Protective Effect of Litchi chinensis Peel Extract-Prepared Nanoparticles on Rabbits Experimentally Infected with Eimeria stiedae

Simple Summary The purpose of this study was to determine how effective nanoparticles made from Litchi chinensis peel extract are at treating rabbit hepatic coccidiosis. Thirty-five rabbits were separated into seven groups: infected, healthy, pretreated rabbits infected after two weeks of treatment with 10 mg/kg L. chinensis peel extract-biosynthesized AgNPs and post-treated rabbits infected before treatment with 50 mg/kg amprolium. The findings showed that L. chinensis peel extract-biosynthesized AgNPs are a novel and safe therapy for Eimeria stiedae infection in rabbits. Abstract The present study used Litchi chinensis peel extract to synthesize silver nanoparticles (AgNPs). This technique is eco-friendly and can be performed in a single step; thus, it has attracted great attention for NPs biosynthesis. Herein, we biosynthesized AgNPs with L. chinensis peel extract and examined their anticoccidial activity in rabbit hepatic coccidiosis induced by E. stiedae infection. Thirty-five rabbits were allocated into seven groups: a healthy group (G1), an infected control group (G2), four groups infected before treatment with 10 mg/kg L. chinensis peel extract-biosynthesized AgNPs (G3, G5) or 50 mg/kg amprolium (G4, G6), and rabbits infected after two weeks of pretreatment with 10 mg/kg L. chinensis eel extract-biosynthesized AgNPs (G7). In this study, both pre-and post-treatment with AgNPs produced a substantial reduction in fecal oocyst output, liver enzyme levels, and histopathological hepatic lesions relative to the infected group. In conclusion, L. chinensis peel extract-prepared AgNPs should be considered harmless and efficient in the cure of hepatic coccidiosis in rabbits.


Introduction
Eimeria stiedae is one of the most pathogenic coccidian protozoans in domestic rabbits (Oryctolagus cuniculus), causing severe hepatic coccidiosis, a reduced growth rate, decreased feed conversion, and increased mortality [1][2][3][4][5][6]. The limitations of certain chemical drugs and weight promoters have increased digestive disorders and the occurrence of death in rabbits. However, many herbs are known to have anticoccidial and antioxidant effects in rabbits and can reduce their consumption of chemicals [7].
Upon postmortem examination, rabbits with hepatic coccidiosis display milky spots on the hepatic surface and throughout the parenchyma and an enlarged gall bladder.

Plant Material and Extract Preparation
Three kilograms of fresh L. chinensis Sonn fruit was purchased from a supermarket in Riyadh, Saudi Arabia. The Department of Botany and Microbiology, College of Science, Helwan University, Cairo, Egypt, validated the plant identification. To remove dust particles, the fruit was rinsed numerous times with deionized water. Then, L. chinensis Sonn peel extract was produced using the standard technique described in [25].

Total Phenolics
The total phenolic content in the L. chinensis extracts was determined using the Folin-Ciocalteu technique as previously described by Abdel Moneim [26]. In brief, 0.1 mL of sample extract was combined with 2.5 mL of distilled water in a test tube, and then 0.1 mL of undiluted Folin-Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) was added. After thoroughly mixing the solution, it was allowed to stand for 6 min before adding 0.5 mL of a 20% sodium carbonate solution. The solution was equilibrated for 30 min at room temperature (20 • C) to develop the color, and the absorbance of the solution was measured at 760 nm with a spectrophotometer (PD 303 UV spectrophotometer, Apel Co., Limited, Saitama, Japan). A blank sample was made using 0.1 mL of methanol instead of the extract. The measured value was compared to a calibration curve built with gallic acid solutions, and the results are given as mg gallic acid per gram of dry weight extract.

Total Flavonoids
The total flavonoid content in the L. chinensis extract was determined using the aluminum chloride colorimetric method published by Abdel Moneim [26]. Fifty microliters of the extract were combined with 4 mL of distilled water in a test tube, followed by 0.3 mL of 5% NaNO 2 solution and 0.3 mL of 10% AlCl 3 ·6H 2 O. After 6 min, 2 mL of 1 mol/L NaOH was added to the mixture, and the total volume of the solution was raised to 10 mL using distilled water. After standing for another 15 min, the absorbance at 510 nm was measured. The total flavonoid content was calculated using a calibration curve and is reported as mg rutin per gram dry weight.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Activity
The ability of the L. chinensis extract to scavenge DPPH radicals was determined using Karakaya and Akillioglu's technique [27]. Fresh DPPH radical solution (0.08 mM) in methanol was produced, and 950 mL of DPPH solution was combined with 50 mL of extract and incubated for 5 min. Five minutes later, the absorbance of the mixture was measured at 515 nm (PD 303 UV spectrophotometer, Apel Co., Limited, Saitama, Japan). The antioxidant activity (AA) is expressed as the percent suppression of DPPH radicals using the equation AA = 100 -(100(A sample/A control)), where A sample is the absorbance of the sample at t = 5 min, and A control is the absorbance of the control. The ABTS + test was carried out using the Gouveia and Castilho method [16]. Fifty milliliters of 2 mM ABTS + solution was mixed with 200 L of 70 mM potassium persulfate solution to make the ABTS + radical solution. This mixture was stored in the dark for 16 hours at room temperature and was stable for two days. The ABTS + solution was diluted with pH 7.4 phosphate-buffered saline (PBS) for each analysis to an initial absorbance of 0.700-0.021 at 734 nm. For each series of analyses, this solution was freshly produced. An aliquot of 100 L of the methanolic solution was combined with 1.8 mL of ABTS + solution to assess the radical scavenging activity, and the decrease in absorbance at 734 nm (PD 303 UV spectrophotometer, Apel Co., Limited, Saitama, Japan) was recorded over 6 min. As determined using a Trolox calibration curve, the results are presented as mol Trolox equivalents per gram of dried extract (mol Trolox/g).

Ferric Reducing Antioxidant Power (FRAP)
The FRAP assay was carried out according to Abdel Moneim's procedure [28]. The FRAP reagent was composed of 300 mM acetate buffer with a pH of 3.6, 10 mM 2,4,6-tris(2-pyridyl)s-triazine (TPTZ) in 40 mM HCl and 20 mM FeCl 3 in a 10:1:1 (v/v/v) ratio. In a test tube, three milliliters of the FRAP reagent were combined well with 100 L of L. chinensis extract and shaken at 37 • C for 30 min in a water bath. After 4 min of reduction of ferric-TPTZ to the ferrous complex, the absorbance was measured with a UV-vis spectrophotometer (PD 303 UV spectrophotometer, Apel Co., Limited, Saitama, Japan) at 593 nm. The results are given as mol Trolox per gram of dried material (mol Trolox/g).

Synthesis of Silver Nanoparticles (AgNPs)
Crude L. chinensis peel extract (0.25 g) was mixed thoroughly with 50 mL of distilled water. To optimize AgNPs synthesis in the pH range of 5-7, an aliquot of 0.1 mM silver nitrate solution was added. The mean size of the silver NPs was determined using a Zetasizer (Nano series, HT Laser, ZEN 3600, Malvern Instruments, Malvern, UK), whereas transmission electron microscopy (TEM; JEM-1011, JEOL, Tokyo, Japan) with an accelerating voltage of 100 kV was used to determine the shape, size, and morphology of the synthesized AgNPs.

Biochemical Analysis
At 28th day PI, blood samples (n = 7) were obtained from each rabbit's ear veins. Colorimetric measurement of liver enzyme markers was performed on the separated serum samples using commercially available kits (Sigma-Aldrich Co. St. Louis, MO, USA) according to the manufacturer's instructions. Serum AST and ALT levels were determined using the method established by Reithman and Frankel in 1957 [29].

Experimental Design
Thirty-five domestic rabbits were separated into 7 groups of 5 rabbits each ( Table 1). The first rabbit group (G1) served as the negative control group; these rabbits were neither infected nor treated. The rabbits in the second group (G2) were orally inoculated with 5 × 10 4 oocysts of a local field isolate of E. stiedae per rabbit and served as the infected untreated positive control group. The rabbits in the third group (G3) were infected with the same dose of E. stiedae per rabbit as the G2 rabbits, but they were also given 10 mg/kg BW of the AgNPs biosynthesized with L. chinensis [30]. Treatment began on day 10 PI and lasted three days, followed by four days of relaxation, three days of treatment, and ten days of daily fecal testing. The rabbits in the fourth group (G4) received the same dose of E. stiedae per rabbit as the G2 rabbits, but they were also given amprolium at a concentration of 50 mg/kg BW. Treatment began on day 10 PI and lasted three days, followed by four days of relaxation, three days of treatment, and ten days of daily fecal testing. The rabbits in the fifth group (G5) were infected and treated with AgNPs biosynthesized with L. chinensis [31] in the same doses as G3 rabbits, but treatment began on day 18 PI and followed the same protocol. The rabbits in the sixth group (G6) were infected and treated with amprolium in the same doses as G4 rabbits, but treatment began on day 18 PI and followed the same protocol. The rabbits in the seventh group (G7) were infected and treated with AgNPs biosynthesized with L. chinensis in the same doses as G3 rabbits, but treatment was administered for three days beginning on the 14th day before infection and continued for 28 days PI. All groups were monitored daily for clinical symptoms and death. The mean BW and BWG were measured at 0 and 28 days PI. At 0, 21, and 28 days PI, feces were collected to determine the number of oocysts per gram (OPG). Blood samples were taken from the rabbits (n = 5) at 28 days PI for hematological analysis. Furthermore, rabbits from the experimental groups (n = 5) were euthanized for liver weight measurements, relative liver weight calculations, and gross liver lesion assessment. In addition, liver tissues were taken for histological analysis. All rabbits were humanely terminated at the end of the experiment using CO 2mediated oxygen deprivation. All parameters were compared across the seven groups.

Experimental Infection of Rabbits with Isolated and Identified E. stiedae
The E. stiedae field strain was obtained from naturally infected rabbits with uneven yellowish-white, milky spots distributed on their liver tissue. The oocysts were collected, measured for concentration, purified, and sporulated using the flotation procedure previously reported [32]. Sporulated oocysts were kept at 4 • C in potassium dichromate (2.5%).
The rabbits were infected by administering the suspension orally through a stomach tube at a dose of 5 × 10 4 oocysts for each rabbit [33]. As the most essential means of evaluating product efficacy, the rabbits were evaluated twice daily PI until the trial ended (28 days PI) to record the primary clinical symptoms, Animals 2022, 12, 3098 6 of 17 postmortem lesions, and mortality rates associated with the hepatic coccidiosis, and these parameters were compared across all the groups.

Body Weight (BW) and Relative Liver Weights (RLW)
The average BWs of the rabbits were calculated at the beginning (0 days PI) and the end of the trial (28 days PI). At 28 days PI, the body weight gain (BWG) was also calculated. The average BW of the rabbits in each group was determined on day 0 before experimental infection. The rabbit weights were used to calculate the average BW and average BWG in each group by subtracting the BW value at 0 days PI from the equivalent value at 28 days PI [33][34][35]. Furthermore, the livers from the euthanized rabbits (n = 5 per group) were obtained for weighing and determining relative liver weights, as previously described by Gómez-Bautista et al. [34].

Gross Liver Lesion Scores
To measure the success of the herbal product treatment, the severity of each gross liver lesion caused by E. stiedae infection was scored as described by Peeters and Geermos [35]. The scores ranging from 0 to +4 were assigned.

Fecal Oocyst Count (FOC)
The fecal samples were obtained from infected rabbits for the detection of Eimeria oocysts at the start of the experiment (0 days PI) and 21 to 28 days PI. On each chosen day, 10 freshly expelled fecal pellets were deposited into a collection bag (one for each group) and weighed in the morning (10:00). The flotation technique was used to concentrate the samples, and the number of Eimeria oocysts was counted. The sensitive McMaster method [36,37] was used to determine the oocysts per gram (OPG) in the feces, and the oocyst reduction % was calculated [38] as follows: Reduction% = OPG of the infected group − OPG of the treated group/OPG of the infected group × 100 2.12.5. Histopathology and Lesion Scoring At 28 days PI, the liver tissues (n = 5 per group) were taken for histological evaluation. Following good practices, the samples were examined blindly. The tissues were fixed in 10% formaldehyde and stained with hematoxylin and eosin (H&E) on paraffin-embedded sections [39]. The slides and photos were captured using a Nikon microscope at a magnification of 400×. The liver tissue sections from all experimental groups were examined histopathologically and scored for lesions. The scores ranging from 0 to +5 were assigned.

Statistical Analysis
The collected growth performance data; fecal oocyst counts; and biochemical, antioxidant, and hematological parameter values were analyzed using F tests and the SPSS software program (v. 20.0, IBM SPSS Inc., Chicago, IL, USA) to produce mean values, standard errors, and p values [40]. The histological liver lesion scores were statistically analyzed using one-way analysis of variance (ANOVA), followed by Tukey's multiple comparison test for pairwise comparisons. In the experimental model, each experimental group was compared to the control group and to every other group.

Determinations of the Total Phenolic and Flavonoid Contents in the L. chinensis Extract
The total phenolic and flavonoid contents present in the investigated extract are shown in (Table 2) and were found to be 10.457 ± 0.843 mg gallic acid/g and 0.819 ± 0.045 mg rutin/g, respectively. Furthermore, the results revealed that the extract has potent free radical scavenging activity. The results from the DPPH, ABTS and FRAP assays were 39.91 ± 1.86, 6.887 ± 0.053, and 0.375 ± 0.0033 µmol Trolox/g, respectively. Table 2. Experimental determinations of the total phenolic and flavonoid contents in the L. chinensis extract and the results from the antioxidant capacity assays (ABTS, DPPH, and FRAP).

Parameter
Mean ± SD Total phenolics (mg eq. gallic acid/g sample) 10.457 ± 0.843 Total flavonoids (mg eq. rutin/g sample) 0.819 ± 0.045 DPPH (%) 39.91 ± 1.86 ABTS (µmol eq. Trolox/g sample) 6.887 ± 0.053 FAB (µmol eq. Trolox/g sample) 0.375 ± 0.003 Figure 1 depicts the average AgNPs size as calculated by dynamic light scattering with Zetasizer equipment (ZEN 3600, Malvern, UK). The size distribution profile of L. chinensis with AgNPs was determined using this technique, and the size was determined to be 91. 38 nm. This result demonstrated that the NPs had a homogeneous size distribution and variation without agglomeration, as evidenced by the emergence of a single peak. Furthermore, the TEM image ( Figure 2) revealed that the majority of the AgNPs had a spherical morphology. The color of the AgNPs in the aqueous solution did not change indicating their stability.
Animals 2022, 12, x FOR PEER REVIEW Figure 1 depicts the average AgNPs size as calculated by dynamic light sca with Zetasizer equipment (ZEN 3600, Malvern, UK). The size distribution profi chinensis with AgNPs was determined using this technique, and the size was dete to be 91. 38 nm. This result demonstrated that the NPS had a homogeneous size d tion and variation without agglomeration, as evidenced by the emergence of a sing Furthermore, the TEM image ( Figure 2) revealed that the majority of the AgNP spherical morphology. The color of the AgNPs in the aqueous solution did not indicating their stability.

Effects of the Different Treatments on Clinical Symptoms, Postmortem Lesions, and Mortality Rate
Throughout the experiment, there were no clinical symptoms in the rabbits in the control (G1) and infected treated (G3-G7) groups; the animals ate well and showed no evidence of diarrhea. The rabbits in the infected untreated group (G2) showed various clinical symptoms at 28 days PI including decreased appetite, and rough coat (Figure 3a), and diarrhea (Figure 3b). Postmortem examination revealed no identifiable gross lesions in the rabbits in the control group (G1). The livers of untreated infected rabbits (G2) were substantially enlarged, with dilated bile ducts and yellow to white colored nodules of different sizes on the liver surface (Figure 3c). There were no macroscopic lesions on the livers of the rabbits in the infected treatment groups (G3-G7).

Effects of the Different Treatments on Clinical Symptoms, Postmortem Lesions, and Mortality Rate
Throughout the experiment, there were no clinical symptoms in the rabbits in the control (G1) and infected treated (G3-G7) groups; the animals ate well and showed no evidence of diarrhea. The rabbits in the infected untreated group (G2) showed various clinical symptoms at 28 days PI including decreased appetite, and rough coat (Figure 3a), and diarrhea (Figure 3b). Postmortem examination revealed no identifiable gross lesions in the rabbits in the control group (G1). The livers of untreated infected rabbits (G2) were substantially enlarged, with dilated bile ducts and yellow to white colored nodules of different sizes on the liver surface (Figure 3c). There were no macroscopic lesions on the livers of the rabbits in the infected treatment groups (G3-G7).

Effects of the Different Treatments on Body Weight and Relative Liver Weight
Table 3 reveals significant differences between the infected group and the other experimental groups. The mean oocyst value was significantly higher on all days in this group, and the mean number of oocysts was 110.40 ± 1.72 on day 28 (p ≤ 0.05). While the experimental groups did not differ significantly from the control group or each other, AgNPs treatment on day 10 had the lowest mean values, and the mean number of oocysts was smaller (0.40 ± 0.24) (99.64%) on day 28. The oocyte reduction mean values were significantly higher on days 27 and 28 in each experimental group, and the mean value on all days was the highest in the protected group compared with those in the other experi-

Effects of the Different Treatments on Body Weight and Relative Liver Weight
Table 3 reveals significant differences between the infected group and the other experimental groups. The mean oocyst value was significantly higher on all days in this group, and the mean number of oocysts was 110.40 ± 1.72 on day 28 (p ≤ 0.05). While the experimental groups did not differ significantly from the control group or each other, AgNPs treatment on day 10 had the lowest mean values, and the mean number of oocysts was smaller (0.40 ± 0.24) (99.64%) on day 28. The oocyte reduction mean values were significantly higher on days 27 and 28 in each experimental group, and the mean value on all days was the highest in the protected group compared with those in the other experimental groups. Except for days 27 and 28, the mean oocyst reduction values ranged from 85.77 ± 1.13 (83.69%) on the 21st day in the amprolium group and on day 18 to 100.00 (100%) on all days in the protected group. Table 3. Effects of the different treatments on the oocyst count/gram feces (OPG) and reduction percent (%) in rabbits infected with E. stiedae.      Table 4 shows that there were no significant differences in BW on the first day and relative liver weight (p ≤ 0.05) between the experimental treatments. However, the remaining parameters showed substantial variations depending on the experimental treatment. Treatment with AgNPs on day 10 was shown to be the best experimental treatment, with the greatest mean BW on day 28 (1934.00 ± 42.50) and BWG (626.00 ± 45.56). Treatment with AgNPs on day 10 also showed an average liver weight (64.56 ± 0.85) that did not differ from that in the other experimental treatment groups. The mean liver weight was significantly higher in the infected group (105.10 ± 0.55), followed by the experimental groups, and finally the negative control group (35.30 ± 0.26). Table 4 also shows no significant differences between the means of the three aforementioned traits (BW on day 28, BWG, and liver weight) in all of the experimental treatment groups except for the AgNPs on the day 10 treatment group at p ≤ 0.05. Table 4. Effects of the different treatments on the mean body weight (BW), body weight gain (BWG), liver weight (LW), and relative liver weight (RLW) of the normal, infected, and treated rabbits. The mean RLW percentage values revealed a significant difference between the infected group, the negative control group, and the other experimental groups. The mean value of the RLW percentage was significantly higher in the infected group and significantly lower in the negative control group.

Treatment BW (g) Day 1 BW (g) Day 28 BWG (g) LW (g) RLW (g) (%)
Concerning the lesion score, there was a significant difference in lesion score between all treatment groups and the negative control group and between the protected group and all experimental groups, except the infected group. On the other hand, there was no significant difference between the lesion score in the infected group and those in all treatment groups, except the protected group. Gross lesion trait analysis confirmed that treatment with AgNPs on day 10 was the best treatment (2.4 ± 1.5), as this value differed significantly (p ≤ 0.05) from that in the infected group (35.0 ± 14.35). Although the gross lesion results of the rabbits receiving NPs treatment on day 10 did not differ significantly from the results of the rabbits receiving the other experimental treatments, they had the lowest average (Table 5).

Effects of the Different Treatments on the Histopathological Lesions
The samples from the negative control group (G1) showed normal hepatocyte, portal area, and bile duct tissue structures on day 28 PI (Figure 4a). The samples from the infected untreated group (G2) showed different E. stiedae developmental stages (macrogametocytes, microgametocytes, and oocysts beginning to form) posterior to the nuclei of the epithelial cells, and moderate eosinophilia in subepithelial cells was also observed (Figure 4b,c). Lesions of the rabbits infected with E. stiedae and treated with AgNPs biosynthesized with L. chinensis at a dose of 10 mg/kg BW on day 10 PI (G3) were examined before and after treatment. Before treatment, the early developmental stages of E. stiedae (possibly early schizonts) posterior to the nuclei of the epithelial cells (along with some that were anterior to the nuclei), eosinophilia in the subepithelial cells, and a healthy epithelial lining (Figure 4d) were observed. After treatment, the hyperplasia of some epithelial cells and no E. stiedae developmental stages are present (Figure 4e). Moreover, the lesions of rabbits treated with amprolium (50 mg/kg BW) on day 10 PI were observed. Before treatment, infiltration by various developmental stages of E. stiedae (possibly trophozoites) posterior to the nuclei, desquamation of some epithelial cells, and eosinophilic infiltration in the subepithelial cells was found (Figure 4f). After treatment, a few E. stiedae trophozoites were present posterior to the nuclei of the epithelial cells, and eosinophils were noted (Figure 4g). In addition, the different developmental stages of E. stiedae were completely absent from the examined tissues of the treated rabbits with AgNPs on day 18 PI (G5) and a small amount of hyperplasia (Figure 4h). The G6 rabbits showed few E. stiedae trophozoites posterior to the nuclei and little eosinophilic infiltration into the subepithelial cells and healthy epithelial cells (Figure 4i). The G7 rabbits displayed few eosinophilic infiltrations in the subepithelial cells and healthy epithelial cells (Figure 4j). Therefore, significantly more severe histopathological hepatic lesions were observed in G2 than in G1 and G3-G7.
in the subepithelial cells was found (Figure 4f). After treatment, a few E. stiedae trophozo-373 ites were present posterior to the nuclei of the epithelial cells, and eosinophils were noted 374 (Figure 4g). In addition, the different developmental stages of E. stiedae were completely 375 absent from the examined tissues of the treated rabbits with AgNPs on day 18 PI (G5) and 376 a small amount of hyperplasia (Figure 4h). The G6 rabbits showed few E. stiedae tropho-377 zoites posterior to the nuclei and little eosinophilic infiltration into the subepithelial cells 378 and healthy epithelial cells (Figure 4i). The G7 rabbits displayed few eosinophilic infiltra-379 tions in the subepithelial cells and healthy epithelial cells (Figure 4j). Therefore, signifi-380 cantly more severe histopathological hepatic lesions were observed in G2 than in G1, G3-381 G7.

Effects of the Different Treatments on the Biochemical and Hematological Parameters
E. stiedae infection caused rabbit hepatotoxicity, which was indicated by elevated activity of serum ALT and AST, whereas supplementation with the green AgNPs yielded a significant (p < 0.05) decrease in the levels of these enzymes and restored them to the control values, as shown in ( Figure 5). Interestingly, our data indicate that these AgNPs biosynthesized with L. chinensis were more effective than amprolium in both treatment groups.

Discussion
Green synthesis nanoparticles are of significant concern because of their wide-ranging usage in nanomedicine, as they show antioxidant, antiparasitic, and anti-inflammatory actions once synthesized by green technologies in the size range of 1-100 nanometers. A distinguishing feature of nanoparticle synthesis using plants is called (photosynthesis), because it has a higher rate of nanoparticle formation and consists of a diverse range of biomolecules such as (poly) phenolic and flavonoid compounds [41,42].
The plant species of Litchi extract was a source for the synthesis of (AgNPs) in this

Discussion
Green synthesis nanoparticles are of significant concern because of their wide-ranging usage in nanomedicine, as they show antioxidant, antiparasitic, and anti-inflammatory actions once synthesized by green technologies in the size range of 1-100 nanometers. A distinguishing feature of nanoparticle synthesis using plants is called (photosynthesis), because it has a higher rate of nanoparticle formation and consists of a diverse range of biomolecules such as poly phenolic and flavonoid compounds [41,42].
The plant species of Litchi extract was a source for the synthesis of AgNPs in this study, as evidenced by the change in color and the stability of the solution. The TEM analysis revealed that the particle size range was approximately 100 nm and that they were spherical in shape. The poly phenolic and flavonoid compounds in the Litchi extract, as well as other constituents, act as surface-active stabilizing molecules for the synthesis of AgNPs with antioxidative and anti-parasitic properties.
The current work examined how well AgNPs biosynthesized with L. chinensis controlled hepatic coccidiosis in rabbits infected with E. stiedae. The oocysts were given to the rabbits in their drinking water 7 days before the treatment, which lasted 28 days. The treatment with AgNPs biosynthesized with L. chinensis efficiently suppressed E. stiedae infection in rabbits in this study and enhanced development and liver function. When given continuously before and during infection, AgNPs biosynthesized with L. chinensis reduced the clinical, parasitological, and production-related effects of the experimental hepatic coccidiosis. On the 28th day of PI, the rabbits infected with E. stiedae developed a variety of clinical symptoms including a lack of appetite, diarrhea, and emaciation. The infected rabbit livers were also substantially enlarged, with dilated bile ducts and yellow to white-colored nodules of varying sizes on the surface. Cam et al. [3], Al-Mathal [4], and Eladl et al. [33] observed varied white nodules on the liver surface of E. stiedae-infected rabbits and reported similar clinical symptoms and postmortem lesions. Disease symptoms and postmortem lesions were absent in rabbits treated with AgNPs biosynthesized using L. chinensis in this investigation.
Disease symptoms were absent in the treated rabbits in our study, and BW increased, similar to the negative control group, indicating recovery from E. stiedae infection. This effect could be attributed to the composition of L. chinensis, as herbal extracts have been shown to increase food intake and BWG. The lack of obvious liver lesions in the group treated with AgNPs with L. chinensis suggests that the herbal ingredients were highly effective. Similarly, Singh et al. [43] established that an ethanolic extract of Aegle marmelos Linn leaves stimulates a natural immunological response. Eladl et al. [33] also observed a significant decrease in oocyst output and the elimination of fecal oocysts in rabbits treated with Herba Cox®. E. stiedae oocysts were found in rabbit feces 21-28 days after infection. Hassan et al. [44] discovered E. stiedae in feces for the first time at 18 days PI, and the maximum OPG in feces was found between 17 and 21 days PI [31]. The reduction in the oocyst yield and the disappearance of fecal oocysts in the treated rabbits in this study may have been due to the anticoccidial effects of the AgNPs biosynthesized with L. chinensis, which may have reduced the growth and development of E. stiedae stages, resulting in a reduction in oocyst formation and fecal shedding.
The hepatotoxicity induced by E. stiedae is the most commonly used model system for screening the hepatoprotective activity of plant extracts/drugs. Infection with E. stiedae induced significant increases in serum AST and ALT levels, which reflect the severity of liver injury [28,45]. These marker enzymes are cytoplasmic in origin and are released into circulation after cellular damage [28,46]. A rise in the AST level is usually accompanied by an elevation in the level of ALT, which plays a vital role in the conversion of amino acids to keto acids [46]. The leakage of large quantities of enzymes into the bloodstream is associated with centrilobular necrosis and ballooning degeneration of the liver. However, the increased levels of these enzymes were significantly decreased by treatment with the L. chinensis-generated AgNPs, implying that the green AgNPs prevented liver damage; this conclusion was confirmed by the reduced number of histopathological injuries.
Seddiek and Metwally [38] reported similar results, demonstrating that rabbits administered Nigella sativa seed oil had considerable decreases in liver enzyme levels.

Conclusions
Our findings suggest that AgNPs biosynthesized from L. chinensis peel extract could be used to create a new candidate therapeutic agent with greater efficacy in suppressing hepatic coccidiosis in rabbits by modulating body weight, liver weight dynamics, and decreasing fecal oocyst output, liver enzyme levels, and histopathological hepatic lesions. Our findings show that these AgNPs biosynthesized with L. chinensis were more effective than amprolium.

Limitations
The obvious limitations are the small sample size, the lack of controls on extract or AgNPs alone, and the need to detect mediated immunity (T cells, B cells, and other cells) to determine the efficacy of these treatments.