Natural products play an important role as environmentally friendly agents that can be used against parasitic diseases. Many Eimeria species cause eimeriosis in poultry. The negative effects of synthetic anti-coccidiosis medications necessitate the quest for alternative treatments derived from medicinal plants in the treatment of eimeriosis. The study was conducted to evaluate the effects of Calotropis procera leaf extract (CPLE) (Madar) on the sporulation of Eimeria oocysts and sporozoites that affect mammalian jejunum and to obtain the best concentration for sporulation inhibition and infection prevention. Extracts were tested in vitro to prevent oocyst sporulation, wall deformity, and anti-sporozoite activity with Eimeria papillata. The plant-chemical compounds analysis of CPLE some active compounds were shown as well as CPLE in vitro effects at various concentrations (200, 100, 50, 25,12.5, and 6.25 mg/mL), while potassium dichromate solution 2.5% and Toltrazuril 25 mg/mL were administered as the control groups. C. procera leaf extract showed the highest inhibitory percentage on E. papillata oocyst at 200 mg/mL of extract, approximately 91%. In addition, CPLE showed the sporozoite highest viability inhibitory percentage on E. papillata at 200 mg/mL of extract, approximately 88%, and the lowest efficacy was 5% at 6.25 mg/mL. Also, we noticed the deformation and destruction of the oocyst wall based on the concentration rate. Sporulation inhibition rate is significantly affected by incubation time and treatment concentration ratio. The results showed that Madar has an effective, inhibitory potential, and protective effect on coccidian oocyst sporulation and sporozoites of E. papillata.
Coccidiosis is one of the more dangerous illnesses that affects a wide range of mammals . It is caused by infection with Eimeria spp. , and causes gut trac problems including diarrhea, performance, and growth decrease, and in extremely severe cases death . Eimeria spp. infections can also promote opportunistic infections with other pathogens such as bacteria . As a result, this disease causes significant economic losses all across the world .
Eimeria oocysts are relatively resistant to environmental conditions, making control challenging . As a result, disrupting the sporulation process is an important control point for this parasite . Furthermore, the widespread use of anti-coccidial feed additives for prophylaxis has resulted in widespread resistance , which has been recorded against currently available medications, e.g., amprolium, sulfonamides, diclazuril, halofuginone, nicarbazin, robenidine, and Toltrazuril . All alternative or indigenous systems of medicine rely heavily on medicinal plant products. Plants have been utilized to treat human and animal illnesses for thousands of years and are regarded as promising sources for the production of new chemical components .
Phytochemicals have demonstrated varying degrees of efficacy against microbial infections, and they are thought to have no or little adverse effects when compared to synthetic anti-microbials . These drugs have organ-protecting characteristics in Eimeria-infected hosts in addition to parasite-targeting properties .
Calotropis procera is used in a large hierarchy of medicinal plants and has been used in the treatment of numerous diseases since antiquity . It is a well-known medicinal plant in the Apocynaceae family. It is a xerophytic perennial shrub with stems that grow 2–6 m tall and tap roots that grow 3–4 m deep in the soil . When the plant’s components are cut or broken, a thick milky sap or latex is released . C. procera has long been recognized as a medicinal plant , and it has been used to treat a variety of ailments, including infectious diseases [9,15]. Amini et al. concluded that C. procera has been shown to have antibacterial, antifungal, antiparasitic, and antiviral activities .
C. procera leaf extract (CPLE) and its constituents include a variety of metabolites, including glycosides and cardenolides, flavonoids, triterpenoids, steroids, saponins, lignans, proteins, different enzymes, hydrocarbons, saturated, and unsaturated fatty acids. C. procera showed a diverse array of biological activities such as anti-microbial, anti-diarrheal, wound healing, anti-inflammatory, anti-cancer or cytotoxic, in vivo immunomodulatory and analgesic, anthelmintic, anti-oxidant, and in vivo anti-hyperglycemic [11,16].
The current study was carried out to investigate the effects of CPLEs on the in vitro sporulation and sporozoite viability of Eimeria papillata oocysts.
2 Materials and methods
2.1 Extract preparation of plant
CPLE was prepared using a wild plant obtained from the desert of Riyadh, Saudi Arabia, flowering herbal plants , and the plant’s vegetarian identification was validated by a taxonomist at the University of King Saud’s Department of Botany. The leaves (250 g) were air dried at 44°C, ground into a powder, and then extracted for 24 h at +4°C with 70% methanol. According to Dkhil et al. , the resulting extract was concentrated and dried in a rotating vacuum evaporator (Yamato RE300, Japan). The powder was dissolved in distilled water for various investigations or experimental studies.
2.2 Phytochemical analysis
The phytochemical analysis of CPLE was performed using direct analysis in real time–time of flight–mass spectrometry (DART–ToF–MS). The high-resolution mass spectrometer was an AccuTOF LC-Plus from JEOL (Japan), and it was used in accordance with the methodology described by Harris et al.  and Cody . The extract’s volatile components were evaporated in a stream of helium heated to 350°C, then ionized by excited metastable helium atoms before entering the time of flight mass spectrometer’s ion source. The molecules are mostly protonated in the positive ionization mode, with no fragmentation; each peak in the spectrum corresponds to a [M + H] + ion. The experimental conditions used for analysis of samples by DART–ToF–MS are listed below.
|Vacuum (Pirani gauge)||1.8 × 102 Pa|
|Vacuum (Analyzer)||1.3 × 10−5 Pa|
|Ring lens voltage||4 V|
|Orifice 1 voltage||5 V|
|Orifice 2 voltage||5 V|
2.3 Oocyst sporulation
Fresh E. papillata unsporulated oocysts were obtained from Prof. Mehlhorn of Duesseldorf University (Duesseldorf, Germany), and the parasite was preserved through passaging in mice until it became capable of injuring later . CPLE was utilized to evaluate in vitro sporulation of parasite oocysts using dichromate at 2.5%.
2.4 Experimental application
The unsporulated oocysts (1 × 103) were incubated in 3 mL of potassium dichromate containing one of the following: CPLE (200, 100, 50, 25, 12.5, and 6.25 mg/mL) and Toltrazuril 25 mg/mL (Veterinary Agriculture Products Company – VAPCO) as a reference control, in addition to using 3 mL of potassium dichromate as an untreated control alone (without the addition of CPLE or Toltrazuril). We used three replicates for each treatment and incubated all the Petri dishes for 24, 48, 72, and 96 h at 27–29°C . The number of abnormal sporocysts was counted (in terms of shape and size) that wall deformity and undistorted by using a McMaster chamber. The sporulated oocysts were counted, and the percentages of sporulated and unsporulated oocysts were calculated using the following equation:
2.5 In vitro anti-sporozoite effect of CPLE
Phosphate-buffered saline (PBS) solution (pH 7.4) was used to wash oocysts that had been stored in K2Cr2O7 many times until K2Cr2O7 was fully eliminated. The oocysts were then incubated in a water bath at 41oC for 60 min while being shaken. The suspension was centrifuged for 10 min at 3,000 rpm before being resuspended in PBS. PBS was used to wash the liberated sporozoites. The McMaster counting chamber was used to count the sporozoites. The in vitro sporozoite activity was assessed using 24 Petri dishes. Test solution of 1 mL was mixed with 1 mL of the parasite suspension (containing 1,000 sporozoites) to make a total volume of 2 mL of each CPLE concentration (200, 100, 50, 25, 12.5, and 6.25 mg/mL). Toltrazuril was utilized as a negative reference drug, while K2Cr2O7 was employed as a positive control group. The setup was checked after 12 and 24 h, and the procedure was performed three times for each treatment and control in the identical settings. The number of viable and non-viable sporozoites was tallied, and the viability% was calculated by counting the number of viable sporozoites out of the total 100 sporozoites. The following is how the viability inhibitory percentage was calculated:
2.6 Statistical analysis
Data were statistically analyzed by two-way ANOVA using the General Linear Models procedure of SPSS. The following model was used:
where γ ijk is the individual observation, μ is the experimental mean, G i is the effect of ith treatment, T j is the effect of jth time of incubation, GT ij is the effect of treatment by time of incubation interaction, and e ijk is the random error.
When significant, Duncan’s multiple range test at 5% probability was used for comparison between means.
The effective phytochemical components were obtained as follows: 2-acetamidobutanoate, 1-(3,4-dimethoxyphenyl)-N methanidylmethanamine, benzoxazolium, 3-ethyl-2-methyl-, iodide, 1-(3,4-dimethoxyphenyl)-N methanidylmethanamine1,3-diphenylpropane, hydroxycadalene, hexanedioic acid, monocyclohexyl ester, 2,3-dihydroxycyclopentane-1-carboxylate, N-1-adamantyl-2-ethylbutanamide, benzyl beta- d-glucopyranoside, dicyclohexyl succinate, cannabioxepane, and 2,4-dinaphthyl pentane (Table 1).
|RT||Experimental mass||Identification||Molecular formula||Unsaturation degree|
|71||162.09011||Benzoxazolium, 3-ethyl-2-methyl-, iodide||C10H12NO||5.5|
|42||230.12366||Hexanedioic acid, monocyclohexyl ester||C12H19O4||3.5|
After incubation for 24–48 h with CPLE at 200 mg/mL and Toltrazuril 25 mg/mL, the E. papillata unsporulated oocysts showed no sporulation. At a concentration of 100 mg/mL, the E. papillata unsporulated oocysts showed a sporulation percentage of approximately 5–10%. Whereas oocysts incubated with potassium dichromate 2.5% and other concentrations (50, 25, 12.5, and 6.25 mg/mL) showed different levels of sporulation (Figures 1 and 2).
The unsporulated E. papillata oocysts showed no sporulation after 72–96 h of incubation with CPLE at 200 and Toltrazuril 25 mg/mL. At a concentration of 100 mg/mL, the sporulation rate increased significantly for E. papillata unsporulated oocysts. Oocysts treated with potassium dichromate 2.5% and various doses (50, 25, 12.5, and 6.25 mg/mL) showed varying amounts of sporulation (Figures 2 and 3).
In vitro investigation, Figure 4 shows the main impacts of sporulation time and experimental groups on sporulation and non-sporulation (%) of E. papillata oocyst sporulation. It shows that the sporulation percentage increased with the increase in incubation time and, conversely, that for non-sporulation percentage. The sporulation inhibition rate increased significantly with incubation time up to 24 h (p < 0.05), indicating that the sporulation inhibition rate did differ significantly between 48, 72, and 96 h exposures (Figures 5 and 6).
After 24 and 48 h of incubation with CPLE at 200, 100, and Toltrazuril 25 mg/mL, the percentage of wall-distorted E. papillata sporulated oocysts increased compared to undistorted oocysts. In contrast, oocysts incubated with potassium dichromate 2.5% (control) and other concentrations (50, 25, 12.5, and 6.25 mg/mL) had a lower percentage of wall distortion compared to undistorted oocysts.
The percentage of wall-deformed E. papillata sporulated oocysts rose after 72–96 h of incubation with CPLE at 200, 100, and Toltrazuril 25 mg/mL, compared to undistorted oocysts. Also, oocysts incubated with potassium dichromate 2.5% (control) and other concentrations (50, 25, 12.5, and 6.25 mg/mL) had a reduced proportion of wall distortion.
It shows the sporozoite viability inhibitory percentage of CPLE on E. papillata as a function of concentrations and incubation time. It follows from the analysis of this figure that, for each time, an increase in concentration seems to have enhanced its efficacy. Thus, inhibition rates significantly increased when concentration was increased. The CPLE therefore has the potential to perform better at 200 mg/mL and probably at higher concentrations (Figure 7). As shown in Figure 7, after 12 h, CPLE at various concentrations inhibited the viability of coccidial sporozoites of E. papillata in a concentration-dependent manner when compared to control groups (K2Cr2O7) but not with Toltrazuril 25 mg/mL. Most concentrations include anti-sporozoite activities against E. papillata at 50, 100, and 200 mg/mL. The highest viability inhibitory percentage was 91% at a concentration of 200 mg/mL of CPLE against the E. papillata strain. The lowest efficacy against E. papillata was 11% at a concentration of 6.25 mg/mL after 24 h.
According to our findings, most concentrations, including the high infusion concentration, displayed anti-sporozoidal activity against E. papillata at 200 mg/mL. With the increase in incubation time, the inhibition rate increased. A high concentration of CPLE with increased time restricted the viability by 96% for E. papillata at a concentration of 6.25 μg/mL of CPLE against E. papillata. The proportion of viability inhibition reduced as the concentration of CPLE dropped (Figure 7).
Eimeriosis is a contagious disease that affects the majority of animal species and causes significant economic loss in many countries . Infections with E. papillata arise when sporozoites from swallowed sporulated oocysts enter the jejunal epithelial cells and rapidly multiply before the oocysts mature. Sporogony happens within the oocysts, outside the host, and they become infectious . Drug-resistant eimeriosis has been observed . Plants produce a wide range of phytochemicals with anti-microbial bioactivities, including phenolics, polyacetylenes, alkaloids, polysaccharides, terpenoids, and essential oils . Herbal medications are a safe alternative to chemical anti-coccidial drugs because they do not leave tissue residue or cause drug resistance [23,24], and because these compounds are more effective, less toxic, and have fewer adverse effects than traditional chemical agents . Six dosages in vitro of CPLE (200, 100, 50, 25, 12.5, and 6.25 mg/mL) were tested as a target natural product against coccidia in this investigation. In terms of anti-coccidial efficacy, this study clearly indicated that 200 mg/mL was the most effective of all the doses examined. The findings of this study demonstrate the efficacy of Madar/CPLE at 200 mg/mL in the treatment of coccidiosis. Identical outcomes were obtained in vitro by Molan and Thomas, who found that the aqueous pine bark extract inhibited the sporulation of Eimeria oocysts , and found that Thonningia sanguinea extracts had a dose-dependent effect on Eimeria tenella and Eimeria necatrix oocysts . Madar leaf extract’s anti-coccidial efficacy may be imputed to its saponin content, which has anti-coccidial properties, which acts on protozoan growth by interacting with cholesterol on the parasitic cell membrane, resulting in parasitic death [25,26], and its flavonoids and phenols contribute to its anti-oxidant capacity and decrease the Eimeria-induced damage to the intestinal wall during the pro-inflammatory reaction, resulting in less harm to the gut [27,28].
Several studies have reported the effects of Calotropis in treating many diseases , and its potential effectiveness as an anti-parasitic agent . In this study, CPLE (200 mg/mL) affected the oocyst sporulation, which is attributable to the presence of numerous bioactive phytochemical constituents . Anti-malarial activity was found in Calotropis gigantea against Plasmodium falciparum and Plasmodium berghei . Madar leaf extract’s anti-diarrheal and anti-ulcer properties also contributed to its anti-coccidial effect . Also, proved the anti-coccidial activity of Allium sativum and turmeric powder against coccidiosis in poultry, which may be attributable to their anti-oxidant qualities . Carica papaya inhibits coccidiosis by proteolytic destruction of Eimeria by papain and anti-inflammatory action by vitamin A . Garlic and its sulfur compounds, i.e., allicin, alliin, and ajoene are shown to have broad anti-microbial efficacy. An in vitro study has indicated that allicin inhibits sporulation of E. tenella activities .
The development of technologies enhances quick knowledge of anti-coccidial pathophysiology in vitro, and measurements of sporulation or inhibition of oocytes in invasion and evolution experiments can shed light on compounds’ test and how they act. The results indicate that Madar leaves are promising in anti-Eimeria as they possess powerful sporulation inhibition and anti-sporozoite activities. Additional studies are needed to verify in vivo its protective effects against intestinal infection caused by E. papillata and determine the active and safe compounds from cheap natural products and their use as a food supplement in animal feed.
The authors sincerely acknowledge the Researchers Supporting Project for funding this work (RSP-2021/3) at King Saud University, Riyadh, Saudi Arabia.
Funding information: The authors extend their appreciation to the Researchers Supporting Project number (RSP-2021/3), King Saud University, Riyadh, Saudi Arabia, for funding this work.
Author contributions: Conceptualization: S.A. and M.M., methodology: M.M., validation: M.A.Q., formal analysis: S.A., investigation: M.M. and M.A.Q., data curation: S.A., writing – original draft preparation: M.M., writing – review and editing: M.M., M.A.Q., visualization: S.A., M.M., supervision: M.A.Q. and M.M. All authors have read and agreed to the published version of the manuscript.
Conflict of interest: The authors declare that they have no conflict of interest.
Ethical approval: All of the experiments were conducted according to the Guidelines for the Institutional Animal Care and Use Committee in parasitology laboratory, Zoology Department, College of Science at King Saud University.
Data availability statement: The authors confirm that the data supporting the findings of this study are available within the article.
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