Evaluating in vitro Efficiency of Calotropis procera against Eimeria flavescens Oocysts, Isolated from Domestic Rabbits

The commercial cultivation of rabbits (Oryctolagus cuniculus) as a source of protein has increased recently. Due to consumer preference for rabbits due to their low fat and cholesterol levels, the production of rabbits has become one most valuable animal resources (Lukefahr et al., 2000; Mensah et al., 2014; Murshed et al., 2024). Because of their prolificacy, low herbage intake and high feed conversion, rabbits can produce tonnes of healthy lean meat in a relatively short period (Beaumont et al., 2003; Birolo et al., 2022). Apart from their commercial worth, these animals serve as valuable models for medical study and are also kept as pets (Mapara et al., 2012). Rabbit production has few constraints; however, parasite infections are a problem. Infections of rabbits have the major intestinal parasite species, including nematodes, trematodes, cestodes and coccidia (Eslampanah et al., 2014; Raue et al., 2017; Marhoon et al., 2018). One widespread protozoal illness that affects rabbits worldwide is coccidiosis, which results in a high death rate among domestic rabbits. The protozoan Eimeria, which causes coccidiosis, is still one of the most important parasites that hinders rabbit production, generating significant economic losses in commercially raised rabbits (Yin et al., 2016). The disease affects organs such as the bile duct, the intestines and the liver and losses may occur as a result of significant morbidity and death (Maertens et al., 2000; Abdel-Megeed et al., 2005; Manjunatha et al., 2019). Eimeria species infesting rabbits are numerous, with varying degrees of pathogenicity. They dwell in a variety of habitats in the intestinal and liver (Pakandl, 2009; Pilarczyk et al., 2020). There are two anatomic forms: Intestinal (induced by Eimeria intestinalis, Emagna, E irresidua, E media, E perforans, E flavescens, or other Eimeria spp.) and hepatic (caused by Eimeria stiedae). Ingestion of the sporulated oocysts, which are typically found in tainted feed or water, is the means of transmission for both the intestinal and hepatic forms (Siotas et al., 2021). The severity of the infection varies according to the species of Eimeria involved, the dose of oocysts used and the age and physiological status of the rabbit. Young rabbits often die at high rates during the early phases of weaning (Chowdhury and Fraser, 2008; El-Ghoneimy A, El-Shahawy, 2017). The widespread spread of Eimeria spp. necessitates continuous monitoring and the implementation of preventive measures to reduce the rate of infestation. Calotropis Procera can be used as an alternate anticoccidial agent to chemotherapy medications for the management of Eimeria species (Zaman et al., 2012). C. Procera is a member of the Asclepiadaceae family and widely grows in many regions of Egypt and other nations. An evergreen perennial shrub with soft wood and few stems, branches and leaves that are mostly concentrated towards the growing point (Mutiso, 2023). The primary components of C. Procera latex are cardenolides, triterpenoids, anthocyanins, alkaloids, resins and proteolytic enzymes (Balekar, 2016).
       
Calotropis procera grows well in dry habitats and is frequently observed in open environments with little competition. It has been discovered that the gigantic milkweed is useful in treating leprosy, fever, menorrhagia, malaria and snake bites (Sharma et al., 2012). Potential uses for the plant include anthelmintic, analgesic, anticoagulant, anticancer, antipyretic, purgative, antibacterial and inflammatory qualities (Aggarwal et al., 2016). In naturally infected, C. procera flowers significantly diminish the egg count % of gastrointestinal nematodes and temporarily paralyse red stomach worms (Iqbala et al., 2005).
       
Therefore, the purpose of the study was to ascertain how C. Procera affected Eimeria flavescens that infected rabbits.

Plant materials and sample gathering
 
Fresh C. procera leaf samples were collected from the desert of Saudi Arabia. The plant was identified by a specialist in botany at the Faculty of Sciences of Riyadh of King Saud University. After cleaning with distilled water to get rid of any dust, the samples were left to dry for a week at room temperature in the shade. Before being analyzed, about 0.5 kg of leaves were ground into a powder in a grinder for five minutes and kept in dark bags to keep out light and moisture.
 
Crude extract preparation
 
The dried powder leaves of 200 gm in 80% methanol were maceration with under stirring at room temperature for 2 days. After filtration (Whatman sheets), the extracts were evaporated to dryness under vacuum (Yamato RE300, Tokyo, Japan) at 40°C., the extract was kept at “20°C until they were employed in an experiment. To dissolve the powder for the various experiments distilled water was used (Zhang et al., 2018).
 
Measurement total phenolic content (TPC)
 
The Folin-Ciocalteu test was used to calculate the extract total phenolic content (Singleton 1999). Gallic acid was used in the external calibration at several concentrations, including 0.00, 0.25, 0.50, 0.75 and 1 mM. 2.0 mL of solution A (mixing 10 mL of 2% Na₂CO₃ with 0.1 mL of CuSO₄ and 0.1 mL of sodium and potassium tartrate) and 200 mL of extracts (1 mg/mL) were combined and after 4 minutes, 0.4 mL of 0.5 M sodium hydroxide was added. 0.2 mL of the Folin-Ciocalteu reagent (1:1 v/v with water) was added after 10 minutes. A UV–Vis spectrophotometer was used to measure the solution’s absorbance at 765 nm after it had been left for 30 minutes. Using the gallic acid calibration curve (y = 0.005-x-0.0088), the total phenolic content was determined in mM of gallic acid equivalent (mM GAE). where (y) is absorbance and (x) is concentration.
 
Measurement content of total tannin (TTC)
 
The total tannin content was determined by utilizing this approach. An overall volume of 0.1 mL of the extract samples was put into an Eppendorf tube with a capacity of 2 mL, which already included 1.5 mL of Milli-Q water and 0.1 mL of the Folin-Ciocalteu phenol reagent. This mixture was incubated for 8 minutes. After that, to neutralize the solution, 0.3 mL of a sodium carbonate solution containing 35% was added to the combination. After that, the ingredients were thoroughly combined and then placed in a dark, ambient temperature area for twenty minutes. The measured value for the wavelength was 700 nm. The following equation, (Y=0.0013x+0.0052 with R² = 9937) was utilized to determine the tannin content total that was calculated was given in units of mg/g dry weight (DW).
 
Measurement content of total flavonoid (TFC)
 
CPLE’s total flavonoids were measured according to the method Ordonez et al., (2006). 2% AlCl₃ (1.0 mL) water solution was combined with 1.0 mL of plant extract (1 mg/mL). Incubation at room temperature for an hour yielded absorbance at 420 nm. The standard solution and standard curve (R²= 0.9996) were prepared using 50-800 g/mL quercetin solution. Flavonoids in the extracts were expressed as quercetin (mg/g DW) using the calibration curve equation, y= 0.0011x+0.0928, where y is absorbance and x is quercetin equivalent concentration (mg/g).
 
Oocysts preparation
 
The experiment was conducted at the King Saud University Parasite Laboratory, Zoology. Samples of rabbit feces were collected and examined for Eimeria infection. Using flotation, oocysts were cleaned and concentrated before experimental use. The McMaster method was used to examine stool for Eimerea spp. oocytes. More than one type was obtained. The oocysts were distinguished based on their morphological characteristics. 10 oocysts were separated for each species, E. flavescens injected was into rabbits free of Eimeria to obtain pure oocysts. Both the prasite oocysts field isolates were kept alive by undergoing periodic transmission (passage) through young rabbits.
 
Application of the experiment
 
After obtaining pure Unsporulated E. flavescens oocysts. A test was conducted on the oocyst’s sporulation in the solution of 2.5% Potassium dichromate K₂Cr₂O₇ (10³ oocysts/mL) with 3 mg/mL of C. procera dissolved in distilled water at 23°C for 96 h. Used was 2.5% potassium dichromate solution as the non-treated control; five different extract concentrations (10, 20, 40, 80 and 100 mg/mL); In addition, 25 mg/ml Toltrazuril as the reference medicine. The mixture’s oocysticidal properties were assessed after 24, 48, 72 and 96 hours. The percentage of sporulated oocysts was assessed by microscopic observation and counting the number of sporulated oocysts in a total of 100 oocysts.  
Statistical analysis
 
The data was analyzed using one-way ANOVA and provided as mean ± SD of three replications. The levels of significance were set at P≤0.05.

Plant extracts have garnered interest as potential anticoccidial agents in the poultry and rabbit industry (MUSA, 2021; Murshed et al., 2023). This is because coccidial infections result in production losses, treatment expenses and the prevention of disease (Abebe and Gugsa 2018). Environmental circumstances that are conducive to infection sporulation, are all factors that contribute to the development of coccidiosis (Knight et al., 2018). To control this parasite, it is necessary to prevent the process of sporulation from occurring by interrupting the life cycle. In addition to elevating the likelihood of contracting coccidiosis, it has the potential to raise the risk of contracting other infectious diseases and parasitic infections (McDougald et al., 2020). CPLE and its metabolites have the potential to serve as an alternate method to anticoccidial medication for coccidiosis administration (Zaman et al., 2012).
       
Fig 1 shows the results of the phenolics TPC, flavonoids TFC and tannin TTC measurements taken of the extract. The total potency concentration of the extract was determined to be 160.22±0.652 mg GAE/g DW, 34.046±0.545 mg QE/g DW and 65.869±0.263 mg TAE/g DW, respectively (Fig 1).
 

       
Microscopic examination and enumerating of oocysts treated with C. procera at different concentrations demonstrated, inhibited, sporulated and lysis, which contained deformed and deteriorated oocysts with serrated walls, as shown in Fig 2.
 

 
The inhibition rates after four days were (89.12 ± 2.11) (78.42 ± 7.34) At the doses of 100 and 80% of extract, compared to the reference drug (91.66 ± 3.13) of 25 mg/ml Toltrazuril. CPLE significantly reduced sporulation rate (P≤0.05) and it stopped at a dose of 100 % AT 96 h.
       
The sporulation rates of CPLE relative to control were 100%, 96±1.55 and 61.85% for 5, 2.5 and 1.25 mg doses, respectively (Table 1). Also, the oocysticidal effect was confirmed by the higher ratio of damaged and ruined oocysts 83.43±7.43, 71.33±5.87 and 50.66±7.02, for concentrations 80, 100% of CPLE doses and 25 mg/ml Toltrazuril, respectively compared to the control.
 

  
The highest rates of inhibition and the lowest rates of sporulation were seen when extract concentrations of 40%, 20% and 10% were compared to the control group. Lower concentrations, on the other hand, did not exhibit any statistically significant changes. It is consistent with the findings of (Murshed et al., 2022), which stated that C. procera has the potential to diminish E. stiedae oocysts when present in high concentrations. In several cases, the inhibition rate was enhanced by increasing the dosages and extending the incubation period. who discovered that the level of oocyst inhibition in potassium dichromate might reach roughly 96% at higher concentrations of C. procera. According to the findings, there was a gradual and dose-dependent reduction in the amount of oocyst being produced. extract inhibited at dosages of 40%, 20% and 10%, with corresponding inhibition rates of 79%, 34.32% and 26%.
       
The results showed variation in sporulation and inhibition with different concentrations during an incubation period of up to 96 hours (four days), during which oocyst test results were taken every 24 hours.
       
The results showed the highest inhibition of oocyst at the concentration of 100 mg/mL and the reference drug, where the rate of inhibition was almost constant during the periods (24, 48, 72 and 96 hours), compared to the other concentrations where the rate of inhibition decreased at lower concentrations, while the increase in sporulation continued in the control with increasing time (Fig 3 and 4).
 

 

 
The results showed the highest sporulation rate of oocysts at 100% in the control group, while the sporulation rate was low at high concentrations despite the increase in incubation periods up to 96 hours, which decreased to a minimum with the reference drug, concentrations 100 and 80% of the extract, while sporulation rates increased at lower concentrations as the incubation period increased (Fig 5 and 6).
 

 

       
The inhibition percentage of the E. flavescens oocysts increased with increasing concentration. The inhibition was highly negatively correlated with concentration R² = 0.9274, p ≤0.01, at 96 h. At Control, 10 mg/ml, 20 mg/ml, 40 mg/ml, 80 mg/ml, 100 mg/ml and Toltrazuril 25 mg/ml (Fig 7).  The overall results of the current study suggest that the leaf extract of C. procera possesses potential oocysticidal properties, which could potentially be employed in coccidial administration.
 

       
The destructed E. flavescens oocyst was highly negatively correlated with concentrations R² = 0.921, p≤0.01(Fig 8).
 

       
Inhibition were found to have a direct relationship over a considerable amount of time (Han et al., 2022). The efficiency of several other extracts varied according to the percentage of concentration and the amount of time spent incubating them. There were no statistically significant differences seen between the low concentrations of C. procera, which were 50%, 25% and 12.5% respectively. These findings agree with the findings of (McDougald et al., 2020), who found that high quantities of sheep bile could inhibit the growth of E. stiedae oocysts. According to (Bezerra et al., 2017), who discovered that larger doses of extract could inhibit oocysts by around 96% in potassium dichromate, the data presented here are consistent with those findings. As a result, a direct connection between inhibition and sporulation was found to exist throughout a considerable amount of time (Murshed et al., 2022). Experimentation revealed that Punica granatum L., Plantago asiatica L., Bidens pilosa L., Acalypha australis L., Pteris multifida Poir and Portulaca oleracea L. extracts, possessed the capacity to prevent the invasion of Eimeria tenella h in vitro and in vivo (href="#han_2022">Han et al., 2022). P. macrophylla extracts may have been responsible for the decreased sporozoite viability, according to a study that was conducted by (Cedric et al., 2017). This occurred because the extracts interfered with calcium-mediated signaling in the sporozoites. The results of the study demonstrate that CPLE, which is an extract of Calotropis Procera leaf, is an efficient method for preventing the formation of oocysts.

The extract of Calotropis Procera 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. More experimental and clinical investigations are needed to better understand the plant’s pharmacological and therapeutic capabilities and isolate active components.
 

We thank the Project of Supporting Researchers No. RSP2024R3, at King Saud University, Saudi Arabia.

The authors declare that there are no conflicts of interest.