies are warranted to optimize extraction methods and evaluate efficacy at lower concentrations under additional in vitro and in vivo conditions. However, the current evidence remains insufficient to determine whether the field isolate exhibits reduced sensitivity to commercially available anticoccidial drugs, and additional studies are needed to clarify this issue.

1. Introduction

Chicken coccidiosis, caused by Eimeria spp., is the most significant protozoal disease in poultry industries worldwide [1,2]. Chickens are a major global protein source [3]; however, their production efficiency is compromised by Eimeria spp., which reduce growth and increase control costs [4,5]. These effects collectively threaten food security and compromise sustainability and economic viability.

The effective control of coccidiosis remains challenging, as seven distinct Eimeria species with differing virulence levels have been identified on chicken farms [6]. Understanding the parasite’s biology is crucial for effective control [5,7]. The Eimeria life cycle consists of three sequential phases: sporogony (an exogenous phase), merogony (an asexual reproduction phase), and gametogony (a sexual reproduction phase) [7]. Sporogony occurs outside the host, where unsporulated oocysts that are shed in feces develop into sporulated oocysts under suitable temperature, oxygen, and humidity conditions. Once sporulated oocysts are ingested, digestive processes release sporozoites that invade intestinal epithelial cells, initiating merogony that proceeds through at least two cycles of schizont and merozoite development [8]. In gametogony, merozoites differentiate into gametes, and fertilization yields zygotes that develop into thick-walled oocysts, which are excreted to initiate sporogony and complete the cycle [5]. Environmental oocyst persistence ensures continuous transmission and disease maintenance in chicken flocks [1].

Eimeria tenella (E. tenella) exhibits the highest prevalence worldwide among the seven Eimeria species identified in chickens, with infection rates over 90% in certain areas [9,10,11,12]. The high pathogenicity of this species, combined with the persistence of resistant oocysts in the environment and efficient fecal–oral transmission, results in recurrent outbreaks that pose a significant threat to intensive poultry industries [5]. The pathogenesis of E. tenella involves cecal epithelial invasion, barrier disruption, and inflammatory responses, causing hemorrhage and potentially death [5,13]. The consistent disease patterns caused by E. tenella infection make it a reliable model for testing new therapeutic treatments [8].

Currently, the prolonged or improper use of antibiotics and chemotherapeutic anticoccidials has imposed strong selective pressure on Eimeria populations, facilitating the emergence and spread of drug-resistant strains and reducing treatment efficacy [14]. In response to the global shift toward antibiotic-free poultry production [3], several medicinal herbs have been tested, showing efficacy in controlling coccidiosis, reducing mortality, and improving growth performance, highlighting their potential as alternative control measures [6,13,15]. When integrated with vaccination programs, these herbal products may help mitigate issues related to drug resistance and residue contamination [16,17].

Among these natural alternatives, Lonicera japonica (LJ), a member of the family Caprifoliaceae, is a perennial, semi-evergreen climbing shrub that is broadly distributed throughout East Asia [18,19]. Its dried flowers, Lonicera japonica Flos (LJF), characterized by their fragrant white blossoms that turn pale yellow over time, have long been used to treat infectious diseases [18,20]. The whole LJ plant contains over 140 bioactive compounds, including essential oils, organic acids, flavones, iridoid glycosides, and saponins [18,19]. While the plant is rich in bioactive constituents, the specific chemical composition of LJF extracts varies considerably depending on extraction methods, which can affect their bioactive profiles [19]. The chemical diversity of these compounds supports LJF’s anti-inflammatory, antioxidant, antiviral, and antibacterial effects, with extraction methods affecting their chemical profiles [18].

This study was conducted to address the knowledge gap regarding the anticoccidial potential of LJF against E. tenella through phytochemical characterization and in vivo efficacy evaluation. Dietary LJF powder (LJFp) was administered to E. tenella-infected chickens, and its effects were assessed by measuring cecal lesion scores (LSs), oocysts per gram of feces (OPG), relative body weight gain (rBWG), mortality, and the anticoccidial index (ACI). In parallel, gas chromatography–mass spectrometry (GC–MS) analysis was performed to identify the chemical constituents of LJF that may contribute to its anticoccidial bioactivity. This integrated approach evaluated LJF as an alternative anticoccidial agent against E. tenella field isolates while providing compositional insights for future mechanistic investigations.

2. Materials and Methods

2.1. Oocyst Preparation

The E. tenella isolate (designated as PT-Te003) was originally obtained from a commercial broiler farm in Pingtung, Taiwan, and was maintained in our laboratory by serial passage. For challenge infection, oocysts were prepared by inoculating chickens with the stock isolate, and fecal samples were collected 6–7 days post-infection (dpi), following the method described by Molan et al. (2009) with some modification [21]. Briefly, the fecal samples were filtered through a 60-mesh sieve (250 μm), subjected to flotation separation using saturated sodium chloride solution, and centrifuged (KUBOTA Model 4000, Tokyo, Japan) at 340× g for 10 min. After separation, the supernatant containing oocysts was collected, washed with distilled water to remove residual flotation solution, and subsequently washed three times with PBS. Oocysts were then resuspended in 2.5% potassium dichromate solution and incubated at room temperature for 24–48 h with constant aeration. Oocyst counts and quality were assessed using the McMaster method and molecular techniques [6,22], and the purified sporulated oocysts were stored in 2.5% potassium dichromate at 4 °C until use.

2.2. Lonicera japonica Flos Powder Preparation

Freshly dried Lonicera japonica flowers and buds, excluding the calyx, were obtained from a local medicinal herb farm and initially processed on-site to achieve a uniform particle size distribution. The flower materials were subsequently ground in the laboratory using high-speed grinders (RT-04A, Shin-Kwang Machinary Industry Co., Taichung, Taiwan) at 25,000 rpm and passed through a 60-mesh sieve. The resulting powder, with an average particle size of less than 250 μm, was stored at room temperature in sealed containers prior to experimental use.

2.3. Ethics of Animal Experimentation

All in vivo procedures complied with the ARRIVE 2.0 guidelines [23] and were approved by the Institutional Animal Care and Use Committee of National Pingtung University of Science and Technology, Taiwan (IACUC; NPUST-106-009, approval date: 16 June 2017). Animal health and welfare parameters were routinely monitored and recorded by a licensed veterinarian to mitigate potential clinical signs, distress, or mortality during the experiment, particularly after Eimeria infection.

2.4. In Vivo Experimental Design

Seventy-two one-day-old, coccidia-free layer chickens were purchased from a commercial Hendrix breeder farm (Yong Kong Farms, Tainan, Taiwan). The breeder flock was maintained under strict biosecurity, and chicks were hatched in a biosecure facility; neither breeders nor chicks were vaccinated against coccidiosis. On the day of hatching, chicks were transported in disinfected containers to a clean, isolated experimental chicken house and randomly allocated to six groups, with four replicate cages per group (three birds per cage). The entire trial was conducted under high-level biosecurity isolation with strict procedural monitoring.

The six groups included three given different doses of LJFp (LJFp-H, 50,000 mg/kg; LJFp-M, 10,000 mg/kg; LJFp-L, 5000 mg/kg), a maduramicin-supplemented group (MDM, 6 mg/kg; China Chemical & Pharmaceutical Co., New Taipei City, Taiwan), an infected unmedicated control (IUC), and an uninfected unmedicated control (UUC). Dietary treatments began upon chick arrival (Day 0) and continued throughout the 28-day experimental period. All birds had ad libitum access to feed and water. No antibiotics or anticoccidial agents were administered during rearing except for LJFp or MDM in the respective treatment groups. Prior to infection, fecal samples from each group were examined microscopically to confirm the absence of field oocyst contamination and cross-contamination among groups.

At 21 days old (D21), the chickens in all the groups except the UUC were subjected to an oral E. tenella challenge (PT-Te003, 2.0 × 10⁴ oocysts/bird). Treatment efficacy was evaluated using cecal LS, rBWG, OPG, survival rate, and ACI parameters. The growth of each group was evaluated by measuring body weight (BW, g) or body weight gain (BWG, g) and calculating rBWG. rBWG (n = 4, each cage comprising 3 birds) was calculated based on BW measurements at D21 and 28 days old (D28) in all groups using the following formula: rBWG = 100 × (BW at D28 − BW at D21)/BW at D21. Fecal samples were collected when the birds were 27 days old (D27) (6 dpi) to assess fecal oocyst shedding through OPG (n = 4, each cage comprising 3 birds) using the McMaster method [22]. At D28 (7 dpi), all chickens were euthanized via one-time electrical stunning for gross pathological examination, with the cecal LS (n = 12) determined according to Johnson and Reid’s scoring system (1970) [24]. The cecal LS was determined independently by two well-trained evaluators who were blinded to the treatment group assignments. The LS ranges from 0 to 4 on a scale with 4 indicating severe lesions or the death of the chicken.

The ACI was calculated as follows: ACI = [survival rate (%) + RWG (%) − (10 × lesion score + 0.4 × oocyst index) [6]. Survival rate (%) = [(total number of birds − number of dead birds)/total number of birds] × 100; RWG (%) = (average BWG_treated group/average BWG_UUC group) × 100; lesion score = mean lesion score per group; oocyst index (%) = (OPG_treated group/OPG_IUC group) × 100. Treatment effectiveness was categorized as very effective (>160), moderate (140–159), partial (120–139), or ineffective (<120) [6,25].

2.5. Chemical Characterization

The ethanol extract of LJF (with a solvent-to-sample ratio of 10:1) was analyzed via GC-MS (Shimadzu GCMS-QP2010, Kyoto, Japan) at National Sun Yat-Sen University, Taiwan. Volatile compounds were separated on a DB-WAX capillary column (29.3 m × 0.25 mm i.d., film thickness of 0.25 µm) using helium as the carrier gas at a constant flow rate of 1.49 mL/min and a linear velocity of 44.5 cm/s. The injector temperature was set to 200 °C, with a split ratio of 20:1 and an injection time of 0.75 min. The oven temperature was initially maintained at 40 °C for 1 min and then increased at 10 °C/min to 200 °C and held for 10 min, amounting to a total run time of 27 min. The mass spectrometer was operated in electron ionization (EI) mode at an ion source temperature of 200 °C and an interface temperature of 250 °C. Data were acquired in full-scan mode (Q3 scan). Compounds were tentatively identified based on the National Institute of Standards and Technology (NIST) Mass Spectral Library (NIST17-1) [26].

2.6. Statistical Analysis

Data were analyzed using IBM SPSS Statistics 27 (IBM, Armonk, NY, USA) with Shapiro–Wilk tests to determine parametric or non-parametric distribution. Parametric data underwent a one-way ANOVA with Tukey’s post hoc test, while non-parametric data used Kruskal–Wallis with Dunn’s procedure. Lesion scores, which represent ordinal data, were analyzed using non-parametric statistical methods. Significance was set at 95% confidence.

3. Results

3.1. Cecal Lesions

Following the E. tenella challenge, the UUC group showed no cecal abnormalities (score = 0), confirming the absence of environmental contamination, whereas the IUC group exhibited the most severe damage with a median LS of 4 (Figure 1). The MDM reference group showed a median LS of 3, which did not differ significantly from the IUC (p > 0.05), suggesting that maduramicin appeared to exhibit limited therapeutic efficacy against infection induced by E. tenella (PT-Te003). In contrast, all LJFp treatment groups demonstrated significant therapeutic efficacy compared to both the IUC and MDM groups: LJFp-H with median LS = 2, p = 0.001 vs. IUC, p = 0.029 vs. MDM; LJFp-M with median LS = 1.5, p < 0.001 vs. IUC, p = 0.017 vs. MDM; and LJFp-L with median LS = 1, p < 0.001 vs. IUC, p = 0.004 vs. MDM. No significant differences were observed among the three LJFp dosage groups, indicating that all tested concentrations were equally effective in mitigating cecal lesions caused by E. tenella.

3.2. Oocyst Shedding

No oocysts were detected in the UUC group, confirming the absence of Eimeria contamination during the experiment (Figure 2). The IUC group exhibited the highest infection level with a median of 3.55 × 10⁶ OPG. The MDM group showed a lower median oocyst count (2.03 × 10⁶ OPG), but the difference from the IUC was not statistically significant (p = 0.2513). In contrast, all LJFp treatment groups demonstrated significant reductions in oocyst shedding compared to the IUC: LJFp-H with 1.70 × 10⁶ OPG, p = 0.004; LJFp-M with 1.71 × 10⁶ OPG, p = 0.004; and LJFp-L with 1.76 × 10⁶ OPG, p = 0.014. No significant differences were observed among the three LJFp dosage groups or when compared to MDM (LJFp-H vs. MDM, p = 0.0786; LJFp-M vs. MDM, p = 0.0786; LJFp-L vs. MDM, p = 0.1936), indicating that all LJFp groups possess potent inhibitory activity against E. tenella replication comparable to or exceeding the reference anticoccidial.

3.3. Growth Performance

Following the E. tenella challenge, the IUC group showed a significant reduction in rBWG compared to the UUC group (median: 32.26 vs. 44.09; p = 0.012), confirming that infection adversely affected growth performance (Figure 3). Compared with the UUC group, the MDM and LJFp-H treatment groups exhibited significantly lower rBWG values (median: 32.38 vs. 44.09; p = 0.008 and median: 34.22 vs. 44.09; p = 0.048, respectively), whereas the LJFp-M and LJFp-L groups did not differ significantly from the UUC group (median: 34.8 vs. 44.09; p = 0.211 and median: 38.74 vs. 44.09; p = 0.510, respectively). However, none of the treatment groups showed significant differences compared with the IUC group (p > 0.05). Overall, following infection with E. tenella (PT-Te003), although the LJFp-L group exhibited significantly higher rBWG than the MDM group (median: 38.74 vs. 32.38; p = 0.048), rBWG values in all treatment groups remained numerically lower than those of the uninfected control birds.

3.4. Anticoccidial Index

The ACI values revealed differences between treatment groups. No mortality was observed throughout the experiment; therefore, the survival rate parameter in the ACI calculation was assigned a value of one hundred. The IUC (91.93) and MDM (106.96) groups showed poor anticoccidial performance, with ACI values below 120 (

Table 1. Evaluation of anticoccidial index after E. tenella challenge. This analysis evaluates anticoccidial effectiveness using ACI values as comprehensive parameters.

Group	Survival Rate	RWG	Lesion Score	Oocyst Index	ACI 1	Interpretation
IUC	100	67.53	3.56	100	91.93	ineffective
MDM	100	61	3	60.1	106.96	ineffective
LJFp-H	100	71.78	2.22	48.6	130.14	partial
LJFp-M	100	77.04	1.78	48.225	139.95	moderate
LJFp-L	100	81.54	1.56	49.475	146.15	moderate

Abbreviations: IUC, infected unmedicated control; MDM, maduramicin (6 mg/kg); LJFp-H, high-dose Lonicera japonica Flos powder (50,000 mg/kg); LJFp-M, medium-dose Lonicera japonica Flos powder (10,000 mg/kg); LJFp-L, low-dose Lonicera japonica Flos powder (5000 mg/kg); RWG, relative body weight gain; ACI, anticoccidial index. ACI = [survival rate + RWG − (10 × lesion score + 0.4 × oocyst index)] [6]. ¹ Very effective (ACI > 160), moderate (ACI: 140–159), partial (ACI: 120–139), or ineffective (ACI: <120).

). In contrast, all LJFp treatments achieved higher ACI values, with LJFp-L demonstrating the highest efficacy (146.15), followed by LJFp-M (139.95); both results indicated moderate anticoccidial efficacy. Meanwhile, LJFp-H exhibited partial anticoccidial activity (ACI = 130.14). Overall, the ACI results indicated that the anticoccidial efficacy of the three LJFp doses against E. tenella ranked, in descending order, as follows: LJFp-L, LJFp-M, and LJFp-H. In contrast, maduramicin, administered within the manufacturer-recommended dosage range, showed minimal efficacy.

3.5. Compositional Analysis

In the GC–MS analysis, a total of 31 peaks were detected in the LJFp ethanol extract following comparison with mass spectral databases (NIST17-1) (Figure 4). However, peaks 2, 10, and 14 were ascribed to siloxane-related contaminants originating from column bleed or exhibited poor mass spectral match quality and were therefore excluded from reliable compound identification. Consequently, 28 compounds were considered to be meaningfully identified.

The identified compounds were primarily composed of essential oils (15/28, 53.57%), organic acids (10/28, 35.71%), and other compound classes (3/28, 10.71%). Among these, linoleic acid (RT = 19.3 min) exhibited the highest signal intensity (TIC = 5.82 × 10⁶ counts) and was thus designated as the most abundant component, with its relative intensity set to 100%, followed by glycerol 1-palmitate (39.52%), linalool (37.80%), and caryophyllene oxide (34.36%). Detailed information on all identified compounds is summarized in

Table 2. The characterization of the compounds in the Lonicera japonica Flos ethanol extract identified via gas chromatography–mass spectrometry (GC–MS).

Major Class	Peak No a	Compound Name	Retention Time	Intensity	Rel. Int. (%) b	Subclass	Reference
Essential Oils	19	Trans-Farnesol (E,E-Farnesol)	18.5	1.00	17.18	Sesquiterpenes	[19]
	29	Nerolidol	22.8	0.98	16.84		[19]
	4	Caryophyllene oxide	10.0	2.00	34.36
	9	β-Caryophyllene	14.8	0.61	10.48		[27]
	16	Spathulenol	17.4	0.40	6.87		[19]
	22	Linalool	8.0	2.20	37.80	Monoterpenes	[19]
	6	α-Terpineol	12.2	0.20	3.44
	3	Linalool oxide	9.5	0.21	3.61		[18]
	8	Benzyl alcohol	13.7	0.80	13.75	Aromatic compounds
	25	Benzaldehyde	20.6	1.21	20.79		[18]
	12	Eugenol	16.6	0.38	6.53
	1	3-Hexenyl benzoate	8.6	0.41	7.04	Esters	[18]
	13	Methyl palmitate	16.8	0.92	15.81		[18]
	15	Ethyl palmitate	17.3	0.79	13.57
	20	Dihydroactinidiolide	18.7	0.22	3.78	Terpenoid degradation product
Organic Acids	5	Isovaleric acid	11.5	0.70	12.03	Short-chain organic acids	[18]
	7	Tiglic acid	13.3	0.82	14.09		[28]
	23	Lauric acid	20.3	0.81	13.92	Saturated fatty acids
	31	Myristic acid	25.0	0.70	12.03		[18]
	30	Palmitic acid	30.3	0.20	3.44		[19]
	21	Linoleic acid	19.3	5.82	100.00	Unsaturated fatty acids and their derivatives	[18,28]
	27	α-Linolenic acid	21.7	0.58	9.97
	26	Ethyl α-linolenate	21.2	0.21	3.61
	28	α-Linolenic acid	22.3	0.21	3.61		[19]
	24	Methyl α-linolenate	22.4	0.60	10.31		[19]
Other Compounds	18	Glycerol 1-palmitate	18.3	2.30	39.52	Fatty acid esters
	17	5-Hydroxymethylfurfural	17.7	0.90	15.46	Carbohydrate degradation products
	11	5-Methyluracil	16.3	0.30	5.15	Pyrimidine derivatives

^a Peaks 2, 10, and 14 are not presented in this table. These peaks correspond to siloxane contaminants from column bleed or that exhibited poor MS match quality, precluding reliable compound identification. ^b Relative intensity (Rel. Int. (%)) was calculated based on the most abundant peak (linoleic acid = 100%). These values represent peak intensities normalized to this base peak and do not reflect normalized area percentages relative to the total peak area.

. These results reflect the volatile and semi-volatile compositional profile of the LJFp ethanol extract under the analytical conditions applied.

4. Discussion

Widespread anticoccidial resistance on commercial chicken farms has increased the economic costs of coccidiosis and driven the search for sustainable control alternatives [1,29]. Among them, medicinal herbs present viable therapeutic options that align with modern chicken production methods and regulatory standards supporting antibiotic-free systems [30]. Building on this growing interest in natural compounds, this study aimed to provide a preliminary evaluation of LJFp’s anticoccidial activity against E. tenella, generating foundational data that may provide insight into strategies addressing emerging anticoccidial challenges [31].

In in vivo studies, the cecal LS and OPG are commonly used to evaluate the pathogenicity of E. tenella [32]. The LS mainly reflects the asexual stage, during which massive merogony leads to epithelial cell destruction, inflammation, hemorrhage, and mucosal erosion [5]. Therefore, a lower LS indicates reduced parasite replication and intestinal damage. In contrast, OPG primarily reflects the sexual stage; thus, a lower OPG value implies the suppression of parasite reproduction, which reduces reinfection intensity and transmission [6,33]. In this study, all three concentrations of LJFp significantly suppressed both the LS and OPG, suggesting that it has inhibitory effects on E. tenella pathogenicity. Based on previous reports indicating that LJF possesses antimicrobial and antiviral activities [18] and considering the life cycle of E. tenella, we hypothesize that LJF may directly compromise the structural integrity of sporozoites and merozoites, as these are the only extracellular stages in which LJF can be encountered in the intestinal lumen [5]. However, future studies should incorporate additional in vitro assays and histopathological examination to provide tissue-level or cellular-level evidence of intestinal pathology, as Eimeria replicates intracellularly within epithelial cells and internal mucosal damage cannot be fully captured by gross lesion scoring alone. Such analyses would help clarify whether LJF exerts direct parasiticidal effects or primarily attenuates host inflammatory responses.

The relative body weight gain in the untreated infected group was significantly lower than that in the uninfected control group, confirming that E. tenella infection, even at moderate levels, compromises digestive and absorptive functions. Interestingly, although the rBWG values in the LJFp-M and LJFp-L groups were not significantly higher than those in the IUC group, they were also not significantly lower than those in the UUC group. These findings may suggest that, at the two tested concentrations, LJF could partially mitigate the growth suppression associated with E. tenella infection. Such effects, if present, might be related to the reported antioxidant, anti-inflammatory, and gut microbiota-modulating properties of LJF [6,34]. Further investigations including larger-scale feed conversion trials are required to clarify the underlying anticoccidial mechanisms.

The LS, OPG, and rBWG are all reliable indicators of anticoccidial efficacy; however, discrepancies among these parameters are sometimes observed because of their distinct biological bases [35,36]. In this study, all three LJFp concentrations significantly reduced the LS and OPG compared with the IUC group [37], while rBWG was slightly higher in the LJFp-L and LJFp-M groups. However, no significant differences were found among the three LJFp doses for any single parameter, making it difficult to determine their relative efficacy.

In addition, the sample size determined in this study was in accordance with the 3R principles (replacement, reduction, and refinement) of animal ethics, aiming to achieve meaningful statistical insights while minimizing the number of experimental animals. Although the replicate structure provided sufficient data for analysis, the smaller sample size may reduce statistical power and result in less stable variance estimates, which should be considered when interpreting the findings. To provide a comprehensive and objective evaluation of anticoccidial efficacy, the anticoccidial index (ACI) was employed. Previous studies have established a standardized formula for the ACI [25,32], which inherently integrates survival rates, lesion scores, and relative weight gain to allow for an objective classification of anticoccidial efficacy. In this study, the ACI was determined following this conventional framework, utilizing RWG as the primary indicator of growth performance. This approach is widely recognized for providing a comprehensive and precise evaluation of anticoccidial performance, particularly in small-scale cage experiments where changes in weight gain can be captured more accurately without the complexities of measuring individual feed intake. Because body weight measurements were not systematically recorded, the feed conversion ratio (FCR) was not included in the present experimental design. Future studies incorporating large-scale trials are warranted to evaluate the FCR under field conditions. Such studies would provide a more definitive validation of anticoccidial efficacy in commercial poultry production and further clarify the potential of LJF as a viable alternative.

In this study, the calculated ACI values ranked in the following order: LJFp-L > LJFp-M > LJFp-H. The inclusion of a higher level (5%) was designed and intended to facilitate the evaluation of a potential dose, and the dosage range was established with reference to the experimental framework reported by Yang et al. (2015) [37]. The comparatively reduced efficacy observed in the high-dose group relative to the medium- and low-dose groups may partly reflect alterations in the overall nutritional characteristics of the diet at higher inclusion levels. Plant-derived powders typically contain complex mixtures of polysaccharides, fibers, and other bioactive constituents that may influence the physicochemical properties of intestinal digesta [19]. In particular, the increased dietary inclusion of plant materials may elevate digesta viscosity, which has been reported to interfere with effective interactions between digestive enzymes and substrates and consequently reduce nutrient digestion and absorption efficiency [38]. Such nutritional effects may partially offset the pharmacological benefits of the herbal additive at higher inclusion levels. Therefore, the relatively favorable responses observed in the low- and medium-dose groups may mainly reflect the pharmacological activity of LJF, whereas the response in the high-dose group may involve a combination of pharmacological and nutritional influences. Therefore, we propose that LJF at low and medium dose levels may exhibit anticoccidial potential, with these effects more likely attributable to pharmacological activity rather than nutritional interference.

Similar non-linear dose–response patterns have also been reported in other phytogenic studies. For instance, Kasem et al. (2019) observed that rosemary extract exerted maximal sporicidal inhibition at 0.001%, while the effect diminished at 1.0% (p < 0.05) [39]. Such unexpected patterns may also arise from LJF‘s complex molecular interactions involving antagonistic, synergistic, or competitive pharmacological mechanisms [18]. These findings highlight the need for further research to determine the optimal and economically feasible dosage.

Widespread resistance to conventional anticoccidial agents, including maduramicin, has been reported globally [40,41]. Consistent with these reports, the E. tenella (PT-Te003) used in this study exhibited a potential reduction in sensitivity to MDM, as suggested by the LS, OPG, rBWG, and ACI results. Although these phenotypic outcomes seem to align with resistance characteristics, the present study lacked other chemical drug positive controls and further validation, such as large-scale in vivo broiler FCR trials, histopathologic evidence, in vitro sporozoite invasion assays in Madin–Darby bovine kidney (MDBK) cells, or molecular verification via real-time PCR. Therefore, a conservative conclusion must be drawn that MDM treatment was relatively ineffective against PT-Te003 under the limited conditions of this study; additional research is warranted to definitively confirm its status as an MDM-resistant strain. Collectively, these preliminary findings may support the exploration of LJF as a potential alternative agent, consistent with global efforts to reduce chemical agents in poultry production.

In this study, the GC–MS analysis of the LJFp ethanol extract revealed a diverse range of constituents, primarily classified as essential oils and organic acids. Although this study did not directly investigate the specific biological activities or anticoccidial mechanisms of these individual components, their potential roles can be inferred from the previously reported literature. For instance, the most abundant compound, linoleic acid, has been documented to partially reduce oocyst shedding when supplemented in poultry feed [41]. Similarly, the second most abundant constituent, glycerol 1-palmitate, classified as a fatty acid ester, has not been directly linked to anticoccidial activity; however, non-ionic surfactant vesicles formulated with monopalmitoyl glycerol have been shown to suppress NF-κB transcript expression in lipopolysaccharide (LPS)-stimulated macrophages, indicating a potential anti-inflammatory effect [42]. The third most abundant compound, a citrus-derived essential oil (linalool), may interfere with parasite protease activity or modulate gut microbiota, thereby enhancing host resistance [43]. The fourth major compound, caryophyllene oxide, reported as a major and characteristic constituent of Korean LJF essential oil [44], is also a dominant component in several essential oils (Lippia alba, Aframomum sceptrum, and Toona sinensis) with documented antiparasitic activity against protozoa and the poultry red mite [27,28,45].

Additional essential oil components, including eugenol and nerolidol, have led to high anticoccidial index values [46,47] and improved body weight gain in infected birds [48]. Linalool may exert similar benefits by modulating parasite protease activity or gut microbiota, thereby reducing oxidative stress and intestinal inflammation [43]. Within the organic acid fraction, isovaleric acid has been reported to modulate gut microbiota composition, which may indirectly influence mucosal immune responses including IgA production [49]. Such immunomodulatory effects could potentially contribute to reduced Eimeria sporozoite invasion and oocyst output, although these mechanisms remain poorly explored in the current literature and warrant further investigation. Lauric acid has also shown antimicrobial activity, particularly against necrotic enteritis (NE)-associated pathogens that can synergize with Eimeria infection [50,51]. These bioactive constituents, identified by GC-MS, provide a phytochemical basis for the observed anticoccidial activity and warrant further mechanistic investigation.

The in vivo anticoccidial effects observed are likely attributable to synergistic interactions among multiple LJF constituents rather than a single dominant compound [52]. Combinations of organic acids and essential oils have been reported to achieve efficacy comparable to that of conventional anticoccidial drugs while improving weight gain and feed conversion [3]. GC–MS analysis provides insight mainly into volatile and semi-volatile constituents, and methodological differences such as extraction protocols and cultivation conditions may account for discrepancies with previous reports [17].

Although conventional coccidiosis control still relies on chemoprophylaxis and vaccination, interest in plant-derived alternatives has increased due to concerns over drug resistance [3,4,53]. Natural medicinal herbs have shown promising activity against resistant strains, and the present findings support the therapeutic potential of LJF as a plant-based candidate for sustainable coccidiosis control [3]. Further research should investigate solvent-based extracts of LJF, test across multiple Eimeria species and strains, and incorporate expanded in vitro and in vivo assays to clarify its mechanisms of action and validate its anticoccidial efficacy.

5. Conclusions

The findings of this study suggest that the whole flower of Lonicera japonica exhibits potential as a natural alternative agent against E. tenella infection in chickens. The in vivo results indicate that LJF may exert anticoccidial effects against a field isolate of E. tenella, while phytochemical analysis provides preliminary compositional insights. These results provide a scientific basis for the further exploration of Lonicera japonica Flos in the development of sustainable anticoccidial strategies, although further studies are warranted to optimize extraction methods, explore lower effective concentrations, incorporate systematic histopathological examination, achieve larger sample sizes with improved statistical power, record feed utilization measurements, and evaluate efficacy under additional in vitro and in vivo conditions. Moreover, given the limitations of the present study, the anticoccidial efficacy of high-dose (5.0%) LJF and the sensitivity of the field isolate toward conventional anticoccidial drugs cannot be fully characterized at this stage, necessitating further investigation.