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An in vitro evaluation of the inhibitory effects of an aqueous extract of Acacia nilotica on

Competing interest: The authors have declared that no competing interest exists Abstract Eimeria tenella is one of the most important species of Eimeria that infect domestic fowl, causing coccidiosis in the poultry industry associated with drastic economic loss. Alternative treatment options are often necessary since anticoccidial drugs are prohibitively expensive, have serious side effects, or develop resistance. The role that herbal therapy plays in basic healthcare has been rediscovered worldwide. Consequently, our research assessed the in vitro inhibitory effect of escalated concentrations (6.25 mg, 12.5 mg, 25 mg, 50 mg, and 100 mg/ml) of Acacia nilotica aqueous extract (ANAE) on Eimeria tenella sporulation. Statistical analysis revealed that ANAE decreased the percentage of oocyst sporulation in a dose-dependent manner. Furthermore, ANAE showed abnormal sporulation and morphological deterioration of E. tenella oocytes. Area Under the Curve (AUC) calculation was used to determine the efficacy of ANAE and revealed that ANAE concentrations significantly reduced the coccidial score index. At 100 mg/ml, ANAE completely suppressed the sporulation of E. tenella oocysts, with obvious changes to their morphology and size. The phytochemical analysis of ANAE has shown that ANAE contains several active principles that possess anthelmintic activities. These compounds include tannins, saponins, flavonoids, terpenoids, and alkaloids, which can be attributed to the anticoccidial activity of ANAE. Considering our findings, we recommend that ANAE be used to prevent and control Eimeria.


Introduction
In the industry of poultry, Coccidiosis is a main economic disease (Williams, 2005). Coccidiosis is the most prevalent disease among growing chickens all over the world. Its spread causes a restriction on the development of the poultry industry. Coccidia has nine species, but seven Eimeria are known among chickens, which are E. maxima, E. acervulina, E. tenella, E. brunetti, E. praecox, E. necatrix, and E. mitis (Conway and McKenzie, 2007). Avian coccidiosis is categorized as intestinal and cecal forms. E. tenella is highly pathogenic, causing bloody cecal coccidiosis (Kaufmann, 1996). Most anticoccidial drugs, such as Amprolium, inhibit the normal growth, metabolism moreover reproduction of coccidia. When anticoccidial drugs are overused, there is a risk of resistance and residues in the poultry (Chapman et al., 1997;Tajick and Shohreh, 2006). Thus, finding new drugs with fewer drug residues and low drug resistance is urgent. Herbal therapy and their byproducts have many advantages, such as few drug residues, side effects, less drug resistance, and low prices. Moreover, they have bioactive components, for instance, tannins, alkaloids, phenolic acids, flavonoids, and terpenes (Abbas et al., 2017a). These components have antioxidant and anticoccidial activity (Abbas et al., 2017b) more than synthetic drugs (Mohiti-Asli and Ghanaatparast-Rashti, 2015). Acacia nilotica has various phytoconstituents, including alkaloids, volatile essential oils, phenols, phenolic glycosides, and terpenes.
These phytoconstituents have a high therapeutic potential and can be used to prevent and treat various infectious diseases and harmful conditions (Sadiq et al., 2015). Therefore, the study aims to evaluate in vitro inhibitory effect of Acacia nilotica pods aqueous extract (ANAE) on Eimeria tenella sporulation.

Materials and methods Collection and processing of plant samples
Taxonomy of Acacia nilotica was conducted by the Botany Department, Faculty of Science, South Valley University, Egypt. Acacia nilotica's pods were collected from Qena Province. After a thorough cleaning, they were shaded and dried for 5-7 days at 32-35°C and 50-60% relative humidity.
The dried pods were mechanically ground in a commercial stainless-steel blender.

Aqueous extract preparation
The aqueous extract of the Acacia nilotica pods sample was prepared via adding distilled water (2 L) to 200 g of powdered pods sample and boiling it for 15 minutes, then filtered by a muslin cloth and Whatman® filter papers. The extracts were concentrated by a laboratory rotary vacuum evaporator at 40°C. The dried extract (chocolate-colored crystals) was weighed, labeled, stored in a clean glass bottle, and kept in the refrigerator at 4°C until needed for usage. The yield was estimated concerning the powdered pod's sample.

Preliminary qualitative phytochemical screening of ANAE
To identify secondary metabolites, present in ANAE, a preliminary qualitative phytochemical analysis was conducted (Harborne, 1998;Trease and Evans,1983).

In vitro inhibitory study:
Eimeria tenella oocysts were obtained from the infected chicken cecum. Feces obtained from the cecum of infected broilers were dissolved in a saturated salt solution for flotation. Oocysts were then collected and washed with saline before being purified, indicating that they were sedimented by centrifugation at 1300 x G for eight minutes, discarding the supernatant and making a total amount of 6 ml. Using Mc-Master Slides, 13000 oocysts were counted in every 1 ml. we added 1 ml of different concentrations of ANAE (12.5,25,50,100,and 200 mg) and 1 ml of the oocyst solution in potassium dichromate 2.5% to each well. For 5 days, oocyst sporulation was examined and counted daily in each well. A 26well plate incubated in a humidified chamber was utilized in a modified protocol for Eimeria sporulation. A Beaker was filled with water as a humidity source. The oocysts were counted in each well (Williams, 2006). The counting of oocysts in each well was performed as follows: (Williams, 2006). The suspension in a well was properly mixed, and then 1 mL of this sample was diluted with 9 mL of distilled and deionized water before being centrifuged at 400 g for 10 minutes. Using a pipette, 9 mL of the supernatant was withdrawn, and 1 mL of the remaining supernatant was resuspended with the sediment. A 100 L subsample was immediately extracted, deposited on a clean, grease-free-standard glass microscope slide, and covered with a 64 × 22 mm no. 1 glass coverslip. The slide was inspected with low and high magnifications by an inverted microscope.
Oocysts with/without sporulation were counted. Every oocyst observed beneath the coverslips corresponded to one oocyst mL-1 of the suspension in the well. This process was continued until no suspension remained in the well. The total number of oocysts (unsporulated and sporulated) in the well suspension was measured by the average count of two subsamples.

Statistical analysis
GraphPad Prism software was used to perform the One-Way ANOVA and to generate the primary graphs.
The Percentage-Maximum-Possible Effect (% MPE) was calculated as the percentage difference between the measured (treated sample) and baseline response (control sample) and divided by the difference between the maximum response and the baseline response. All data were presented as Mean ± SD.

Results
The phytochemical profile of ANAE confirms its presence of tannins, saponins, flavonoids, terpenoids, steroids, proteins, amino acids, carbohydrates, and the absence of alkaloids and anthraquinones (Table 1).
Sporulation index recorded a highly significant decline (P < 0.0001) with ANAE 25, 50 and 100 mg/m. In contrast, the index was found to be insignificant with ANAE 6.25 and 12.5 mg/ml compared to the control ( Fig  1A). Data analysis revealed that ANAE inhibited oocyst sporulation with the inhibition concentration 50% (IC50) was 40.27 mg/ml (Fig 1B).

The impact of ANAE on sporulated oocysts count:
The number of sporulated oocysts in ANAE 50 and 100 mg/ml decreased on day 3 (P < 0.01, P < 0.001) and significantly on day 5 (P < 0.0001) compared control (Fig 2A). Coccidial score of all concentration of ANAE 6.25, 12.5, 25, 50, and 100 mg/ml recorded a highly significant (P < 0.001) decrease compared to control group (Fig 2B). The Percentage Maximum Possible Effect (%MPE) was calculated as the percentage difference between the measured and baseline response and divided by the difference between the maximum response and the baseline response. The percentages 10, 20, 53, 73, 98% for 6.25, 12.5, 25, 50, and 100 mg/ml concentrations of ANAE respectively, as represented in (Fig. 3). presented as the mean ± S.D. of 3 for each group. ***p < 0.001 compared with control group. The number of sporulated oocysts in ANAE 50 and 100 mg/ml decreased on day 3 (P < 0.01, P < 0.001) and significantly on day 5 (P < 0.0001) compared control. Coccidial score of all concentration of ANAE (6.25, 12.5, 25, 50, and 100 mg/ml) recorded a highly significant (P < 0.001) decline compared to control group.

Fig. 3. The Percentage Maximum Possible Effect (% MPE)
was calculated as the percentage difference between the measured and baseline response and divided by the difference between the maximum response and the baseline response. The percentages 10, 20, 53, 73, 98% for 6.25, 12.5, 25, 50, and 100 mg/ml concentrations of ANAE, respectively.

Effect of different concentrations of ANAE on morphology and size of Eimeria tenella oocyst:
ANAE at 6.25, 12.5, and 25 mg/ml showed morphological alterations in sporocysts and sporozoites, with aberrant sporulation, while the oocyst sporulation process is completely inhibited by damaging of oocyst wall in ANAE 50 and 100 mg/ml as shown in (Fig 4A). ANAE 38 50 mg/ml induced a significant decrease in oocyst size (P < 0.05), with a highly significant decrease in the size (P < 0.0001) in ANAE 100 mg/ml (Fig 4). sporocysts, and normal sporozoites (untreated control); (ANAE 6.25, 12.5, 25 mg/ml) exhibited significant morphological alterations in sporocysts and sporozoites, with aberrant sporulation. (ANAE 50 mg/ml) Showed significant changes in oocysts morphometry, and morphological alteration in Sporocysts and Sporozoites, the sporulation process came to a halt and did not proceed. (ANAE 100 mg/ml) The oocyst sporulation process is completely inhibited, with visible morphological alterations.

Discussion
The present study investigated the in vitro inhibitory effect of ANAE on the sporulation of E. tenella oocyte, which is one of the most crucial factors affecting the epidemiology of coccidiosis since poultry infected by  -Tawab et al., 2020;Balta et al., 2021;Yang et al., 2019;Yong et al., 2020). Acacia is native to Egypt and widely distributed worldwide in tropical and subtropical countries like Asia, Australia, Africa, and America (Spicer et al., 2007). The plants have many secondary metabolites (Alli et al., 2011;Brenan, 1983)  Research showed tannins, flavonoids, and saponins are known to have anticoccidial activity by decreasing sporulation (Anteneh et al., 2021). Tannins and saponins penetrate/s the coccidia's oocyst wall and destroy the cytoplasm, as they inactivate the endogenous enzymes responsible for the cycle of sporulation in chickens (Molan et al., 2004;Mwale et al., 2006;Yim et al., 2011) and (Sanchez-Hernaandez et al., 2019) reported sporozoite damage and growth inhibition of Eimeria by the effect of saponins. Saponin also binds to the 4-sterol molecules on the Eimeria cell membrane and causes lipids disturbance leading to cell death (Wang et al., 1998;Cheeke, 2000;Alfaro et al., 2007). Du and Hu, 39 (2004) also demonstrated the inhibiting effect of saponins and Triterpenoid glycosides on the maturing of unsporulated and killing effect on the sporulated oocysts. Polyphenols and flavonoids alter the oocyst wall formation process and inhibit sporulation by destroying the sporozoites (Pop et al., 2019). In this work, we illustrated the ability of ANAE to inhibit Eimeria oocyst sporulation by different concentrations of ANAE at 6.25, 12.5, 25, 50, and 100 mg/ml for 5 days. Sporulation inhibition study showed an increase in sporulation inhibition with increasing concentration of the extract, and sporulation inhibition is commonly used to evaluate anticoccidial activity (Molan et al., 2009). It is possible that the phytochemicals had an antisporulation effect by interfering with sporulation-related physiological processes such as oxygen availability and suppressing the sporulation enzyme (Zaman et al., 2012). Various concentrations of ANAE not only inhibit the sporulation of oocyst but also produce some morphological changes (shape and size). Similar observations were obtained by (Fatemi et al., 2015), who found that petroleum ether (PE) and ethanol Artemisia extracts (E) inhibited sporulation of Eimeria oocysts in 2 and 5 ppt concentrations, many oocysts in PE and E groups were wrinkled and contained abnormal sporocysts at the same concentration. The mechanism is unclear, but PE and E extracts of the plant may penetrate the oocyst wall and damage sporozoite in 2 and 5 ppt. Considering that the deformed oocysts are not infective, the impact of reducing the infective oocysts is, thus, doubled (McDougald, 2013;Conway and McKenzie, 2007). Also Wondimu et al., (2021) showed that the exposure to a higher concentration of crude extract (100 mg/ml) produced a greater proportion of oocyst wall deformation, which was 6,000, 9,600, and 5,400 for Vernonia amygdalina, Croton macrostachyus, and Azadirachta indica, respectively. Swelling and corrugation of curcuminexposed sporozoites at concentrations of 25, 50, 100, 200, and 400 μM may reflect a drastic osmotic disturbance due to affected cell membranes of treated sporozoites (Khalafalla et al., 2011). In conclusion the present study provides insight into the therapeutic potential of ANAE as an effective inhibitor of Eimeria tenella sporulation and a promising agent against chicken coccidiosis.