Methanolic extract of neem (Azadirachta indica) leaves mitigates experimentally induced coccidiosis challenge in Japanese quails

ABSTRACT

Eimeria infection is a common parasitic disease in Japanese quails. A total of 150 male Japanese quails were equally assigned to five experimental treatments as follows: control (basal diet only), negative control (Eimeria challenged), amprolium treated, 130 mg/l methanolic extracts of neem leaves (NMLE 130) and 190 mg/l methanolic extracts of neem leaves (NMLE 190). All the E. tenella-infected quails were orally dosed with sporulated oocysts (n = 5.5 × 10⁴). The results showed that compared to the negative control, supplementation of NMLE increased feed consumption, body weight and feed conversion ratio in Eimeria-infected quails. Mortality percentage and lesion score were significantly (P < .01) decreased in quails supplemented with NMLE. Moreover, oocysts per gram and histology of cecum in infected birds were restored in NMLE. The histological findings of NMLE-supplemented quails showed mild sloughed epithelium of cecal villi. In conclusion, NMLE is recommended as a naturally effective alternative to an anticoccidial drug to mitigate Eimeria infection in Japanese quails.

KEYWORDS:

• Eimeria tenella
• growth
• lesion score
• mortality
• quail

Introduction

During their brief life, Japanese quails lay several eggs and develop high resistant to different life-threatening infections (Berto et al. 2008). Eimeria infection is a serious parasitic disease threatening all poultry species (Dalloul et al. 2007). Eimeria invades the digestive tract and causes severe morbidity and mortality in broilers (Hafeez et al. 2020) and quails (Khan and Naz 2013). Anticoccidial drugs are usually administered to the infected birds to reverse the life-threatening condition (Ahmad et al. 2020). However, extensive and uncontrolled use of anticoccidial medicines against Eimeria has resulted in during resistance (Ali et al. 2019) and meat residues in poultry meat. The issues outlined above highlight the urgent need for natural alternatives to medications and disinfectants that can decrease Eimeria growth, strengthen immune systems, and increase animal production. Several plants have recently been studied to see whether they can help with coccidiosis control.

Neem (Azadirachta indica), for example, has anticoccidial qualities and has been used to treat coccidiosis. Neem is known for many biological and pharmacological properties (Landi et al. 2011). Nimbanene, nimbin, quercetin, nimbolide, 6-desacetylnimbinene, n-hexacosanol, nimbandiol, amino acid, ascorbic acid, 17-hydroxyazadiradione, 7-desacetyl-7-benzoylgedunin, and nimbiol are some of the chemical elements found in neem leaves, which have antibacterial and anticoccidial effects (Alzohairy 2016; Mohammed et al. 2021). Flavonoids found in neem leaves are potent antioxidants that prevent damage by reducing reactive oxygen species formation. They are also powerful anti-inflammatory drugs, inhibiting prostaglandin production, and enzymes, including protein kinases and phosphodiesterase, known for inflammatory response (Batista et al. 2018).

The search for anticoccidial drug alternatives has resulted in a long list of different feed additives (Tanweer et al. 2014; Chand et al. 2016, 2021). These supplements have improved the immune system against diseases and decreased stress level leading to better performance (Landi et al. 2011). Neem leave extracts have been used in Eimeria challenge in rabbits (Mohammed et al. 2021), pigeons (Qudoos et al. 2020) and broilers (Onyiche et al. 2021), however, it has not been tried in Japanese quails infected with Eimeria challenge. Therefore, the objective of the present study was to evaluate the anticoccidial effect of methanolic extract of neem leaves on the growth performance, oocytes shedding and lesion score in seca in Japanese quails under the Eimeria tenella challenge.

Materials and methods

Preparation of methanolic extract of neem leaves

Methanolic extract of neem leaves was prepared, as reported by Roy and Sharma (2020). Briefly, to make the concentrated methanolic extract, fresh neem leaves (Azadirachta indica) were taken from the neem tree and dried at room temperature. In a nutshell, 18 and 27 g of leaf powder were mixed in 200 and 400 mL methanol to prepare 130 and 190 mg/l doses, respectively. The extract was filtered and kept at 4°C. The plant extract was distilled at 80°C for 5 h. After distillation, the extract was crystallized at 60°C with continuous stirring. Crystals were formed on cooling and collected in a plastic container and stored at room temperature.

Animals

A total of 150 Japanese quails (9 ± 0.5 g) were reared on a concrete floor. The birds were divided into five groups, each with three replicates (10 birds/replicate) using a paper sheet, which was replaced when needed. Quails were free of coccidial infection, and anticoccidial drug vaccine was not given to them. The first group was provided only a basal diet with no coccidial infection (control); the second group was challenged with Eimeria tenella (negative control); the third group was experimentally infected with E. tenella and treated with amprolium at the rate of 1 g/kg in water; the fourth group was challenged E. tenella and supplemented with 130 mg/l methanolic extracts of neem leaves (NLME 130), the fifth group was infected and supplemented with 190 mg/l methanolic extracts of neem leaves (NLME 190).

Chicks were brooded for the first week at a temperature above 30°C and dropped to 22–23°C in the next week. Continuous light for 16 h was maintained at the rate of 21 Lux. Birds had free access to the basal diet (Table 1), as recommended by National Research Council (1994), and clean drinking water.

Table 1. Basal composition of feed during the starter and finisher phases.

Ingredients	Starter (1–21 days)	Finisher (22–35 days)
Corn	53.21	60.75
Soybean meal	37.92	25.00
Corn gluten meal	2.00	7.10
Corn oil	2.20	2.80
Dicalcium phosphate	2.30	2.05
Limestone	0.83	0.68
Salt	0.45	0.50
VM Mix^a	0.50	0.50
DL-Methionine	0.20	0.10
Lysine-HCl	0.22	0.37
Threonine	0.11	0.10
Choline chloride	0.05	0.05
Chemical composition
ME, kcal/kg	3000	3150
Crude protein, %	23.5	21.30
Methionine, %	0.55	0.44
Lysine, %	1.42	1.23
Sulphur amino acids, %	0.96	0.80
Threonine, %	0.95	0.85
Calcium, %	1.05	0.90
Phosphorus, %	0.50	0.45

^a Vitamin-mineral premix contains in the following per kg: vitamin A, 2,400,000 IU; vitamin D, 1,000,000 IU; vitamin E, 16,000 IU; vitamin K, 800 mg; vitamin B1, 600 mg; vitamin B₂, 1600 mg; vitamin B₆, 1000 mg; vitamin B₁₂, 6 mg; niacin, 8000 mg; folic acid, 400 mg; pantothenic acid, 3000 mg; biotin 40 mg; antioxidant, 3000 mg; cobalt, 80 mg; copper, 2000 mg; iodine, 400; iron, 1200 mg; manganese, 18,000 mg; selenium, 60 mg, and zinc, 14,000 mg.

Coccidial infection

Except for negative control, all the quails in the remaining four groups were challenged at a dose rate of 5.5 × 10⁴ sporulated oocysts of freshly prepared E. tenella orally on the eighth day of the experiment.

Growth performance and carcass traits

Different growth traits, such as feed consumption and weight gain, were measured and used the data to calculate the feed conversion ratio (FCR). On day 35, two birds/replicates were randomly slaughtered. The dressing percentage was estimated on a live body weight basis. Mortality was recorded whenever occurred.

Lesion score

Lesion score in the ceca of two birds per replicate was estimated using the formula described by Khan and Naz (2013): 0 = No lesion, += Mild lesion, ++ = Moderate lesion, +++ = Severe lesion.

Number of oocysts per gram (OPG)

At 5, 7, and 10 days post-infection (dpi), OPG was assessed using the technique described by Ali et al. (2019) using a McMaster chamber and microscope (Nikon, Japan).

Histopathological evaluation

Samples from the seca were aseptically removed and preserved at −20°C. Randomly a piece of 1 cm² was cleaned in a buffer solution of formalin and then dehydrated in a graded series of alcoholic solutions. Using a microtome, sections were cut and blocks were prepared, stained and examined under a microscope.

Statistical analysis

Using a randomized complete block design, data were analyzed using analysis of variance (ANOVA) and then subjected to the Tukey test for significance with the help of STATISTIC-2010 statistical software.

Results

Growth performance

Feed consumption, weight gain, and FCR of infected and supplemented Japanese quails are given in Table 2. Compared to the control, feed intake in negative control was significantly (P < .01) lower. Amprolium, NLME 130 and NLME 190-supplemented quails showed no significant differences. Weight gain in the negative control was significantly (P < .01) lower compared to that of the negative control. Amprolium-treated and NLME 190 quails showed no significant difference. Based on these parameters, poor FCR was calculated for the negative control.

Table 2. Effect of methanolic extract of neem leaves on feed consumption, weight gain and feed conversion ratio in quails challenged with coccidiosis

	Feed intake (g)			Weight gain (g)			Feed conversion ratio (g/g)
Groups	Starter phase	Finisher phase	Overall mean	Starter phase	Finisher phase	Overall Mean	Starter phase	Finisher phase	Overall mean
Control	131.85^a ± 3.29	302.95^a ± 2.05	434.80^a ± 4.53	69.25^a ± 1.82	93.10^a ± 0.92	162.36^a ± 0.91	1.90^b ± 0.01	3.25^b ± 0.04	2.67^c ± 0.01
Negative control	120.96^c ± 0.73	238.45^c ± 2.15	359.41^c ± 2.04	57.96^d ± 2.03	66.31^c ± 0.31	124.28^d ± 2.12	2.09^a ± 0.08	3.60^a ± 0.01	2.89^a ± 0.05
Infected + Amprolium	127.04^b ± 1.32	292.13^b ± 4.07	419.17^b ± 0.39	65.13^b ± 0.78	89.20^b ± 0.61	154.34^b ± 1.17	1.95^b ± 0.00	3.27^b ± 0.04	2.71^bc ± 0.02
Infected + NLME 130	124.06^b ± 0.66	288.60^b ± 0.70	412.66^b ± 0.70	62.70^c ± 0.63	86.29^b ± 0.65	149.00^c ± 0.51	1.98^b ± 0.02	3.34^b ± 0.09	2.77^b ± 0.03
Infected NLME + 190	125.11^b ± 0.47	289.10^b ± 2.60	414.21^b ± 2.58	63.48^bc ± 0.06	87.22^b ±0.16	150.70^bc ± 0.21	1.97^b ± 0.01	3.31^b ± 0.03	2.75^b ± 0.02
P-value	.0002	.0000	.0000	.0000	.0000	.0000	.0038	.0001	.0001

Mean values bearing different superscripts in a column differ significantly (P < .05); NLME 130: Neem leaves’ methanolic extract at the rate of 130 mg/l; NLME 190: Neem leaves’ methanolic extract at the rate of 190 mg/l.

Carcass weight, lesion score and mortality

The dressing percentage in NMLE supplemented quails was significantly (p < .01) high compared to the negative control (Table 3). On the other hand, the lesion score was higher significantly (P < .01) in the negative control compared to the control. Compared to the negative control, the lesion score was lower (P < .01) in NMLE-supplemented quails. In the negative control, the mortality percentage was significantly (P < .01) higher than the control and amprolium-treated quails.

Table 3. Effect of methanolic extract of neem leaves on dressing percentage, lesion score and mortality of Eimeria tenella-infected quails.

Groups	Dressing percentage	Lesion scores	% Mortality
Control	69.71^a ± 1.71	6.66^b ± 5.77	0.00^d ± 0.00
Negative control	59.27^c ± 0.25	30.00^a ± 0.00	3.50^a ± 0.50
Infected + Amprolium	67.25^b ± 0.11	10.00^b ± 10.00	1.00^c ± 0.00
Infected + NLM 130	66.89^b ± 0.96	13.33^b ± 5.77	2.00^b ± 0.00
Infected + NLM 190	67.24^b ± 1.04	13.33^b ± 11.54	1.50^bc ± 0.50
P-value	.001	.0330	.0000

Mean values bearing different superscripts in a column differ significantly (P < .05); NLME 130: Neem leaves’ methanolic extract at the rate of 130 mg/l; NLME 190: Neem leaves’ methanolic extract at the rate of 190 mg/l.

Oocysts shedding (OPG)

Supplementation of NMLE and treatment of amprolium significantly (p < .05) affected the OPG of feces in broilers on the target days (5, 7, and 10) post-infection (dpi), as shown in Table 4. Negative control had significantly (p < .01) higher oocysts count at 5, 7 and 10 dpi. Significantly (p < .01) lower OPG was found in NMLE 190 compared to the negative control.

Table 4. Effect of methanolic extract of neem leaves on oocyst per gram of feces (OPG) in coccidiosis-challenged quails.

Groups	5 DPI	7DPI	10DPI
Control	0.00^e ± 0.00	0.00^e ± 0.00	0.00^d ± 0.00
Negative control	749.00^a ± 6.00	827.00^a ± 3.00	380.40^a ± 5.50
Infected + Amprolium	379.50^d ± 1.50	396.50^d ± 1.50	150.00^c ± 8.00
Infected + NLME 130	494.00^b ± 4.00	533.50^b ± 3.50	223.50^b ± 1.50
Infected + NLME 190	465.50^c ± 10.50	510.50^c ± 1.50	162.50^c ± 12.50
P-value	.0000	.0000	.0000

Mean values bearing different superscripts in a column differ significantly (P < .05); NLME 130: Neem leaves’ methanolic extract at the rate of 130 mg/l; NLME 190: Neem leaves’ methanolic extract at the rate of 190 mg/l.

Histopathological alterations

The findings of cecum histological changes of Eimeria challenged quails are shown in Table 5. The results indicated that histological dimensions were significantly (P < .01) higher in the negative control than the control. Similar observations were found in amprolium-treated and NMLE-supplemented quails. The negative control showed highly broken villi, diffused haemorrhages and short of the crypt. In response to supplementation of NMLE, the intestinal villi were restored except few sloughed patches. The numbers of Eimerian gametocytes were significantly reduced in NMLE-supplemented quails, as shown in Figure 1.

Figure 1. Histological features of Eimeria tenella-infected quails (A) negative, (B) negative control, (C) infected + Amprolium treated, (D) infected + methanolic extract of neem leaves at the rate of 130 mg/l, (E) infected + methanolic extract of neem leaves at the rate of 190 mg/l.

Table 5. Effect of methanolic extracts of neem leaves on histopathological changes of the cecum of coccidiosis-challenged quails.

Groups	Villus length (mm)	Villus width (mm)	Crypt depth	VH: CD	Villus surface area
Control	0.41^ab ± 0.00	0.17^a ± 0.01	0.09^a ± 0.01	2.44 ±0.15	0.11^a ±0.02
Negative control	0.31^c ± 5.77	0.11^b ±1.00	0.05^b ± 0.00	2.87 ± 0.26	0.05^b ± 0.00
Infected + Amprolium	0.40^ab ± 5.77	0.19^a ± 0.01	0.07^a ± 5.77	2.05 ± 0.11	0.09^a ± 0.01
Infected + NLME130	0.38^b ± 0.02	0.16^a ± 0.01	0.07^a ± 0.01	2.38 ± 0.13	0.09^a ± 0.01
Infected + NLME 190	0.44^a ± 0.03	0.20^a ± 0.04	0.07^a ± 5.77	2.29 ± 0.65	0.10^a ± 5.77
P-value	0.0001	0.0042	0.0158	0.1207	0.0024

Mean values bearing different superscripts in a column differ significantly (P < .05); NLME 130: Neem leaves’ methanolic extract at the rate of 130 mg/l; NLME 190: Neem leaves’ methanolic extract at the rate of 190 mg/l.

Discussion

Novel techniques are urgently needed to combat the rising of drug-resistant coccidia strains in poultry houses. As a result, NMLE was investigated as a preventive and therapeutic method for coccidiosis management. In the present study, it is clear that NMLE supplementation had a positive impact on the restoration of body performance and alleviation of egg shedding, cecal lesion and histological dimensions in the Eimeria-infected quails. On the other hand, infected untreated quails showed poor body weight and feed efficiency. Reduced body weight is common in the presence of coccidiosis because severe coccidiosis produces substantial intestinal damage, resulting in loss of nutrients, and therefore, impaired performance. We also observed that the beneficial impact was greater in response to the combined doses of NMLE compared to individual effects. Improved growth performance was reported by Eimeria challenge in rabbits (Mohammed et al. 2021), pigeons (Qudoos et al. 2020) and broilers (Onyiche et al. 2021) in response to aqueous extract of A. indica. Previous studies have documented that Eimeria infections damaged development and feed utilization, producing nutritional imbalances and disruptions in feed digestion and absorption (Tanweer et al. 2014; Chand et al. 2016, 2021; Ali et al. 2019; Ahmad et al. 2020; Hafeez et al. 2020). The pharmacological effects of neem extract can be linked to the polyphenols it contains, as rutin is the predominant phenolic component. Flavonoids, polyphenols, tannins, gallic acid, catechins, quercetins, and saponins are highly water-soluble phytochemicals found in neem that have higher health-promoting and biological effects (Heyman et al. 2017). It is expected that neem leaf extract will be effective against drug-resistant bacteria when targeted at areas other than those employed by anticoccidials (Lahiri et al. 2021).

In the current study, lesion score, mortality percentage and OPG were significantly decreased when NMLE was added to the diet of quails. Neem leaves’ extract has been shown to have therapeutic efficacy against coccidiosis in several research investigations (Qudoos et al. 2020; Mohammed et al. 2021; Onyiche et al. 2021). For determining the severity of coccidiosis infection, lesion grading is critical. Infected birds with fewer lesion scores in the gut had less damage and a higher chance of recovery (Ritzi et al. 2014). Before the dormant oocysts are created and expelled, neem prevents the parasite from growing in the intestinal cells. The neem plant has the potential to deconstruct external and internal parasites; it includes hormones that disrupt the parasite’s life cycle, inhibiting the parasite's ability to feed, and finally preventing the egg from hatching. According to Luong et al. (2014), neem leaf slurry is an organic substance that serves as a larvicidal and is utilized in Africa. Landi et al. (2011) reported that 7 g/kg neem produced favourable effects on the immune response of broilers. It’s thought that azadirachtin’s interference and dominant effect on protozoan egg embryonic development is responsible for ovicidal activity.

Growth retardation due to low feed consumption and weight increase is a common indication of Eimeria parasite infection, which causes major economic losses to the poultry industry. Morphological destructions of the intestines are the most common findings of Eimeria infection resulting in poor absorption of nutrients and poor growth (Ali et al. 2019). In the present study, the deteriorated intestinal dimensions, such as villus height, width, crypt depth, villus surface area and crypt depth, were significantly improved with the supplementation of NMLE. The composition and compactness of the intestinal mucosa and its microstructures are useful predictors of the tiny nutrients. Higher intestinal villi features are considered to be associated with higher digestion and absorptive capacity. Scarce research reports are available in the published literature on the alleviation effects of methanolic extract of neem leaves on the intestinal microstructures in quails. It is inferred that methanolic extract of neem leaves improves the defensive mechanism of gut cells against Eimeria through antioxidant, antimicrobial and antiparasitic effects. Birds’ absorption ability is improved by larger enteric villi, and their crypts are involved in the creation or replacement of intestinal cells in response to inflammation or infection.

Conclusion

Methanolic extract of neem leaves exhibited substantial oocysticidal activity against Eimeria tenella in an experimental model of Japanese quails by restoring weight gain and reduction of harmful effects on intestinal ceca.

Animal welfare statement

The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received. The authors confirm that they have followed EU standards for the protection of animals used for scientific purposes and all the procedures with animals were approved by the Local Ethics Committee of Animal Experiments of the University (Protocol no. 2020-1-134).

Consent to participate and consent to publish

All the authors have equally participated in this study and agreed to publish this work in this journal.

Disclosure statement

No potential conflict of interest was reported by the author(s).