In Vitro: The Effects of the Anticoccidial Activities of Calotropis procera Leaf Extracts on Eimeria stiedae Oocysts Isolated from Rabbits

Hepatic coccidiosis is an infectious and mortal disease that causes global economic losses in rabbits. The research aimed to assess the efficacy of Calotropis procure leaf extracts on the inhibition of Eimeria stiedae oocysts and to determine the optimal dosage for suppressing the parasite’s infective phase. In this experiment, oocyst samples per milliliter were tested, and 6-well plates (2 mL) of 2.5% potassium dichromate solution containing 102 non-sporulated oocysts on Calotropis procera leaf extracts were exposed after 24, 48, 72, and 96 h, and the treatments were as follows: a nontreated control, 25%, 50%, 100%, and 150% of C. procera for oocyst activities. In addition, amprolium was utilized as a reference drug. The Calotropis procera was analyzed by GC-Mass, and results showed that the botanical extract contained 9 chemical components that were able to inhibit the oocysts of E. stiedae at 100% and 150% concentrations by about 78% and 93%, respectively. In general, an increase in the incubation period and a greater dose resulted in a decrease in the inhibition rate. The results showed that C. procera has an effective ability, inhibitory potential, and protective effect on the coccidian oocyst sporulation of E. stiedae. It can be used in the disinfection and sterilization of poultry and rabbit houses to get rid of Eimeria oocysts.


Introduction
In recent years, the intensive breeding of rabbits has played an important role in covering people's needs for meat. Rabbit products are the main diet components, thus making them a suitable and important source of protein. In addition, they are devoid of fat. The effectiveness of obtaining products derived from rabbits is impaired when the animals are infected with diseases, including intestinal parasites such as Eimeria spp. [1,2].
Eimeria stiedae is the name given to the parasite that is responsible for causing hepatic coccidiosis [3,4]. Since it was found more than a hundred years ago, it has been known to be one of the most common parasites that affect rabbit colonies, which can cause big problems. Its pathogenicity may vary from moderate to severe. It is accompanied by a loss of weight, light intermittent to severe diarrhea, and stools that include mucus or blood, which can lead to dehydration and a reduction in rabbit breeding. There is a possibility of an increase in the death rate [5,6]. Adults are asymptomatic carriers of coccidioidomycosis, and they are a source of acute infection with clinical indications that can lead to infection and the death of young rabbits [7][8][9]. It is one of the most dangerous intracellular and extraintestinal coccidia species that parasitizes liver cells. It belongs to the fourteen different Eimeria species that affect rabbits all over the world [10,11]. Coccidiosis costs rabbit production USD 127 million per year, and comparable losses might occur universally [12]. The appearance of anticoccidial drug resistance catalyzed research for alternative products [13,14].
In order for intensive rabbit farming to work, live vaccinations and anticoccidial drugs put into the feed must be used to prevent coccidiosis. Over the years, drug resistance has made it so that coccidiostats have lost some of their effectiveness. Subclinical coccidiosis is caused by drug-resistant strains of Eimeria, high costs, and worries about residues in food and the environment. These are also to blame for the subsequent drop in economic performance, as measured by body weight gain and feed conversion ratio [15,16]. Natural compounds derived from plants were employed by researchers to combat parasites because these products have the potential to be useful sources for innovative anti-parasite candidate drugs. These drugs are not only effective against the parasites, but they may also protect organs in the parasite-infected target hosts [17].
Calotropis procera leaf extracts (CPLE), which are sometimes called "Madar," are a medicinal herb that is used in many different herbal mixtures to treat many different illnesses and physical problems. Madar is also a common name for the plant [18]. There have been several studies about the possible remedial qualities of CPLE, including its anti-cancer, anti-radiation, antimicrobial, anti-aging, and anti-stress capabilities. It has been shown that the aqueous extract of the flower has properties that are analgesic, antipyretic, and anti-inflammatory [19][20][21]. It is a well-known tribal plant that is used in a wide variety of traditional medicines to treat a variety of illnesses, including skin conditions, elephantiasis, toothaches, asthma, leprosy, and rheumatism [22]. Another essential component of Ayurvedic medicine is the C. procera plant. It is included in the treatment of many different disorders in the form of polyherbal medicines. One of the plants, Ayurveda, has been investigated for its potential anticonvulsant activity [23]. The dried plant may be used as a tonic and has expectorant and anthelminthic properties [24,25].
Calotropis procera has phytochemical components such as saponins, alkaloids, tannins, flavonoids, glucosides, and terpenoids. According to several studies, Table 1 shows the chemical compounds available in CPLE shrubs. These components have therapeutic features, including antimicrobial, antioxidant, anti-inflammatory, analgesic, spasmolytic, anti-fertility, and anti-ulcer activity. According to the results, CPLE has a favorable impact, has an inhibitory capacity, and protects against the sporulation of coccidian oocysts and sporozoites when tested in vitro [26]. Recently, many plant extracts, such as garlic [27], Rumex nervosus, and Vitis vinifera, have been used against Eimeria spp. [28,29].  [40] The purpose of this study is to assess the effectiveness of Calotropis procera leaves in vitro as a bioindicator of inhibition and the sporulation oocysts of E. stiedea isolated from naturally infected rabbits.

Results
The botanical chemical analysis obtained by GC-Mass ( Figure 1) showed that CPE contained nine chemically active compounds at different peak areas and retention times.
The proportion of inhibition oocysts was calculated for the control, reference therapy, and experimental groups at different times (24,48,72, and 96 h). It is important to note that oocysts began to sporulate after 24 h of incubation. Concentrations (25 and 50 mg/mL) resulted in lower oocyst inhibition percentages. The concentrations of the Madar leaf extracts were inversely proportional to the sporulation inhibition percentage of E. stiedae oocysts and directly proportional to the increase in time ( Figure 3). The follow-up to the inhibition of sporulation continued after 24 h of E. stiedae oocyst incubation in different concentrations with CPLE, and we noticed that sporulation was low in the control group and the concentrations (100, 50, and 25 mg/mL), while at 150 mg/mL and amprolium at 28 mg/mL, there was no sporulation ( Figure 4). However, a concentration of 150 mg/mL showed less than 97% inhibitory oocysts. Other concentrations showed different levels of sporulation with time increasing. While the control group (2.5% potassium dichromate solution) remained continuously sporulation throughout the incubation time until 96 h, we note significant differences between treatments with all concentrations of CPLE compared with the reference drug and 2.5% potassium dichromate control group. CPLE (150 mg/mL and amprolium 28 mg/mL) inhibited E. stiedae oocysts at marked levels after a 48-h incubation period. At the same time, the control showed high levels of sporulated oocysts, whereas concentrations of 100, 50, and 28 mg/mL of CPLE showed low levels of sporulation ( Figure 5).  The results also showed that incubation after 72 h with CPLE at concentrations of 150 mg/mL and 28 mg/mL of amprolium inhibited sporulation by nearly 68-79%, while incubation with CPLE at the same concentration and amprolium for 96 h inhibited sporulation by nearly 89% compared to the control group with potassium dichromate (2.5%), which reached 1% (non-sporulation oocysts). In addition, the increased inhibition of oocysts was at various concentrations throughout 72-96 h for CPL extracts compared with reference therapy ( Figure 6).   The experimental groups generally had a substantial impact on the rates of inhibition (%) and sporulation (%). The greatest inhibition rates and lowest sporulation rates were observed for concentrations of CPLE at 150 and 100 mg/mL (p ≤ 0.05). The lowest suppression of sporulation was seen at concentrations of 50 and 25, respectively, of CPLE (p ≤ 0.05) (Figure 8). A cumulative comparison was made between the different times (24,48,72, and 96 h) for all concentrations (25, 50, 100, and 150 mg/mL) with the control group and the reference drug that was tested (Table 4). Additionally, a cumulative comparison was made between the inhibited and sporulated oocysts (Table 5).

Discussion
Eimeria stiedae, a pathogenic species that inhabits the liver and intestine, causes significant economic losses in several countries around the world. It is identified in the examined fecal samples containing oocysts. The in vitro anticoccidial properties of C. procera extracts were assessed based on the quantity and percentage of Eimeria oocysts that were destroyed, in addition to the number of oocysts that were inhibited. As sporozoites from sporulated oocysts that have been ingested are released, they penetrate the hepatic cells, where they quickly proliferate and lead to infections caused by E. stiedae before oocysts are generated [12]. The progeny develops outside of the host inside the oocysts, and then those progenies become infectious. Eimeriosis medication resistance has been shown in several investigations [47,48]. Because of this, there is a growing interest in the use of natural products as antiparasitic agents since, in comparison to traditional chemical agents, they are more effective, less toxic, and have fewer negative side effects [17].
In recent years, a number of plant extracts have been utilized to prevent and cure infectious diseases in animals and poultry, such as coccidiosis. Several studies have shown that the Madar plant's methanolic extract possesses anticoccidial effects [49,50]. Our research showed that oocysts and damage in Eimeria were inhibited in a dose-dependent manner. The used C. procera extracts had more active chemical classes that could predict the inhibition of oocysts. The effect of the CPLE on the oocyst's inhibition of the parasite E. stiedae was tested in vitro with various concentrations (25%, 50%, 100 mg/mL, and 150 mg/mL), compared with control (2.5% potassium dichromate solution) and reference drug (Amprolium), over various time periods (48,72, and 96 h), which demonstrated the inhibitory potential of CPE on the coccidian oocysts.
According to our results, concentrations of 100 and 150 mg/mL exhibited activity against oocysts. It is shown to have maximum sporulation inhibition activities and is observed to be more effective against E. stiedae. Studies on the components of CPE against Eimeria oocysts are very few. To our knowledge, these are among the scarce reports in Saudi Arabia using CP extracts to prevent the coccidiosis oocyst sporulation in the parasite E. Stiedae. Similar to the present findings observed in vitro sporulation inhibition with Psidium guajava leaf extracts in four Eimeria species [51]. Since condensed grape tannins have been shown to inhibit endogenous enzyme activities (such as mannitol-1 phosphate dehydrogenase, mannitol-1 phosphatase, mannitol dehydrogenase, and hexokinase), it is possible that CPLE reduced the rate of sporulation by inhibiting or inactivating the enzymes responsible for the sporulation by inhibiting or inactivating the enzymes responsible for the sporulation process as in helminth eggs [52].
This supports the results of other writers who asserted that some plants, including Pinus radiata [53], Aloe vera, and Saccharum officinarum [54], exhibited anticoccidial characteristics [55]. Moreover, oocysts displayed varying degrees of damage, including damage to their cell walls. In some cases, the oocyst wall almost entirely vanished, leaving just free sporocysts visible [56]. The Madar crude leaf extract may have generated anaerobic conditions that led to these results. This could be a result of the plant extract's active ingredients. The pathogens are negatively impacted by anaerobic digestion, which is a series of microbial activities that convert the organic material in waste into biogas. They were contrasted with controls, which were different developmental phases of regular E. stiedae oocysts. Throughout a 96 h period, a high inhibitory oocyte count was also seen at different dosages of the plant extract when compared to amprolium and the control group.
We figured that the dosage of 150 mg/mL of the extract of CPLE had an effect on the process of oocysts inhibition [57], and this effect was caused by the presence of bioactive phytochemical components. That anti-coccidiosis efficacy demonstrated by CPLE led to a significant elevation in the number of inhibited oocysts from E. stiedae oocysts; this corresponds to leaf extracts used from Vitis vinifera in vitro, as proven in decreased oocyst output in the feces of mice infected by Eimeria papillate [29]. These results are consistent with previous research projects that looked into potential anticoccidial agent sources, such as Azadirachta indica [58] Punica granatum plant [59], Ziziphus Spina-Christi extract [60], mulberry extract [61], and grape [56].
Herbal extracts and other extracts from natural products are often added to poultry feed to help them grow and stay healthy by getting rid of microbes and parasites, especially coccidia (Eimeria spp.). In a great number of studies, plant extracts have been shown to improve chicken diets, as well as their performance and the efficiency with which they convert feed to meat [62][63][64]. Also, CPLE may be used as a dietary ingredient in poultry feed because of its anticoccidial activity. Similar in vitro outcomes were discovered by [52], who discovered that an aqueous pine bark extract prevented the sporulation of Eimeria oocysts. The ethnomedicinal herbs' in vitro anticoccidial effectiveness was shown to be concentration dependent.
Plant extracts with larger dilutions (i.e., 150 and 100 mg/mL) may have higher quantities of the bioactive components, suggesting that the action may be pharmacologically mediated. This corresponds with similar conclusions reached [65,66], and it was also shown that Thonningia sanguine and Aloe vera extracts had effects on the oocysts of E. tenella that depended on concentration [67]. P. biglobosa extracts, however, were less effective in preventing the formation of coccidia. This might be due to the absence or presence of only a few amounts of bioactive chemicals. The present analysis discovered that the herbal extracts exhibit anticoccidial activity against E. stiedae oocysts, which is contrary to [26], who claimed that there was anticoccidial action shown in vitro of K. senegalensis. The appearance of aberrant sporocysts in oocysts subjected to 150 and 100 dilutions of the majority of herbal extracts may be the result of fundamental plant components penetrating the oocysts' cell walls and damaging the cytoplasm [68], which claimed that there was anticoccidial action shown in vitro of K. senegalensis. Furthermore, the appearance of aberrant sporocysts in oocysts subjected to 150 and 100 mg/mL dilutions of the majority of herbal extracts may be the result of fundamental plant components penetrating the oocysts' cell walls and damaging the cytoplasm [29].
Findings indicate that Madar leaves have a strong effect on the inhibition of E. stiedae oocysts and anti-sporulation. It has a potential protective role in the disinfection of poultry and rabbit wards, as well as the sterilization of straws in case of the emergence of infection in chicken and rabbit wards.

Ethical Approval
The research was conducted in accordance with the ethical guidelines for animal use established by the Kingdom of Saudi Arabia (Ethics Committee, King Saud University, Ethics Agreement ID: KSU-SE-21-86).

Preparation of Extract
The process of preparing the extracts from the C. procera leaves in accordance with the technique that was presented by [69]. Fresh leaves and flowers were collected from herbal plant in the desert near Riyadh, Saudi Arabia, and the identity of the plant was validated by a classification scientist at King Saud University's Botany Department. The leaves were dried for 72 h in an incubator at 45 • C before being ground into a powder and extracted for 24 h at +4 • C by percolating with 80% methanol at room temperature. The extract was then stored for 24 h at 4 • C with intermittent stirring. After this, the extract that was produced was concentrated and dried using a rotating vacuum evaporator (Yamato RE300, Tokyo, Japan) at 40 • C and under decreased pressure. When the crude extracts were produced, they were weighed and then kept at −20 • C until they were employed in an experiment. In order to dissolve the powder for the various experiments, a solution of potassium dichromate (K 2 Cr 2 O 7 ) and distilled water was used.

Analysis of Phytochemical Compounds Extracted from C. procera Leaves Using GC-MS
Trace GC-ISQ Quantum mass spectrometer system (Thermo Scientific, Austin, TX, USA) was used to analyze the extract of C. procera. The used flow rate was 1 mL.mn −1 /min. About 1 µL from the sample was injected into a GC-MS equipped with a TG-5MS column (30 m × 0.25 mm ID, 0.25 µm film thickness). Helium gas was used as a carrier at a constant flow of 1.0 mL min −1 . The mass spectra were observed between 50-500 m/z. The temperature initially started at 50 • C for 10 min, then increased at a rate of 5 • C min −1 to 250 • C, and isothermal was held at 300 • C for 2 min, and finally, isothermal was held for 10 min at 350 • C. NIST, Adams, Terpenoids, and Volatile Organic Compounds libraries were used to identify the phytochemical constituents by comparing the recorded mass spectra for each compound with the data stored in the previous libraries. The relative percentage amount for each component was calculated using Retention time index and comparing its average peak area with total peak areas [70].

Oocyst Sporulation
Oocysts of E. stiedae were isolated from feces and gallbladders of rabbits that had spontaneously contracted the infection. Flotation was used as a means of concentrating the collected oocysts after they had been collected [71]. Oocyst sporulation was carried out in Petri dishes in a moist chamber with temperatures ranging from 25-28 • C, and a humidity of approximately 65% (ETI 6102 Therma-Hygrometer-Indian) in a solution of 2.5% potassium dichromate for 7 days until sporulation. Then, oocyst was washed to get rid of dichromats and pass them via young rabbits. The rabbit is followed for 5-10 days. Fresh feces samples were collected, and they were washed and concentrated according to the method [29], and then they were tested with the extracts (CPLE).

In Vitro Effect of Extracts on Inhibition of Sporulated Oocyst
A suspension of 800 µL of freshly sporulated oocysts of E. stiedea containing 1 × 10 3 oocysts was added to each 24-well plate, and then these plates were incubated in 2 mL of potassium dichromate with 1 of the following concentrations of CPLE: (25%, 50%, 100%, and 150%). Only the negative control group made use of potassium dichromate solution (K 2 Cr 2 O 7 ), whilst the positive control group made use of amprolium 60% (Veterinary and Agricultural Products Company (VAPCO, Amman, Jordan) (EMEA, 2001). We used three different replicates for each different concentration. All 24-well plates for all of the different groups were heated between 25-29 • C, the relative humidity was between 65-75%, and the plates were shaken periodically while they were semi-covered [30]. After 24, 48, 72, and 96 h, a microscopically inspected 10 µL suspension was taken from each group and seen via a light microscope (BX51TF, OLYMPUS, Tokyo, Japan) at a magnification of 40×. Using a light microscope equipped with a McMaster chamber, the viability (oocysts with sporocysts, deformed walls, and inhibitory oocysts) was analyzed, and the percentages of sporocysts and inhibitory oocysts were determined with the use of the following formulae: Sporulation (%) = a number of sporulation oocysts/total number of oocysts × 100. (1) Inhibition of sporulation (SI%) = Sporulation of control-sporulation of treated/sporulation of control × 100, as described [72].

Statistical Analysis
The ANOVA analysis was performed in one direction, and statistical comparisons between the groups then are made using the Duncan technique. At a significance level of ≤0.05, values have been reported as the mean minus the standard deviation. For the purpose of statistical analysis, the Sigma Plot program, version 11, was applied.

Conclusions
Findings showed that Madar leaves have a strong effect on the inhibition of E. stiedae oocysts and anti-sporulation in vitro. More studies are needed to accurately define the active chemical compounds in CPLE and their modes of action and applications to determine plant extracts that may be an option for the formulation of new products as alternative therapies and determine whether they can be used for in vivo treatment and to understand the histological and molecular mechanisms of CPLE and its protective effects against E. steidae-induced hepatic damage. In addition, it can be used to disinfect the floors of poultry and rabbits and fight the fallen oocysts on the floor straw.