Evaluating the in vitro efficiency of grapevine leaves against Eimeria flavescens sporozoites infecting domestic rabbits

[Avaliação da eficiência in vitro das folhas de videira contra esporozoítos de Eimeria flavescens que infectam coelhos domésticos]

ABSTRACT

Coccidiosis is a common disease in rabbits and is caused by Eimeria spp., which poses a threat to its survival. The research aims to assess the effect of the Vitis vinifera extract on Eimeria flavescens sporozoites for inhibition viability. Twenty-four-well plates with a volume of 3 mL each, each containing one thousand sporulated oocysts/mL, were subjected to seven different treatments: control of 2.5% potassium dichromate solution; five different extract concentrations 125, 250, 500, 750, and 1000μg/mL concentrations for anti-sporozoites activities. in addition, 25mg/ml of Toltrazuril was as a reference medicine. The mixture's anti-sporozoite properties were assessed, and it was able to block the E. flavescens oocysts at 125g/mL, Vitis vinifera showed the lowest inhibition of 9% and the largest suppression of 87% of the viability of E. flavescens sporozoite at 1000g/mL. Longer incubation times and higher doses usually increase the inhibition rate for sporozoite viability. Findings showed that V. vinifera leaf extract has effective activity in vitro sporozoite viability.

Keywords:
anticoccidial; Eimeria species; sporozoiticidal

RESUMO

A coccidiose é uma doença comum em coelhos e é causada por Eimeria spp. que representa uma ameaça à sua sobrevivência. A pesquisa tem como objetivo avaliar o efeito do extrato de Vitis vinifera sobre os esporozoítos de Eimeria flavescens quanto à viabilidade de inibição. Vinte e quatro placas de poços com um volume de 3 mL cada, cada uma contendo mil oocistos esporulados/mL, foram submetidas a sete tratamentos diferentes: controle de solução de dicromato de potássio a 2,5%; cinco concentrações diferentes de extrato 125, 250, 500, 750 e 1000μg/mL para atividades antiesporozoítas. As propriedades antiesporozoíticas da mistura foram avaliadas, e ela foi capaz de bloquear os oocistos de E. flavescens a 125g/mL, a Vitis vinifera apresentou a menor inibição de 9% e a maior supressão de 87% da viabilidade do esporozoíto de E. flavescens a 1000g/mL. Tempos de incubação mais longos e doses mais altas geralmente aumentam a taxa de inibição da viabilidade do esporozoíto. Os resultados mostraram que o extrato da folha de V. vinifera tem atividade eficaz na viabilidade de esporozoítos in vitro.

Palavras-chave:
anticoccidiano; espécies de Eimeria; esporozoiticida

INTRODUCTION

Rabbits are animals rich in protein, and with the increase in human population, the need for animal protein from the five principal livestock species (cattle, sheep, goats, and poultry) is necessary (El-Sabrout, 2020; Siddiqui et al., 2023). This has led to a growing interest in micro livestock like rabbits, whose production holds great promise for addressing the issue of animal protein scarcity in developing nations (El-Sabrout, 2020). Due to its low fat and cholesterol content and high concentration of vital amino acids, rabbit meat is one of the white meats that people eat the most (Siddiqui et al., 2023). Rabbits' essential characteristics as micro livestock include their small body size, short generation interval, capacity to use fewer competing diets, growth fast, possibility for genetic improvement, and production of high-quality meat and beneficial by-products (Olowofeso et al., 2012). Despite the increasing interest in rabbit production, the rabbit industry is faced with several devastating. One of the most challenging inhibition constraints to rabbit production is coccidiosis, caused by Eimeria (Cedric et al., 2017). It includes 11 species of Eimeria in rabbits (Oryctolagus cuniculus), including ten intestinal and one hepatic (Al-Mathal, 2008). The majority of these Eimeria species have an impact on rabbit production, and depending on how pathogenic they are, they may also result in a decreased growth ratio, decreased feed conversion, and higher death rate. Thus, in commercial rabbit systems, coccidiosis is most likely the most costly and common infectious disease. Eimeria flavescens is a protozoal parasite, it is one of the species responsible for causing coccidiosis in rabbits and affects the intestines (Hamad et al., 2014).

The majority of anti-coccidian medications on the market today have detrimental side effects and poor efficacy (Cedric et al., 2017). Drug-resistant parasites and drug residues in animal meat have resulted from the widespread use of chemical anti-coccidian medications to treat this illness (Al-Mathal, 2008). Researchers everywhere are turning to different strategies to manage parasite issues (Hamad et al., 2014). The disease coccidiosis and oxidative stress are closely related. As to (Cedric et al., 2017), sporulated oocysts that are consumed by a healthy animal produce inflammation in the bowel. Free radicals are produced excessively because of the inflammatory process. By oxidizing lipids and harming proteins and DNA, these free radicals are not only harmful to the parasites but also to the host. Thus, antioxidants show potential as an alternative for controlling coccidiosis today when used as anticoccidial treatments (Cedric et al., 2017).

Infective Eimeria oocysts are extremely resistant to environmental stress and frequently contaminate feed and water due to weak biosecurity measures and poor farm management methods (Abbas et al., 2008; Shahzad et al., 2012). Furthermore, the tactics employed to control coccidiosis in rabbits have been impeded by the introduction of widely resistant Eimeria strains, no accessible vaccinations for rabbit coccidia, and the toxicity of synthetic medicines and disinfectants for rabbits and people (Pakandl, 2005).

The aforementioned difficulties highlight the critical need to discover natural substitutes for medications and cleaning agents that can stop the spread of Eimeria, strengthen rabbits' immune systems, and boost animal productivity (Ogolla et al., 2018; Pakandl, 2009). Finally, numerous plants were examined for their ability to control coccidiosis (Tipu et al., 2006). Among these plants, the grapevine (Vitis vinifera), belongs to the family Vitaceae, and is one of the most versatile medicinal plants, with a broad spectrum of biological and pharmacological activities, and has anticoccidial properties (Salehi et al., 2019). V. vinifera leaves contain a variety of chemical constituents, including oleic acid; linoleic acid, n-Hexadecanoicacid, 5-hydroxymethylfurfural, octadecanoic acid, β-D-glucopyranose, 1,6-anhydrous, phytol, glycidyl oleate, 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl, 9,12-octadecadienoyl chloride, (Z, Z), hexanedioic acid, and mono(2-ethylhexyl) ester (Murshed et al., 2023).

The research was carried out to evaluate the efficacy of grapevine leaf extract and its effects on E. flavescens sporozoites viability in vitro.

MATERIAL AND METHODS

Fresh grape leaves from Riydah farms, Saudi Arabia, were obtained to prepare the extract according to the protocol described by (Manikandan et al., 2008), and the botanical identity of the plant was confirmed by a classification scientist at King Saud University's Botany Department. After the 300g of air-dried leaves were ground into a powder, they were extracted by allowing 70% methanol to percolate at room temperature. The mixture was then stored for 24 hours at 4°C while being stirred periodically. Extract put in the rotating vacuum evaporator (Yamato RE300, Tokyo, Japan) was under reduced pressure at 40◦C, and the resulting extract was concentrated and dried. The extracts were saved at −20◦C until using. Distilled water was employed to dissolve the extract for the experiments.

Un-sporulation E. flavescens oocysts were obtained from droppings of a flock of rabbits positive for coccidia. Oocysts were purified via flotation in a saturated salt solution. The droppings were combined with tap water and filtered through a 250μm filter. The suspension was centrifuged (3000 rpm for 7 minutes), and the pellet was resuspended in a salt-saturated solution (excess NaCl in water; density 1.18). The suspension was centrifuged at 1800 rpm for 7 minutes, then the supernatant was collected and centrifuged three times in water at 3000 rpm for 7 minutes each. Following the final centrifugation, the pellet was put in a 2.5% potassium dichromate solution and examined under a microscope to determine the presence and cleanliness of the oocyst. The oocysts were then incubated at 28 °C for three days to sporulate before being kept at 4 °C until needed. The parasite was kept alive through passage in young rabbits (Murshed et al., 2022). The pure strain (E. flavescens) was first purified from field material by isolating a single oocyst and growing it in coccidia-free young rabbits.

Multiple washing procedures were carried out on oocyst samples that were stored in potassium dichromate solution. These procedures were carried out with phosphate-buffered saline (PBS) that had a pH of 7.4. for 10 min, we repeated the procedure of centrifuging the Falcon tubes with 15mL of liquid at a speed of 1008 gm until all of the K₂Cr₂O₇ was removed. While the oocysts were being incubated in a water bath at a temperature of 41 degrees Celsius, they were shaken for one hour. Following the resuspension of the pellet in PBS and the application of a single pass in PBS, the suspension was centrifuged at a speed of 1008g for ten minutes. To carry out the function of counting the sporozoites, a McMaster chamber was utilized. To evaluate the sporozoite activity in vitro, plates well (24) were taken into consideration. To generate a total volume of 2mL for each dose of Vitis vinifera (125, 250, 500, and 1000μg/mL), a test solution of 1mL was combined with mL of the parasite suspension that contained 1000 sporozoites. This mixture was then used to construct the test solution. Positive control was the medication toltrazuril, which was administered at a dose of 25μg/mL to make a comparison. Under the same circumstances, the potassium dichromate solution was used as the control for the negative outcome. The experiment was carried out three times, with each dose and control treatment being subjected to the same conditions each time. After 6, 12, 18, and 24 hours, the findings were evaluated. The percentage of viable sporozoites was determined by counting the number of viable sporozoites among a total of one hundred sporozoites. This was done after the number of viable and nonviable sporozoites had been determined. To calculate the viability % inhibition, the formula that you described (Il Oh and Hoff, 1986).

$Inhibitionofviability\left(Vi\right)\%=\left(Vi\%control-Vi\%bile\right)/\left(Vi\%control\right)\times 100$

The data was analyzed with one-way ANOVA and presented as the mean ± SD of three replications. The criterion of significance was fixed at P ≤ 0.05.

RESULT

Parasite eradication is vital to organismal health, which can help design sustainable production methods that protect consumer health. The extract of Vitis vinifera leaves was found to play a vital function in suppressing sporozoites and inhibiting their viability, as well as having a damaging effect on sporozoite oocysts.

As a function of concentration, incubation period, and the kind of plant extract, the sporozoite viability inhibitory percentage of E. flavescens is shown to be a function of V. vinifera extract. Based on the findings of the analysis of Figure 1 (6 and 12 h), it can be deduced that an increase in the concentration of the extract seems to have increased its effectiveness. Therefore, when the concentration was raised, there was a considerable rise in the inhibition rates after 18 and 24 (Figure 2). As a result, the extract has the potential to exhibit improved performance when administered at a concentration of 1000μg/mL. Additionally, it is evident from Figures 3 and 4 that there was a rise in the inhibition rate with an increase in the amount of time that the incubation process was carried out. The viability percentage of E. flavescens was restricted by 92±2.87 %due to the presence of a high concentration of methanolic extract from V. vinifera. As the concentration of extracts fell, the percentage of viability inhibition likewise decreased proportionally (Table 1).

General, all times (6, 12, 18, and 24 hours), in the extract exhibited anti-sporozoite activity against E. flavescens, at 1000μg/mL (Figures 5).

The sporozoite activities percentage of the E. flavescens decreased with increasing exposure periods. The sporozoite viability inhibition % was highly negatively correlated with exposure time (R² = 0.9343, P=0.05; at 6, 12 hours., R² = 0.9448 and P=0.05; at 18, 24 hours) at 1000µl/mL VVE and CON/Positive Toltrazuril treatment, respectively (Figures 6, 7, 8 and 9). Where the correlate efficiency at lower concentrations was moderated negatively and weak at 125µl/mL VVE, 250µl/mL VVE 500µl/mL VVE, and 750µl/mL VVE concentrations.

Figure 1
Effects of Vitis vinifera on the anti-sporozoite activities of E. flavescens oocysts in vitro throughout periods 6 and 12 hours. Significance (*): p < 0.05.

Figure 2
Effects of Vitis vinifera on the anti-sporozoite activities of E. flavescens oocysts in vitro throughout periods 18 and 24 hours. Significance (*): p < 0.05.

Table 1
Effect of different doses of Vitis vinifera on Viability Sporozoite % and Un-viability Sporozoite rates of E. perforans as a function of concentration, incubation time, and plant extract type

Concentration µg/mL	Viability inhibition %	Viability Sporozoite %
Control	00	100
125 µg/mL VVE	09 ± 2.44e	89.33 ± 6.42a
250 µg/mL VVE	19 ± 4.89d	58.33 ± 6.66b
500 µg/mL VVE	31 ± 5.35db	67.66 ± 7.02bc
750 µg/mL VVE	22 ± 9.27bc	78.33 ± 3.05cd
1000 µg/mL VVE	93± 3.54a	6.13 ± 2.87a
Toltrazuril 25 µg/mL	94 ± 0.99a	6.66 ± 5.13a

Figure 3
Effect of the different times (6 and 12 h), with Viability inhibitory percentage of Vitis vinifera extract on E. flavescens, Significance (*): p-value < 0.05.

Figure 4
Effect of the different times (18 and 24 h), with Viability inhibitory percentage of Vitis vinifera extract on E. flavescens, Significance (*): p-value < 0.05.

Figure 5
Viability inhibition rate of grapevine leaf extracts against E. flavescens sporozoites at various exposure periods (6,12, 18, and 24 hours).

Figure 6
Mean Viability inhibition Eimeria flavescens Sporozoites % of grapevine leaf at different concentrations at 6 h.

Figure 7
Mean Viability inhibition Eimeria flavescens Sporozoites % of grapevine leaf at different concentrations at 12 h.

Figure 8
Mean Viability inhibition Eimeria flavescens Sporozoites % of grapevine leaf at different concentrations at 18 h.

Figure 9
Mean Viability inhibition Eimeria flavescens Sporozoites % of grapevine leaf at different concentrations at 24 h.

The overall results of the current study suggest that the leaf extract of V. vinifera may possess potential properties in sporozoite viability inhibition, which could potentially be employed in the management of the prerequisites that one should take much care of when handling and applying the compound.

DISCUSSION

The findings showed that methanolic extract concentrations have sporozoiticidal action against Eimeria flavescens. After 24 hours, the V. vinifera methanolic extract was shown to be more effective. It exhibited maximum sporulation inhibitory activity at 1000 mg/ml. Comparable to the current discovery, Calotropis procera leaf extract was found to suppress sporozoite in vitro vitality in Eimeria papillae by (Murshed et al., 2022). Condensed grape tannins have been demonstrated to inhibit the activities of endogenous enzymes, including mannitol-1 phosphate dehydrogenase, mannitol dehydrogenase, and hexokinase (Horigome et al., 1988; Il Oh and Hoff, 1986). Therefore, it is plausible that the tannin-containing V. vinifera extract decreased the rate of viability by blocking or deactivating the enzymes involved in the sporulation process, just like in helminth eggs (Molan et al., 2003).

According to the findings of the current investigation, the Vitis vinifera extract may have broken through the oocyst wall and caused damage to the cytoplasm (sporont). This was demonstrated by the presence of aberrant sporocysts in oocysts that were subjected to higher concentrations of the extract. Since potassium dichromate is also a bactericidal agent, it is possible that it killed the bacteria that were present, which increased the sporulation of oocysts. This could be the explanation for the observation that potassium dichromate was unable to have an inhibitory effect on sporulation. According to (Hamad et al., 2014), the use of potassium dichromate to kill bacteria in a sample that contained coccidian oocysts increased the incidence of sporulation of coccidia oocysts (Murshed et al., 2023). As a result, it is possible that bacteria, if they were there, may have impeded the process of oocyst sporulation. This could have occurred either through the competition for resources or by the consumption of the oocysts themselves (Terral et al., 2010).

According to (Hamad et al., 2014), the percentage of sporozoites that were viable under control conditions (K₂Cr₂O₇) in this investigation was equivalent to comparable percentages in prior studies that used rabbits of the Eimeria species (Hamad et al., 2014). The results of our investigation agree with the findings of another study that investigated the inhibitory effect of Curcuma longa on the activity of E. tenella sporozoites (Khalafalla et al., 2011). It has been proven by (Schubert et al., 2005), that the signaling of extracellular calcium and Ca2+ is necessary for the invasion of E. tenella sporozoites into host cells (Schubert et al., 2005). Research has demonstrated that extract can both activate and desensitize receptors in calcium channels (Sárközi et al., 2007). The extract of Vitis vinifera may be responsible for the observed decrease in sporozoite viability. This inhibition may occur because of the disruption of calcium-mediated signaling present in the sporozoites.

More experimental and clinical investigations are needed to better understand the plant's pharmacological and therapeutic capabilities and isolate active components.

ACKNOWLEDGMENTS

This work was supported by the Researchers Supporting Project (RSP-2024R3).

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Publication Dates

• Publication in this collection
  21 Feb 2025
• Date of issue
  Mar-Apr 2025

History

• Received
  20 Mar 2024
• Accepted
  21 June 2024