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Full Research Article

Efficacy of Vitis vinifera Leaves Extracts against Eimeria stiedae Infections in New Zealand Rabbit

M
Mohammed M. Mares1,*
R
Rewaida Abdel-Gaber1
S
Saleh Al-Quraishy1
1Department of Zoology, College of Science, King Saud University, Riyadh 1145, Saudi Arabia.

ABSTRACT

Background: Coccidiosis is considered one of the most serious diseases affecting rabbits and various other animal species, leading to substantial economic losses worldwide. Plant-derived extracts have emerged as promising alternatives to conventional chemotherapeutic agents, particularly in light of increasing drug resistance, as well as their advantage of not causing harmful tissue residues.

Methods: This study was conducted to determine the effectiveness of Vitis vinifera leaf extract (VVLE) against Eimeria stiedae in rabbits. The extract was subjected to phytochemical analysis by GC-MS, revealing the existence of 34 biologically active compounds. A total of 35 male New Zealand rabbits were divided into seven groups. Group 1 served as the uninfected, untreated (negative control) group. Group 2 uninfected and treated at 400 mg/kg of extract to test for toxicity. Group 3 represented the infected, non-treated (positive control) group. While, groups 4, 5 and 6 comprised infected treated with at 100 mg/kg, 200 mg/kg and 400 mg/kg of extracts, respectively. Finally, group 7 consisted of infected rabbits treated with Toltrazuril at a dosage of 5 mg/kg.

Result: The findings indicate that 400 mg/kg concentration of VVLE has a significant protective effect against Eimeria stiedae. It relieves clinical symptoms, reduces oocyst production, improves biochemical parameters and decreases the level of iron (Fe), copper (Cu), magnesium (Mg) and zinc (Zn) in the liver of Eimeria stiedae-infected rabbits. This refers to the effectiveness of Vitis vinifera leaf extracts (VVLE) against coccidiosis. Further investigations are needed to identify the pharmacological ingredients responsible for these effects and their potential as a natural alternative for coccidia prevention in animal feed.

INTRODUCTION

Rabbit farming for meat has made a significant contribution to addressing the global meat shortage, playing an important role in supplying substantial quantities of meat alongside beef and poultry (Trocino et al., 2019). Moreover, rabbit meat is highly nutritious, with abundant protein, low fat and cholesterol and a rich source of calcium and phosphorus. Additionally, it contains high levels of linoleic acid, which is beneficial for overall health (Nistor et al., 2013). In recent years, its production has increased significantly and it has become one of the major animal protein sources in the Kingdom of Saudi Arabia (Al-Mathal, 2008). Coccidiosis, a highly transmissible disease, significantly affects the global domestic rabbit industry, leading to substantial financial losses (Geru et al., 2017). Coccidiosis in rabbits is caused by approximately 15 species of the genus Eimeria that affect the intestinal tract, including E. magna, E. matsubayashi, E. neoleporis, E. nagpurensis, E. irresidua, E. flavescens, E. piriformis, E. intestinalis, E. exigua, E. elongata, E. perforans, E. vejdovskyi, E. coecicola and E. media (Soulsby, 1968). Among these species, Eimeria stiedae is specifically responsible for hepatic and bile duct infections (Li and Ooi, 2009). The disease affects the intestines, liver and bile ducts of animals and is associated with digestive disorders (Manjunatha et al., 2019). The disease significantly affects growth performance parameters, leading to elevated morbidity and mortality rates and resulting in a reduction of carcass weight by over 23% (Lebas et al., 1997). Young rabbits, especially those in the first four months of life, are more susceptible to the disease, while older rabbits tend to show greater resistance to infection (Sivajothi et al., 2014). The treatment of hepatic coccidiosis poses challenges and the disease may persist throughout an animal’s lifetime (Karaer, 2001). The challenges outlined above underscore the critical necessity of discovering natural alternatives to drugs and disinfectants that can effectively inhibit Eimeria growth, enhance rabbit immunity and boost animal productivity (Ogolla et al., 2018). Recently, several plants have been tested for their potential to control coccidiosis (Tipu et al., 2006). Vitis vinifera (L.) is a perennial woody vine in the Vitaceae family that has been studied for potential medicinal use. Leaves are used externally to treat wounds and abscesses (Kallel et al., 2008). Traditional medicine derived from grape leaves has historical use in treating conditions such as diarrhea, hepatitis and abdominal pain (Felicio et al., 2001).  The main compounds in this plant are resveratrol, quercetin, catechin, flavones, flavanols, anthocyanins, gallic acid and epicatechin (Aouey et al., 2016). The extract from the leaves of V. vinifera has antioxidant, anti-inflammatory, analgesic and antipyretic activities (Aouey et al., 2016). The aqueous extract obtained from the leaves of Vitis vinifera exhibits antidiabetic and antioxidant properties, as well as antibacterial activity against Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Vibrio alginolyticus (Orhan et al., 2006; Mansour et al., 2011). Furthermore, it was found that the organic Zn-glycine complex and grape seed powder effectively alleviated the negative effects on growth performance, lesion scores and oocyst shedding in broilers infected with Eimeria tenella (Trocino et al., 2019).  Herbal remedies have proven to be effective and cost-efficient alternatives to preventive anticoccidial medications (Tipu et al., 2006). However, their application against coccidiosis in production rabbits remains limited. Therefore, this study was conducted to evaluate the ameliorative effects of Vitis vinifera leaf extracts on Eimeria stiedae infection in New Zealand rabbits.

MATERIALS AND METHODS

Ethical approval
 
The research experiment was conducted in compliance with Saudi Arabian regulations governing the use of animals. Ethical approval for the study was granted by the King Saud University Research Ethics Committee (REC) under approval number KSU-SE-22-38.
 
Preparation of the Vitis vinifera extract
 
Vitis vinifera leaves were obtained from local markets in Riyadh, Saudi Arabia. The leaves were air-dried and then ground into a fine powder using an electric blender. Subsequently, cold extraction was carried out using 70% methanol, with continuous agitation on a shaker for 24 hours (Amer et al., 2015). The dissolved contents obtained were homogenized, filtered using filter paper and evaporated at 40°C in a rotary evaporator (IKA, Germany). The solvent was evaporated to obtain extractions (Chikoto and Eloff, 2005).  The extract obtained was kept at -20°C until use.
 
Gas chromatography-mass spectrometry (GC-MS) analysis
 
The GC-MS technique was employed to identify the active components in the extract from Vitis vinifera leaves. GC-MS analysis of this extract was performed using Thermo Scientific, Trace GC Ultra/ISQ single Quadrupole MS, TG 5MS fused silica capillary column (length 30 mL, inner diameter 0.251 mm and film thickness 0.1 mm). For GC/MS detection employed an electron ionization system with an ionization energy of 70 eV, utilizing helium gas as the carrier gas at a constant flow rate of 1 ml/min. The injector and MS transfer line were both maintained at a temperature of 280°C. The oven temperature was ramped up from an initial 40°C (held for 3 minutes) to a final temperature of 280°C, increasing at a rate of 5°C per minute (with a 5-minute hold at the final temperature). The relative peak area percentages were used to quantify all identified components. Additionally, we attempted to identify the compounds by comparing their relative retention times and mass spectra with data from the NIST WILLY library using a GC/MS system (Hussein et al., 2016).
 
Experimental animals
 
A total of 35 male New Zealand White rabbits (Oryctolagus cuniculus) were obtained from a farm in Riyadh, Saudi Arabia. Upon arrival, they were immediately housed individually in disinfected wire cages of uniform size under hygienic conditions at the animal facility of the Department of Zoology, King Saud University. All animals were provided with ad libitum access to feed and water and the diet was free of anticoccidial drugs. The rabbits were acclimatized for one week, during which fecal samples were examined daily for coccidia using a concentrated flotation technique (Heelan and Ingersoll, 2002).
 
Collection and preparation of Eimeria oocysts
 
Oocysts of Eimeria stiedae were collected from the gallbladders of naturally infected rabbits obtained from a bird market in Riyadh, Kingdom of Saudi Arabia. The oocysts were suspended in 2.5% potassium dichromate solution and incubated at 25°C with continuous aeration. Daily monitoring was performed to assess sporulation (Soulsby, 1986). After complete sporulation, the oocysts were repeatedly washed by centrifugation until a clear supernatant was obtained and then stored at 4°C for subsequent use.
 
In vivo infection and experimental design
 
A total of 35 male New Zealand rabbits (Oryctolagus cuniculus) aged 12 weeks were individually weighed and assigned to 7 groups of 5 rabbits per group as the following:
 
Group 1: Non-treated (negative control) (n=5).
 
Group 2: Non-infected and treated (n=5) with the plant extracts at 400 mg/kg of body weight.
 
Group 3: Infected-non-treated (positive control) (n=5).
 
Group 4: Infected and treated (n=5) with the plant extracts at 100 mg/kg of body weight.
 
Group 5: Infected and treated (n=5) with the plant extracts at 200 mg/kg of body weight.
 
Group 6: Infected and treated (n=5) with the plant extracts at 400 mg/kg of body weight. 
 
Group 7: Infected and treated (n=5) with Toltrazuril at 5 mg/kg body weight.
       
All groups except groups 1 and 2 were inoculated with 5×104 sporulated Eimeria stiedae oocysts (Mohammed et al., 2021).  After one hour of infection, the three doses of Vitis vinifera leaf extracts and the reference drug was injected orally into all rabbits according to Aouey et al., (2016) and El-Ghoneimy and El-Shahawy (2017), respectively.
 
Oocyst number in gallbladder
 
Fresh gallbladder samples were collected on day 18 post-infection with Eimeria stiediae after sacrificing rabbits to compare the oocyst rate in gall bladder from both infected and infected treated groups. To determine the average of oocysts number. The Contents gallbladders were suspended in a tube then mixed well and pipetting 10 μL into a glass slide was done and then covered by a coverslip. after that, the total number of oocysts within 10 μL was counted by using the light microscope per Contents gallbladders.
 
Macroscopic examination and lesion scoring
 
On day 18 post-infection, rabbits from each group were euthanized and their livers were subjected to macroscopic examination. Liver lesion scores were evaluated and compared among the infected untreated group, the group treated with VVLE (400 mg/kg) and the control group.
 
Histochemical studies
 
Liver samples were immediately excised and cut into small sections, then fixed in 10% neutral buffered formalin. After fixation, the tissues were processed through dehydration, embedded in paraffin wax and sectioned at a thickness of 5 µm. The sections were subsequently stained using the periodic acid-Schiff (PAS) technique for the detection of total carbohydrates (Hotchkiss, 1948), while mercury bromophenol blue staining was applied to demonstrate total protein content (Maize et al., 1953).
 
Biochemical study
 
In rabbit plasma, the liver enzymes alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were measured using kits provided by Biodiagnostic Co. (Giza, Egypt). ALP and ALT activities were determined according to the methods described by Belfield and Goldberg (1971) and Bergmeyer et al., (1978), respectively.
 
Metal ions in the liver
 
The concentrations of metal ions (Cu, Fe, Zn and Mg) in liver samples from both infected and noninfected rabbits were determined using concentrated nitric acid, following the methodology outlined by UNEP/FAO/IAEA/IOC, (1984).  The concentrations of metal ions were analyzed using the atomic emission spectrometer iCAP-6500 Duo, which utilizes inductively coupled plasma (Thermo Scientific, U.K.).
 
Statistical analysis
 
Statistical significance was assessed using one-way analysis of variance (ANOVA) and multiple comparisons among groups were conducted using Duncan’s test with SPSS software (version 17.0). All data are presented as mean ± standard error of the mean (SEM). A two-tailed p-value of ≤0.05 was considered statistically significant for all analyses.

RESULTS AND DISCUSSION

Gas chromatography-mass spectrometry (GC-MS) analysis of (VVLE).
    
GC-MS analysis was used to examine the phytochemical composition of Vitis vinifera leaf extract (VVLE). The analysis revealed 34 phytochemical components present at varying peak areas and retention times (Table 1).

Table 1: The phytochemical compounds present in Vitis vinifera leaves extract (VVLE) identified using GC-MS.


       
During necropsy, a detailed macroscopic examination of the control group rabbits revealed no detectable lesions. In contrast, the infected untreated group showed marked enlargement of the liver, along with dilation of the bile ducts. On the surface of the liver in this group, nodules of different sizes, ranging in color from yellowish to white, were observed. The cross section of the nodules displayed a fluid that ranged in color from yellowish to cream. No nodules were yellowish in the liver of rabbits in the group infected and treated with Toltrazuril. The macroscopic liver findings in rabbits from the infected group treated with 400 of VVLE were comparable to those observed in the group treated with toltrazuril (Fig 1). These changes were particularly pronounced in the untreated infected group, consistent with previous reports on E. stiedae infection (Abed and Yakoob, 2013).  The observed changes may be due to the proliferation and dilation of the bile ducts, which lead to the formation of raised nodules on the liver surface. This is subsequently associated with an increased secretion of yellowish-white caseous material (Al-Naimi et al., 2012). It has been suggested that the toxic effects of protozoa may play a role in the development of coccidian nodules on the liver surface (Barriga and Arnoni, 1981).

Fig 1: Rabbit livers from different groups.


       
During the examination of gall bladders from rabbits infected with Eimeria stiedae, day 18 post-infection was identified as the peak of infection. The percentage reduction in oocyst output was higher in animals treated with VVLE at a dose of 400 mg/kg compared with those treated with 100 and 200 mg/kg, as well as the infected untreated group. However, the highest suppression of oocyst shedding was observed in the toltrazuril-treated group, which exceeded all VVLE-treated groups (Fig 2). Furthermore, this extract may enhance both innate and adaptive immune responses in rabbits, which is consistent with previous reports by Brisbin et al., (2008) and Chand et al., (2021). The findings of the present study suggest that Vitis vinifera leaf extract, similar to toltrazuril, effectively inhibits the development of the sexual stages of Eimeria stiedae in the liver, thereby reducing oocyst production in the gall bladder. This effect may be partly attributed to the potent antioxidant compounds present in the extract, which could suppress the growth and development of E. stiedae stages, ultimately decreasing oocyst formation and accumulation. In addition, Vitis vinifera leaf extract may induce oxidative stress through the generation of highly reactive oxygen species via the reductive cleavage of endoperoxide bridges mediated by iron complexes or iron-induced free radicals (Levander et al., 1989; Mares et al., 2024). These reactive species are capable of alkylating parasite proteins, leading to parasite death. This proposed mechanism is in agreement with the findings of Levander et al., (1989); Zhang and Gerhard (2008); del Cacho et al. (2010); Murshed et al., (2025) and Mares et al., (2023).

Fig 2: Oocyst number in gall bladder of rabbits infected with Eimeria stiedae and for infected treated groups with 100 mg/kg, 200 mg/kg and 400 mg/kg VVLE on day 18 p.i.


   
In histochemical studies, Eimeria stiedae infection resulted in a reduction in carbohydrate and protein content within hepatic cells, along with an altered distribution in all infected groups on day 18 post-infection compared with the control group. However, treatment of infected rabbits with VVLE (400 mg/kg) led to a restoration and increase of carbohydrate and protein levels in liver cells, approaching those observed in the toltrazuril-treated group (Fig 3 and 4). The reduction in carbohydrate content and its distribution within hepatic cells may be attributed to the increased metabolic burden on the liver resulting from parasitic infection and the presence of intracellular developmental stages, which require substantial energy consumption (Ibrahim, 1999). It may also reflect the maintenance of normal blood glucose levels through enhanced catabolism of carbohydrate reserves stored in tissues (Chapman et al., 1982) and (Mares et al., 2023).

Fig 3: Periodic acid schiff’s-stained sections showing carbohydrates stores in the liver for the rabbits in the infected groups and infected-treated group with toltrazuril.



Fig 4: Mercuric bromophenol blue -stained sections showing proteins stores in the liver for the rabbits in the infected groups and Infected-treated group with toltrazuril.


       
Similarly, the observed decrease in protein content within liver cells may be due to the disruption of lysosomal membranes under the influence of various deleterious factors, leading to the release of proteolytic enzymes (Awasthi et al., 1984). Comparable findings have been reported in previous studies, where reduced hepatic protein levels were observed in chickens infected with Eimeria mitis and Eimeria tenella (Musajev and Surkova, 1970).
    
Eimeria stiedae infection led to a significant increase (P≤0.05) in plasma levels of ALP and ALT in rabbits (Fig 5 and 6). However, treatment of infected rabbits with VVLE (400 mg/kg) reduced ALP and ALT levels to values nearly comparable to those of the control group. Similar effects were observed when compared with the reference drug, toltrazuril. The improvement in plasma enzyme levels may be attributed to the regeneration of hepatic cells and epithelium, as well as the inhibition of the parasite’s sexual stage development. During the recovery phase, marked restoration of damaged liver tissues and bile ducts was observed. In addition, several biochemical parameters, including liver enzymes and plasma proteins, returned to normal values. These findings are consistent with those reported by Cam et al. (2008) and Abdel-Maged et al. (2013).

Fig 5: Plasma alkaline phosphatase level after treatment of Eimeria stiedae-infected rabbits with Vitis vinifera.



Fig 6: Plasma alkaline transaminase level after treatment of Eimeria stiedae -infected rabbits with Vitis vinifera.


    
Rabbits infected with Eimeria stiedae showed a statistically significant increase (P≤0.05) in hepatic concentrations of heavy metals, including iron (Fe), copper (Cu), magnesium (Mg) and zinc (Zn) (Table 2). However, treatment with VVLE (400 mg/kg) reduced the levels of Fe, Cu, Mg and Zn to values comparable to those of the control group, with effects similar to those observed in the toltrazuril-treated group. The observed changes may be attributed to the fact that elevated heavy metal concentrations are often associated with inflammatory hepatic lesions. Inflammatory processes can induce increased ferritin synthesis. Although ferritin is generally regarded as a reliable marker of total iron stores, its role is altered under inflammatory conditions, where it becomes involved in the immune response and may affect lymphocyte function (Kernan and Carcillo, 2017). These findings are in agreement with Schultheiss et al., (2002) and Thornburg (2000), who reported increased hepatic copper and zinc levels in dogs with inflammatory liver lesions.

Table 2: Heavy metal concentrations in rabbits after Eimeria stiedae infection.

CONCLUSION

The methanolic extract of Vitis vinifera leaves appears to be a safe therapeutic candidate for the management of coccidiosis in rabbit production. It has shown efficacy in mitigating the adverse effects of hepatic coccidiosis in rabbits, likely due to its phytochemical constituents with anticoccidial activity. Nevertheless, further investigations are required to evaluate its potential as a prophylactic natural alternative to conventional coccidiostats in animal feed.

ACKNOWLEDGEMENT

This work was funded by Ongoing Research Funding Program, (ORF-2026-3), King Saud University, Riyadh, Saudi Arabia.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

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    Full Research Article

    Efficacy of Vitis vinifera Leaves Extracts against Eimeria stiedae Infections in New Zealand Rabbit

    M
    Mohammed M. Mares1,*
    R
    Rewaida Abdel-Gaber1
    S
    Saleh Al-Quraishy1
    1Department of Zoology, College of Science, King Saud University, Riyadh 1145, Saudi Arabia.

    ABSTRACT

    Background: Coccidiosis is considered one of the most serious diseases affecting rabbits and various other animal species, leading to substantial economic losses worldwide. Plant-derived extracts have emerged as promising alternatives to conventional chemotherapeutic agents, particularly in light of increasing drug resistance, as well as their advantage of not causing harmful tissue residues.

    Methods: This study was conducted to determine the effectiveness of Vitis vinifera leaf extract (VVLE) against Eimeria stiedae in rabbits. The extract was subjected to phytochemical analysis by GC-MS, revealing the existence of 34 biologically active compounds. A total of 35 male New Zealand rabbits were divided into seven groups. Group 1 served as the uninfected, untreated (negative control) group. Group 2 uninfected and treated at 400 mg/kg of extract to test for toxicity. Group 3 represented the infected, non-treated (positive control) group. While, groups 4, 5 and 6 comprised infected treated with at 100 mg/kg, 200 mg/kg and 400 mg/kg of extracts, respectively. Finally, group 7 consisted of infected rabbits treated with Toltrazuril at a dosage of 5 mg/kg.

    Result: The findings indicate that 400 mg/kg concentration of VVLE has a significant protective effect against Eimeria stiedae. It relieves clinical symptoms, reduces oocyst production, improves biochemical parameters and decreases the level of iron (Fe), copper (Cu), magnesium (Mg) and zinc (Zn) in the liver of Eimeria stiedae-infected rabbits. This refers to the effectiveness of Vitis vinifera leaf extracts (VVLE) against coccidiosis. Further investigations are needed to identify the pharmacological ingredients responsible for these effects and their potential as a natural alternative for coccidia prevention in animal feed.

    INTRODUCTION

    Rabbit farming for meat has made a significant contribution to addressing the global meat shortage, playing an important role in supplying substantial quantities of meat alongside beef and poultry (Trocino et al., 2019). Moreover, rabbit meat is highly nutritious, with abundant protein, low fat and cholesterol and a rich source of calcium and phosphorus. Additionally, it contains high levels of linoleic acid, which is beneficial for overall health (Nistor et al., 2013). In recent years, its production has increased significantly and it has become one of the major animal protein sources in the Kingdom of Saudi Arabia (Al-Mathal, 2008). Coccidiosis, a highly transmissible disease, significantly affects the global domestic rabbit industry, leading to substantial financial losses (Geru et al., 2017). Coccidiosis in rabbits is caused by approximately 15 species of the genus Eimeria that affect the intestinal tract, including E. magna, E. matsubayashi, E. neoleporis, E. nagpurensis, E. irresidua, E. flavescens, E. piriformis, E. intestinalis, E. exigua, E. elongata, E. perforans, E. vejdovskyi, E. coecicola and E. media (Soulsby, 1968). Among these species, Eimeria stiedae is specifically responsible for hepatic and bile duct infections (Li and Ooi, 2009). The disease affects the intestines, liver and bile ducts of animals and is associated with digestive disorders (Manjunatha et al., 2019). The disease significantly affects growth performance parameters, leading to elevated morbidity and mortality rates and resulting in a reduction of carcass weight by over 23% (Lebas et al., 1997). Young rabbits, especially those in the first four months of life, are more susceptible to the disease, while older rabbits tend to show greater resistance to infection (Sivajothi et al., 2014). The treatment of hepatic coccidiosis poses challenges and the disease may persist throughout an animal’s lifetime (Karaer, 2001). The challenges outlined above underscore the critical necessity of discovering natural alternatives to drugs and disinfectants that can effectively inhibit Eimeria growth, enhance rabbit immunity and boost animal productivity (Ogolla et al., 2018). Recently, several plants have been tested for their potential to control coccidiosis (Tipu et al., 2006). Vitis vinifera (L.) is a perennial woody vine in the Vitaceae family that has been studied for potential medicinal use. Leaves are used externally to treat wounds and abscesses (Kallel et al., 2008). Traditional medicine derived from grape leaves has historical use in treating conditions such as diarrhea, hepatitis and abdominal pain (Felicio et al., 2001).  The main compounds in this plant are resveratrol, quercetin, catechin, flavones, flavanols, anthocyanins, gallic acid and epicatechin (Aouey et al., 2016). The extract from the leaves of V. vinifera has antioxidant, anti-inflammatory, analgesic and antipyretic activities (Aouey et al., 2016). The aqueous extract obtained from the leaves of Vitis vinifera exhibits antidiabetic and antioxidant properties, as well as antibacterial activity against Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Vibrio alginolyticus (Orhan et al., 2006; Mansour et al., 2011). Furthermore, it was found that the organic Zn-glycine complex and grape seed powder effectively alleviated the negative effects on growth performance, lesion scores and oocyst shedding in broilers infected with Eimeria tenella (Trocino et al., 2019).  Herbal remedies have proven to be effective and cost-efficient alternatives to preventive anticoccidial medications (Tipu et al., 2006). However, their application against coccidiosis in production rabbits remains limited. Therefore, this study was conducted to evaluate the ameliorative effects of Vitis vinifera leaf extracts on Eimeria stiedae infection in New Zealand rabbits.

    MATERIALS AND METHODS

    Ethical approval
     
    The research experiment was conducted in compliance with Saudi Arabian regulations governing the use of animals. Ethical approval for the study was granted by the King Saud University Research Ethics Committee (REC) under approval number KSU-SE-22-38.
     
    Preparation of the Vitis vinifera extract
     
    Vitis vinifera leaves were obtained from local markets in Riyadh, Saudi Arabia. The leaves were air-dried and then ground into a fine powder using an electric blender. Subsequently, cold extraction was carried out using 70% methanol, with continuous agitation on a shaker for 24 hours (Amer et al., 2015). The dissolved contents obtained were homogenized, filtered using filter paper and evaporated at 40°C in a rotary evaporator (IKA, Germany). The solvent was evaporated to obtain extractions (Chikoto and Eloff, 2005).  The extract obtained was kept at -20°C until use.
     
    Gas chromatography-mass spectrometry (GC-MS) analysis
     
    The GC-MS technique was employed to identify the active components in the extract from Vitis vinifera leaves. GC-MS analysis of this extract was performed using Thermo Scientific, Trace GC Ultra/ISQ single Quadrupole MS, TG 5MS fused silica capillary column (length 30 mL, inner diameter 0.251 mm and film thickness 0.1 mm). For GC/MS detection employed an electron ionization system with an ionization energy of 70 eV, utilizing helium gas as the carrier gas at a constant flow rate of 1 ml/min. The injector and MS transfer line were both maintained at a temperature of 280°C. The oven temperature was ramped up from an initial 40°C (held for 3 minutes) to a final temperature of 280°C, increasing at a rate of 5°C per minute (with a 5-minute hold at the final temperature). The relative peak area percentages were used to quantify all identified components. Additionally, we attempted to identify the compounds by comparing their relative retention times and mass spectra with data from the NIST WILLY library using a GC/MS system (Hussein et al., 2016).
     
    Experimental animals
     
    A total of 35 male New Zealand White rabbits (Oryctolagus cuniculus) were obtained from a farm in Riyadh, Saudi Arabia. Upon arrival, they were immediately housed individually in disinfected wire cages of uniform size under hygienic conditions at the animal facility of the Department of Zoology, King Saud University. All animals were provided with ad libitum access to feed and water and the diet was free of anticoccidial drugs. The rabbits were acclimatized for one week, during which fecal samples were examined daily for coccidia using a concentrated flotation technique (Heelan and Ingersoll, 2002).
     
    Collection and preparation of Eimeria oocysts
     
    Oocysts of Eimeria stiedae were collected from the gallbladders of naturally infected rabbits obtained from a bird market in Riyadh, Kingdom of Saudi Arabia. The oocysts were suspended in 2.5% potassium dichromate solution and incubated at 25°C with continuous aeration. Daily monitoring was performed to assess sporulation (Soulsby, 1986). After complete sporulation, the oocysts were repeatedly washed by centrifugation until a clear supernatant was obtained and then stored at 4°C for subsequent use.
     
    In vivo infection and experimental design
     
    A total of 35 male New Zealand rabbits (Oryctolagus cuniculus) aged 12 weeks were individually weighed and assigned to 7 groups of 5 rabbits per group as the following:
     
    Group 1: Non-treated (negative control) (n=5).
     
    Group 2: Non-infected and treated (n=5) with the plant extracts at 400 mg/kg of body weight.
     
    Group 3: Infected-non-treated (positive control) (n=5).
     
    Group 4: Infected and treated (n=5) with the plant extracts at 100 mg/kg of body weight.
     
    Group 5: Infected and treated (n=5) with the plant extracts at 200 mg/kg of body weight.
     
    Group 6: Infected and treated (n=5) with the plant extracts at 400 mg/kg of body weight. 
     
    Group 7: Infected and treated (n=5) with Toltrazuril at 5 mg/kg body weight.
           
    All groups except groups 1 and 2 were inoculated with 5×104 sporulated Eimeria stiedae oocysts (Mohammed et al., 2021).  After one hour of infection, the three doses of Vitis vinifera leaf extracts and the reference drug was injected orally into all rabbits according to Aouey et al., (2016) and El-Ghoneimy and El-Shahawy (2017), respectively.
     
    Oocyst number in gallbladder
     
    Fresh gallbladder samples were collected on day 18 post-infection with Eimeria stiediae after sacrificing rabbits to compare the oocyst rate in gall bladder from both infected and infected treated groups. To determine the average of oocysts number. The Contents gallbladders were suspended in a tube then mixed well and pipetting 10 μL into a glass slide was done and then covered by a coverslip. after that, the total number of oocysts within 10 μL was counted by using the light microscope per Contents gallbladders.
     
    Macroscopic examination and lesion scoring
     
    On day 18 post-infection, rabbits from each group were euthanized and their livers were subjected to macroscopic examination. Liver lesion scores were evaluated and compared among the infected untreated group, the group treated with VVLE (400 mg/kg) and the control group.
     
    Histochemical studies
     
    Liver samples were immediately excised and cut into small sections, then fixed in 10% neutral buffered formalin. After fixation, the tissues were processed through dehydration, embedded in paraffin wax and sectioned at a thickness of 5 µm. The sections were subsequently stained using the periodic acid-Schiff (PAS) technique for the detection of total carbohydrates (Hotchkiss, 1948), while mercury bromophenol blue staining was applied to demonstrate total protein content (Maize et al., 1953).
     
    Biochemical study
     
    In rabbit plasma, the liver enzymes alkaline phosphatase (ALP) and alanine aminotransferase (ALT) were measured using kits provided by Biodiagnostic Co. (Giza, Egypt). ALP and ALT activities were determined according to the methods described by Belfield and Goldberg (1971) and Bergmeyer et al., (1978), respectively.
     
    Metal ions in the liver
     
    The concentrations of metal ions (Cu, Fe, Zn and Mg) in liver samples from both infected and noninfected rabbits were determined using concentrated nitric acid, following the methodology outlined by UNEP/FAO/IAEA/IOC, (1984).  The concentrations of metal ions were analyzed using the atomic emission spectrometer iCAP-6500 Duo, which utilizes inductively coupled plasma (Thermo Scientific, U.K.).
     
    Statistical analysis
     
    Statistical significance was assessed using one-way analysis of variance (ANOVA) and multiple comparisons among groups were conducted using Duncan’s test with SPSS software (version 17.0). All data are presented as mean ± standard error of the mean (SEM). A two-tailed p-value of ≤0.05 was considered statistically significant for all analyses.

    RESULTS AND DISCUSSION

    Gas chromatography-mass spectrometry (GC-MS) analysis of (VVLE).
        
    GC-MS analysis was used to examine the phytochemical composition of Vitis vinifera leaf extract (VVLE). The analysis revealed 34 phytochemical components present at varying peak areas and retention times (Table 1).

    Table 1: The phytochemical compounds present in Vitis vinifera leaves extract (VVLE) identified using GC-MS.


           
    During necropsy, a detailed macroscopic examination of the control group rabbits revealed no detectable lesions. In contrast, the infected untreated group showed marked enlargement of the liver, along with dilation of the bile ducts. On the surface of the liver in this group, nodules of different sizes, ranging in color from yellowish to white, were observed. The cross section of the nodules displayed a fluid that ranged in color from yellowish to cream. No nodules were yellowish in the liver of rabbits in the group infected and treated with Toltrazuril. The macroscopic liver findings in rabbits from the infected group treated with 400 of VVLE were comparable to those observed in the group treated with toltrazuril (Fig 1). These changes were particularly pronounced in the untreated infected group, consistent with previous reports on E. stiedae infection (Abed and Yakoob, 2013).  The observed changes may be due to the proliferation and dilation of the bile ducts, which lead to the formation of raised nodules on the liver surface. This is subsequently associated with an increased secretion of yellowish-white caseous material (Al-Naimi et al., 2012). It has been suggested that the toxic effects of protozoa may play a role in the development of coccidian nodules on the liver surface (Barriga and Arnoni, 1981).

    Fig 1: Rabbit livers from different groups.


           
    During the examination of gall bladders from rabbits infected with Eimeria stiedae, day 18 post-infection was identified as the peak of infection. The percentage reduction in oocyst output was higher in animals treated with VVLE at a dose of 400 mg/kg compared with those treated with 100 and 200 mg/kg, as well as the infected untreated group. However, the highest suppression of oocyst shedding was observed in the toltrazuril-treated group, which exceeded all VVLE-treated groups (Fig 2). Furthermore, this extract may enhance both innate and adaptive immune responses in rabbits, which is consistent with previous reports by Brisbin et al., (2008) and Chand et al., (2021). The findings of the present study suggest that Vitis vinifera leaf extract, similar to toltrazuril, effectively inhibits the development of the sexual stages of Eimeria stiedae in the liver, thereby reducing oocyst production in the gall bladder. This effect may be partly attributed to the potent antioxidant compounds present in the extract, which could suppress the growth and development of E. stiedae stages, ultimately decreasing oocyst formation and accumulation. In addition, Vitis vinifera leaf extract may induce oxidative stress through the generation of highly reactive oxygen species via the reductive cleavage of endoperoxide bridges mediated by iron complexes or iron-induced free radicals (Levander et al., 1989; Mares et al., 2024). These reactive species are capable of alkylating parasite proteins, leading to parasite death. This proposed mechanism is in agreement with the findings of Levander et al., (1989); Zhang and Gerhard (2008); del Cacho et al. (2010); Murshed et al., (2025) and Mares et al., (2023).

    Fig 2: Oocyst number in gall bladder of rabbits infected with Eimeria stiedae and for infected treated groups with 100 mg/kg, 200 mg/kg and 400 mg/kg VVLE on day 18 p.i.


       
    In histochemical studies, Eimeria stiedae infection resulted in a reduction in carbohydrate and protein content within hepatic cells, along with an altered distribution in all infected groups on day 18 post-infection compared with the control group. However, treatment of infected rabbits with VVLE (400 mg/kg) led to a restoration and increase of carbohydrate and protein levels in liver cells, approaching those observed in the toltrazuril-treated group (Fig 3 and 4). The reduction in carbohydrate content and its distribution within hepatic cells may be attributed to the increased metabolic burden on the liver resulting from parasitic infection and the presence of intracellular developmental stages, which require substantial energy consumption (Ibrahim, 1999). It may also reflect the maintenance of normal blood glucose levels through enhanced catabolism of carbohydrate reserves stored in tissues (Chapman et al., 1982) and (Mares et al., 2023).

    Fig 3: Periodic acid schiff’s-stained sections showing carbohydrates stores in the liver for the rabbits in the infected groups and infected-treated group with toltrazuril.



    Fig 4: Mercuric bromophenol blue -stained sections showing proteins stores in the liver for the rabbits in the infected groups and Infected-treated group with toltrazuril.


           
    Similarly, the observed decrease in protein content within liver cells may be due to the disruption of lysosomal membranes under the influence of various deleterious factors, leading to the release of proteolytic enzymes (Awasthi et al., 1984). Comparable findings have been reported in previous studies, where reduced hepatic protein levels were observed in chickens infected with Eimeria mitis and Eimeria tenella (Musajev and Surkova, 1970).
        
    Eimeria stiedae infection led to a significant increase (P≤0.05) in plasma levels of ALP and ALT in rabbits (Fig 5 and 6). However, treatment of infected rabbits with VVLE (400 mg/kg) reduced ALP and ALT levels to values nearly comparable to those of the control group. Similar effects were observed when compared with the reference drug, toltrazuril. The improvement in plasma enzyme levels may be attributed to the regeneration of hepatic cells and epithelium, as well as the inhibition of the parasite’s sexual stage development. During the recovery phase, marked restoration of damaged liver tissues and bile ducts was observed. In addition, several biochemical parameters, including liver enzymes and plasma proteins, returned to normal values. These findings are consistent with those reported by Cam et al. (2008) and Abdel-Maged et al. (2013).

    Fig 5: Plasma alkaline phosphatase level after treatment of Eimeria stiedae-infected rabbits with Vitis vinifera.



    Fig 6: Plasma alkaline transaminase level after treatment of Eimeria stiedae -infected rabbits with Vitis vinifera.


        
    Rabbits infected with Eimeria stiedae showed a statistically significant increase (P≤0.05) in hepatic concentrations of heavy metals, including iron (Fe), copper (Cu), magnesium (Mg) and zinc (Zn) (Table 2). However, treatment with VVLE (400 mg/kg) reduced the levels of Fe, Cu, Mg and Zn to values comparable to those of the control group, with effects similar to those observed in the toltrazuril-treated group. The observed changes may be attributed to the fact that elevated heavy metal concentrations are often associated with inflammatory hepatic lesions. Inflammatory processes can induce increased ferritin synthesis. Although ferritin is generally regarded as a reliable marker of total iron stores, its role is altered under inflammatory conditions, where it becomes involved in the immune response and may affect lymphocyte function (Kernan and Carcillo, 2017). These findings are in agreement with Schultheiss et al., (2002) and Thornburg (2000), who reported increased hepatic copper and zinc levels in dogs with inflammatory liver lesions.

    Table 2: Heavy metal concentrations in rabbits after Eimeria stiedae infection.

    CONCLUSION

    The methanolic extract of Vitis vinifera leaves appears to be a safe therapeutic candidate for the management of coccidiosis in rabbit production. It has shown efficacy in mitigating the adverse effects of hepatic coccidiosis in rabbits, likely due to its phytochemical constituents with anticoccidial activity. Nevertheless, further investigations are required to evaluate its potential as a prophylactic natural alternative to conventional coccidiostats in animal feed.

    ACKNOWLEDGEMENT

    This work was funded by Ongoing Research Funding Program, (ORF-2026-3), King Saud University, Riyadh, Saudi Arabia.

    CONFLICT OF INTEREST

    The authors declare that they have no conflict of interest.

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