In vitro Therapeutic Evaluation of Calotropis procera Extract against Eimeria perforans Oocysts Infecting Rabbits

The Eimeria genus is the predominant cause of coccidiosis, a widespread and impactful parasitic infection affecting animals. Particularly in local rabbit populations, it leads to substantial mortality rates (Madlala, Okpeku and Adeleke 2021). Adult rabbits that are carriers of a coccidial illness and are generally asymptomatic have the potential to produce infections in young, with morbidity and mortality rates reaching up to 90% and 60%, respectively with young (Fang et al., 2019).
       
Eimeria perforans, identified as a slightly pathogenic species developing in the duodenum, adds to the complexity of this issue (Katsui et al., 2022). The life cycle of Eimeria involves both external and endogenous phases and it is classified as an obligatory parasite in the phylum Apicomplexa (López-Osorio  et al., 2020). Entering the host through the oral route, these parasites infect and proliferate within the mucosal epithelia and various digestive tract sections, causing gastrointestinal damage, inflammation, bloody or watery feces, other symptomatic manifestations, morbidity and death (Ananthakrishnan and Xavier, 2020). According to scientific studies, this pathogen has the potential to disrupt the microbiota of the intestinal tract and make its environment of the intestinal tract more conducive to the growth of pathogenic bacteria such as Clostridium perfringens, which can ultimately lead to necrotic enteritis (Madlala et al., 2021).
       
Eimeria infection destroys the mucosal cells in the host, leading to increased cell permeability, leakage of nutrients and plasma proteins, reduced digestion and decreased protein absorption (Madlala et al., 2021). These side effects contribute to the clinical and subclinical manifestation of coccidiosis (Yang et al., 2020). Dehydration, anemia, hypoproteinemia and nutrient malabsorption are among the potential outcomes of induced villus atrophy, which can also lead to electrolyte imbalance.Consequently, commercial rabbits were economic costs (Montoro-Huguet  et al., 2021). Managing these disorders requires costly therapeutic interventions, including chemoprophylaxis and live-attenuated vaccines, both of which have notable disadvantages such as potential parasite resurgence and medication resistance (Madlala et al., 2021). Given existing challenges, the search for novel agents with unique modes of action is imperative (Waters et al., 2023). Natural products, such as Calotropis Procera, have emerged as potential supplementary and alternative means of controlling coccidiosis. Calotropis procera’s anti-coccidial action C. procera suggests that it may be a useful substitute for chemotherapy drugs for Eimeria species (Murshed et al., 2023).
       
Calotropis procera, which belongs to the Asclepiadaceae family, is an evergreen, perennial shrub with unique medicinal properties (MANDILA, 2021). Its latex contains triterpenoids, alkaloids, cardenolides, anthocyanins, resins and proteolytic enzymes (Mali et al., 2019). C. procera thrives in arid environments and open spaces with little competition (Siraj et al., 2023). Treatments for leprosy, fever, menorrhagia, malaria and snake bites have been linked to giant milkweed (McGaw et al., 2022). With known benefits in various ailments, including analgesic, anticancer, anticoagulant, anti-inflammatory (Khan et al., 2020a) and antimicrobial properties, C. procera has demonstrated its efficacy in preventing coccidian and inhibiting oocyst sporulation in Eimeria papillata (Mpofu, 2022; Murshed et al., 2024). Recent studies have highlighted the oocysticidal activity against Eimeria stiedae (Murshed et al., 2023). Considering this background, the current investigation aims to assess the effectiveness of C. procera in handling rabbit Eimeria perforans oocysts and sporozoites through in-vitro trials, providing valuable insights into potential alternative therapies for coccidiosis.

Experiment Implementation
 
The experiment was carried out in the Parasitology Laboratory, Department of Zoology, College of Science (Kingdom of Saudi Arabia). The experiment was carried out 3-4 months in 2024.
 
Collected plant leaf
 
Calotropis procera leaf extracts were obtained from an herbal plant in the desert of Riyadh, Saudi Arabia and the identification of the plant was confirmed by a classification scientist in the Botany Department of King Saud University. Distilled water was used to dissolve the powder in various experiments distilled water was used (Begashaw et al., 2017).
 
Parasite
This study was conducted by Veterinary and Sanitary Expertise (Saudi Arabia) at the King Saud University Parasitology Laboratory of the Department of Zoology. Samples of rabbit feces were collected for analysis when it was discovered that they were naturally infected with Eimeria. Feces were examined for oocysts of Eimerea spp. This strain was obtained from E. perforans. Oocysts are distinguished based on their morphological characteristics (Ogedengbe, 2011).
 
In vitro: E. perforans oocysts sporulation test by C. procera
 
To determine the effects of different concentrations of C. Procera on the sporulation of E. perforans oocysts, an experiment was conducted In vitro. In this particular test, C. procera was introduced into a mixture of 2.5% potassium dichromate, which included 1 x 10³ oocysts. The final concentration of the mixtures ranged from 5 to 1.25%. Oocytes in the control group were not treated. Each treatment was performed thrice to ensure accuracy. After each Petri dish had been incubated at a temperature of 28^oC for four days, the sporulation percentage was determined for each treatment by counting the number of oocysts that had sporulated and those that had not sporulated using the McMaster technique. Morphological alterations, malformations and destruction percentages were evaluated for each therapeutic intervention.
 
Preparation of sporulated oocysts
 
Oocysts of E. perforans were obtained from the duodenum. The method according to (Schito et al., 1996).
 
Statistical analysis
 
One-way analysis of variance (ANOVA) was performed for every experimental group using the statistical program SPSS 22 (SPSS, Chicago, IL, USA), along with a Duncan post-hoc test for multiple comparisons. The significance level was set at p≤0.05. To evaluate the correlations between variables, a straightforward linear correlation study was conducted using Pearson’s approach.

The effects of different doses of C. procera over different periods on the prevention of E. perforans sporulation were tested. The methanolic extract showed that the longer the incubation period, the greater the rate of inhibition; the opposite was true for sporulation rate. As the incubation time increased, the rate of sporulation inhibition continued to vary depending on the extract concentration (p≤0.05) in the treatment groups. In general, the best dose for inhibiting the parasite was 200 mg/ml, compared with the control group (2.5% potassium dichromate) and the reference treatment (Fig 1). The consistent application of natural extracts on animal farms, including rabbits and poultry, could emerge as a therapeutic and preventive strategy to diminish the survival rate of oocysts belonging to the genus Eimeria and prevent them from maturing (Kuralkar and Kuralkar, 2021). The effects of natural product extracts on the oocysts of these parasitic protozoa have been the subject of extensive research. In our previously published study, we investigated the effects of Nerium oleander leaf extract on E. magna and E. exigua oocysts, bile on E. columbae and E. papillate oocysts and Calotropis procera leaf extracts on E. stiedae oocysts (Murshed et al., 2025). In a methanolic extract exposed to oocysts for 96 h, the results demonstrated a significant inhibition ranging from 90% to 98%.


       
Coccidiosis, a globally prevalent disease, significantly hampers rabbit productivity and leads to substantial financial loss. The associated morbidity and mortality from these parasitic infections not only impair livestock productivity but also pose a severe health risk (Sander et al., 2020). Understanding the current species and prevalence of intestinal parasites is crucial for minimizing financial losses in the rabbit sector, assessing infection risks and implementing effective control measures (Pinto et al., 2024). Numerous authors have explored the effects of diverse plant extracts on oocyst viability and sporulation duration (Murshed et al., 2024; Abd-ELrahman  et al., 2022). These extracts have proven effective in inhibiting sporulation and reducing the vitality of Eimeria oocysts (Khan et al., 2020b).
       
The experimental groups showed significantly higher rates of inhibition with increasing doses and the statistical analysis showed that there were statistically significant differences between the 100 mg/mL dose compared to the control group (p<0.01) and between the reference drug toltrazuril (30 µg/mL) compared to the control group (p<0.05). The other doses did not show any significant differences (Fig 2). This study investigated the effect of C. procera extract over varying durations (24, 48, 72 and 96 h) and concentrations (12.5%, 25%, 50% and 100%) on E. perforans oocyst sporulation in vitro. The highest effectiveness of the tested concentrations resulted in approximately 91% inhibition of sporulation after 96 h of exposure to the 100% C. procera extract. In contrast, the control group (K₂Cr₂O₇) exhibited higher levels of oocyst sporulation. The efficacy of different extract concentrations varied depending on the concentration percentage and incubation period of the oocysts. Comparing extract concentrations of 50%, 25% and 12.5% to the control group, the highest rates of inhibition and lowest rates of sporulation were observed, whereas lower concentrations showed no statistically significant differences. These findings align with those of (Murshed et al., 2023b), who reported that C. procera may reduce E. stiedae oocysts at high concentrations. Extended incubation times and doses often improve inhibition rates. Extract inhibited at doses of 50%, 25% and 10%, with corresponding inhibition rates of 71.7%, 33.11% and 19.88%. Sporulation and inhibition are directly related over an extended period (Murshed et al., 2024).


       
Fig 3 apparition the main effects of sporulation time and experimental groups on sporulation and non-sporulation (%) of E. perforans oocysts sporulation in vitro. It demonstrates that the sporulation percentage increased with incubation time and, conversely, the non-sporulation percentage decreased. The rate of sporulation inhibition rose considerably with incubation time up to 96 h (p<0.05), showing that the rate of sporulation inhibition differed significantly between 24, 48 and 72 h exposures (Fig 3).


       
Microscopic examination and counting of the oocysts treated with C. procera at various concentrations of C. Procera revealed deformed oocysts with cracked walls and lysis. The sporulation rate in the control after three days was 898±1.41. At the doses of 100 mg/mL and the reference drug 30 µg/mL of toltrazuril (p£0.05), C. Procera significantly reduced the sporulation rate (p≤0.05) and it stopped at a dose of 100 mg/mL the sporulation inhibition rates of C. procera relative to control were 100%, 99.1%, 71%, 50%, 40%, 27% and 98%, respectively (Table 1).



In vitro experiments show the major impacts of sporulation times and tested groups on the number of E. perforans oocysts that sporulate (%), don’t sporulate (%) and inhibitory (%). The results revealed that as the extract’s concentration ratio grew, so did the proportion of inhibition. In the extract groups, on the other hand, when the concentration went down, the number of non-sporulating oocysts and destroyed oocysts also went down (Fig 4).


       
A negative correlation was observed between the sporulation and concentration. The linear equation had the highest coefficient of determination (R2) p-value. 000244, The result is significant at p<0.05. for sporulation. The p-value is .039412. The result is significant at p<0.05, for destruction (Fig 5).


       
The results align with Remmal’s study, where the effects of eight plant extracts, essential oils and their combination were investigated as potential treatments for coccidial infections, particularly against Eimeria tenella (Arczewska-Włosek  et al., 2023). Remmal independently assessed the primary components of essential oils, including carvacrol, isoeugenol, thymol, eugenol and carvone, were found to be effective against coccidians. In another study, the anticoccidial effects of essential oils from Artemisia argyi and Camellia sinensis, along with extracts from Punica granatum L., Plantago asiatica L., Bidens pilosa L., Acalypha australis L., Pteris multifida Poir and Portulaca oleracea L., were explored for their potential as anticoccidial agents. These substances demonstrated the ability to inhibit Eimeria tenella invasion both in vitro and in vivo (Han et al., 2022). A study by (Murshed et al., 2023) suggested that P. macrophylla extracts might have influenced the decreased sporozoite viability by interfering with calcium-mediated signalling in the sporozoites. Vitis vinifera leaf extracts also affected E papillae oocysts (Murshed et al., 2025), which is consistent with our results. The findings affirm that CPLE (Calotropis procera leaf extract) effectively prevents the formation of oocysts.

The elimination of parasites is critical to the health of organisms, which in turn can contribute to the development of sustainable production methods that maintain the health of consumers. It was concluded that the extract of C. procera leaves plays an important role in inhibiting oocysts and preventing sporulation and it also has a destructive effect against oocysts. Further experimental and clinical studies are needed to understand the pharmacological and therapeutic properties of plants to isolate the active compounds.

The present study was supported by Researchers Supporting Project number (RSP2024R94), King Saud University, Riyadh, Saudi Arabia
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.