Effects of Vitis vinifera Leaves on the Oxidative Status of New Zealand Rabbit Liver Infected with Eimeria stiedae

Rabbit production has become one of the most important animal resources in the world (Cadric et al., 2017). Consumers prefer rabbits for their low cholesterol and fat contents and high levels of the essential amino acid (Cadric et al., 2017). In addition to this commercial value, it is also due to the high prolificacy, early maturity, fast growth rate, high genetic selection potential, efficiency in feed conversion and economic utilization of space by rabbits (Lukefahr and Cheeke, 1990). Coccidiosis constitutes a major parasitic disease in poultry and other domestic animals, including rabbits (Yousif and Abdul-Aziz, 1995). Several risk factors allegedly affect the increased number of coccidiosis cases in rabbits, including the enclosure management, hygiene level, season and age of rabbits (Wang and Tsai, 1991). Eimeria is a genus of Apicomplexan parasites that includes various species responsible for the development of coccidiosis (Barbour et al., 2015).
       
Currently, 15 species of the genus Eimeria are believed to be responsible for rabbit coccidiosis, including Eimeria stiedae, Eimeria magna, Eimeria matsubayashi, Eimeria neoleporis, Eimeria nagpurensis, Eimeria irresidua, Eimeria flavescens, Eimeria piriformis, Eimeria intestinalis, Eimeria exigua, Eimeria elongate, Eimeria perforans, Eimeria vejdovskyi, Eimeria coecicola and Eimeria media (Liu, 2019; Xie et al., 2021). E. stiedae is by far the most pathogenic Eimeria species and mainly parasitizes the liver and bile duct epithelial cells of rabbits, resulting in liver coccidiosis (Hassan et al., 2016; Jing et al., 2016). Most of these Eimeria species affect rabbit production and according to their level of pathogenicity can cause reduced growth rate, feed conversion and increased mortality (El-Shahawi et al., 2012). Hepatic coccidiosis (Eimeria stiedae) is one of the most pathogenic coccidian protozoans in domestic rabbits causing severe coccidiosis and increased mortality (Sivajothi et al., 2016).
       
Vitis vinifera is a perennial woody vine of the family Vitaceae and it has been evaluated for potential medical uses. From different parts of this plant essentially fruits, several preparations used in folk medicine have been derived (Bombardelli and Morazzonni, 1995). Fresh leaves have been used externally to heal wounds and to lance abscesses (Kallel et al., 2008). Grape leaf-based medicines are traditionally used for diarrhea, hepatitis and stomachaches (Kapoor, 1990; Bombardelli and Morazzonni, 1995; Felicio et al., 2001). The most important compounds recorded previously in this plant were resveratrol, quercetin, catechin, flavone, flavonols, anthocyanin, gallic acid and epicatechin (Aouey et al., 2016). Here, we determined the antioxidant activity of Vitis vinifera leaf extracts against coccidiosis induced by Eimeria stiedae in New Zealand rabbits.

Preparation of the Vitis vinifera extract
 
Leaves of Vitis vinifera were collected from local markets in Riyadh, Saudi Arabia. The leaves underwent air-drying and were subsequently ground into powder using an electric blender. Subsequently, cold extraction was performed in 70% methanol, with continuous mixing by 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.
 
Infrared spectroscopy analysis of Vitis vinifera leaves extract
 
Following the processing steps, a small portion of the material was homogenized by blending it with an abundant amount of potassium bromide powder (1: 99 wt%). Subsequently, the material underwent a coarse crushing process before being loaded into pellet-forming equipment. The NICOLET 6700 Fourier-transform infrared spectroscopy (FT-IR), an optical spectrometer from Thermo Scientific, was employed to analyze the infrared spectrum. At a temperature of 25°C, spectra were captured with a 4 cm resolution, spanning from 4000 cm^-1 to 400 cm^-1  (Hussein et al., 2016).
 
Experimental animals
 
A total of 35 male New Zealand white rabbits (Oryctolagus cuniculus) were purchased from a farm in Riyadh, Saudi Arabia. The rabbits were at the age of 12 weeks and non-infection Eimeria stiedae. On arrival, they were immediately placed in disinfected wire cages of the same size and housed individually under hygienic conditions in the Animal House of the Department of Zoology, King Saud University. All rabbits were provided with water and feed. The diet was free from anticoccidial drugs. Rabbits were acclimated for a week and their fecal samples were checked daily for coccidia using a concentrated flotation technique (Heelan and Ingersoll, 2002). The King Saud University Research Ethics Committee (REC) approved this study (KSU-SE-22-38).
 
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 will be injected orally into all rabbits according to Aouey et al., (2016) and El-Ghoneimy and El-Shahawy, 2017, respectively.
 
Body weight and feed consumption
 
Each rabbit’s weight was measured individually and the body weight gain (BWG) was determined by subtracting the initial body weight (recorded on day 0 post-infection) from the final body weight (measured on day 18 post-infection). The feed consumption (FC) for each replicate was determined by subtracting the weight of the remaining feed from the initial offered feed weight at the beginning of the experiment.
 
The liver’s oxidative status
 
Pieces of the rabbit’s liver were weighed and homogenized in a 10% (w/v) phosphate buffer. Subsequently, it was centrifuged for 15 minutes at 5000 g and 4°C. The supernatant was utilized for various biochemical analyses using kits supplied by Biodiagnostic Co. (Giza, Egypt). The liver homogenate was prepared following the method outlined by Dkhil et al., (2012). Glutathione (GSH) and nitric oxide levels were assessed by Paglia et al., (1967) and Montogomery (1961), respectively. Malondialdehyde (MDA) levels were determined using the method described by Ohkawa et al., (1979).
 
Statistical analysis
 
Significance was evaluated by one-way analysis of variance and statistical comparisons between the groups were performed by Duncan’s test, using a statistical package program (SPSS version 17.0). All values were expressed as the mean and the standard error of the mean. All p-values are two-tailed and p≤0.05 is considered as significant for all statistical analyses in this study.

The analysis of Vitis vinifera leaf extracts using Fourier transform infrared spectroscopy (FTIR) showed major bands at 3368.86 cm^-1, 2934.35 cm^-1, 1726.98 cm^-1, 1615.20 cm^-1, 1514.23 cm^-1, 1403.86 cm^-1, 1238.96 cm^-1, 1048.39 cm^-1, 931.17 cm^-1, 890.24 cm^-1, 818.14 cm^-1, 778.23 cm^-1, 711.28 cm^-1, 592.24 cm^-1, 520.21 cm^-1 (Fig  1, Table 1). The 3368.86 cm 2 band corresponds to N-H stretching, confirming the presence of an aliphatic primary amine. Additionally, the 2934.35 cm² band indicates C-H stretching, suggesting the presence of an alkane. The 1726.98 cm² band corresponds to C=O stretching, confirming the presence of an α, β-unsaturated ester compound. Lastly, the 1615.20 cm² band confirms N-H stretching, indicating the presence of an α, β-unsaturated ketone. There was a nitro compound, an alcohol, an alkyl aryl ether, anhydride, an alkene and a 1,3-disubstituted at 1514.23, 1403.86, 1238.96, 1048.39, 931.17 and 890.24 cm^-1, in that order. Other compounds were also present.
 

 

       
The BWG of infected rabbits with Eimeria stiedae decreased significantly (P≤0.05) on day 18 post-infection compared with the non-infected rabbits (Fig 2). However, VVLE-challenged rabbits’ groups without infection showed a significant increase (P≤0.05) in the BWG when compared to the infected rabbits’ group, where the dose of 400 mg/kg VVLE showed a greater increase in the BWG than that occurred with the reference drug Toltrazuril (Fig 2). On day 18 post-infection the feed consumption (FC) of the infected group decreased compared to the non-infected group (Fig 3). This significant decrease (P≤0.05) was improved by the treatment of animals with VVLE in the treated group with 400 mg/kg VVLE (Fig 3). Rabbits treated with Toltrazuril also significantly appeared with increased FC (Fig 3). The beneficial effects observed may be attributed to the presence of phytochemicals, flavonoids and phenolic compounds in Vitis vinifera (grape) leaf extract. These compounds act as potent antioxidants, aiding rabbits in maintaining a healthy commensal microflora. Additionally, they facilitate efficient nitrogen uptake, enhancing food digestion and nutrient absorption. Furthermore, this extract contributes to an improved innate and acquired immune response in rabbits, aligning with findings from previous studies by Brisbin et al., (2008) and Chand et al., (2021).
 

 

       
There has been a remarkable change in the infected with Eimeria stiedae and treated rabbits in glutathione (GSH), nitric oxide (NO) and malondialdehyde (MDA). Eimeria stiedae infection caused a significant decrease (P≤0.05) in the level of glutathione in the liver of rabbits (Fig 4). but after treatment of the infected rabbits with VVLE (400 mg/kg), the level of glutathione increases to nearly the same as in the control groups. Compared to the reference drug, Toltrazuril. While the level of nitric oxide increased significantly (P≤0.05) after infection of rabbits with Eimeria stiedae (Fig 5). VVLE (400 mg/kg) was able to decrease nitric oxide to nearly the same as in the control groups. Compared to the reference drug, Toltrazuril. And Eimeria stiedae infection caused a significant increase (P≤0.05) in the level of malondialdehyde in the liver of rabbits (Fig 6). but after treatment of the infected rabbits with VVLE (400 mg/kg), the level of malondialdehyde decreased to nearly the same as in the control groups. Compared to the reference drug, Toltrazuril. Oxidative stress plays a crucial role in the pathophysiology of various pathological conditions. It is characterized by an increase in reactive oxygen species (ROS) and/or a reduction in antioxidant defense mechanisms (Heyman et al., 2011). The findings align with the research by Çam et al., (2008) and Abdel-Maged et al., (2013). These studies indicate that elevated levels of malondialdehyde (MDA) and nitric oxide in infected rabbits imply that Eimeria stiedae triggers lipid peroxidation. This process arises from damage to the bile duct and, consequently, the hepatic parenchyma. Lipid peroxidation can lead to impaired cell membrane permeability and subsequent cellular reactions, potentially resulting in cell death. Also, the decrease in glutathione production could result from liver damage caused by the infection. The liver plays a crucial role in glutathione synthesis. If the hepatic parenchyma is compromised due to the infection, it may lead to reduced glutathione production.  Also, excessive free radicals: During the infection, these highly reactive molecules can cause damage to cellular components, including enzymes like glutathione. When free radicals overwhelm the antioxidant defense system, it can lead to a decrease in glutathione activity (Kaya et al., 2007; Wang et al., 2008). Regarding VVLE (400 mg/kg) and the toltrazuril supplemented group, the results of lipid peroxidation parameters were close to control values. VVLE (400 mg/kg) and toltrazuril led to a reduction in MDA and nitric oxide levels, while simultaneously increasing glutathione levels. These effects may further validate their antioxidant and anticoccidial properties.
 

 

 

Our findings indicate that Vitis vinifera is an effective agent, influencing both weight and feed intake. Additionally, Vitis vinifera appears to regulate the oxidative status in the liver of infected New Zealand rabbits. However, further studies are necessary to elucidate the precise mechanism of action of Vitis vinifera on both the host and the parasite.

We thank the Researcher Support Program (RSP2024R/3), at King Saud University.

All authors declared that there is no conflict of interest.