Anticoccidial Effect of Herbal Powder “Shi Ying Zi” in Chickens Infected with Eimeria tenella

Xu Song; Yunhe Li; Shufan Chen; Renyong Jia; Yongyuan Huang; Yuanfeng Zou; Lixia Li; Xinxin Zhao; Zhongqiong Yin

doi:10.3390/ani10091484

. 2020 Aug 24;10(9):1484. doi: 10.3390/ani10091484

Anticoccidial Effect of Herbal Powder “Shi Ying Zi” in Chickens Infected with Eimeria tenella

Xu Song ^1,^†, Yunhe Li ^1,^†, Shufan Chen ^1,^†, Renyong Jia ¹, Yongyuan Huang ¹, Yuanfeng Zou ¹, Lixia Li ¹, Xinxin Zhao ¹, Zhongqiong Yin ^1,^*

PMCID: PMC7552158 PMID: 32846893

Abstract

Simple Summary

Herbal medicines are playing an increasingly important role in the control of poultry diseases. The present study demonstrated that the herbal powder “Shi Ying Zi” consisting of Cnidium monnieri (L.) Cuss, Taraxacum mongolicum Hand.-Mazz., and sodium chloride could protect chickens from infection with Eimeria tenella through prophylactic or therapeutic administration. The “Shi Ying Zi” powder could improve the survival rate and relative growth rate with the anti-coccidial indexes of 165 (prophylactic effect) and 144 (therapeutic effect), which were equal to positive controls (monensin and sulfamlopyrazine). The “Shi Ying Zi” powder exhibits the potential to control E. tenella infection.

Abstract

Coccidiosis is one of the most economically important diseases affecting the poultry industry. Currently, anticoccidial drugs used in veterinary clinics show many deficiencies, and new control measures are urgently needed. This study presents an anticoccidial herbal powder “Shi Yin Zi”, which consists of Cnidium monnieri (L.) Cuss, Taraxacum mongolicum Hand.-Mazz., and sodium chloride. In chickens infected with Eimeria tenella, supplementation with “Shi Yin Zi” powder for 3 d prior to infection or treatment with “Shi Yin Zi” powder after infection could improve the survival rate and relative growth rate and alleviate the pathological changes in the cecum, liver, and kidney. “Shi Yin Zi” powder could recover the levels of alanine aminotransferase, creatinine, albumin, and triglycerides in serum. The hemorrhage occurrence and total number of oocysts in feces were reduced. The anti-coccidial indexes reached 165 for the prophylactic effect and 144 for the therapeutic effect. The anti-coccidial effects were equal to positive controls (monensin and sulfamlopyrazine). These results suggest that “Shi Ying Zi” powder possesses a potent anticoccidial effect and exhibits the potential to control E. tenella infection.

Keywords: anticoccidial drug, coccidiosis, “Shi Yin Zi” powder

1. Introduction

Coccidiosis, which is caused by seven species of intracellular protozoan parasites of the genus Eimeria, is one of the most detrimental and lethal diseases of commercial poultry flocks [1]. It is a rapidly developing intestinal disease that presents with bloody diarrhea and listlessness and can cause high levels of mortality in affected flocks [2]. Eimeria oocysts are highly infectious and resilient [3]. The primary sources of the spread of oocysts are usually rodents, insects, and wild birds, and their development is favored by moisture and heat, which are not lacking in poultry farms [4]. Eimeria oocysts typically invade intestinal epithelium cells and cause destruction of the infected cells, resulting in a reduction of feed conversion, body weight gain, and egg production, and increased morbidity and mortality [5]. Eimeria tenella is one of the most frequent and destructive pathogens of the genus Eimeria. It parasitizes chicken cecum and induces bloody excrement and body weight reduction [6]. Therefore, the management of coccidiosis and maintenance of the immune functions for ensuring maximum performance, growth, and production in the poultry industry are fundamental requirements for profitable farming [7]. In the prevention of coccidiosis, good sanitary conditions of poultry farms, such as maintaining cleanliness, good ventilation, and the regular replacement of litter, are important for eliminating dampness and the appearance of mold, which create an ideal environment for protozoa to thrive. Furthermore, nets that prevent rodents, insects, and wild birds from entering the farms also play an important role. The regular administration of coccidiostats in feed and preventive vaccinations are typical methods used to control the disease, but coccidial oocysts gradually show drug tolerance and the production capacity of current vaccine lines is limited [8,9]. To prevent the emergence of drug resistance, new drugs have been developed and administered on a rotational basis with existing drugs. However, this has resulted in an increased cost of poultry products. Furthermore, anticoccidial drug residues in poultry products are potentially an annoyance to consumers [8]. In recent years, anticoccidial drug development has been slower than the emergence of drug-resistant Eimeria spp., which has stimulated the search for alternative control methods for avian coccidiosis [10].

There are many plant-derived drugs with anticoccidial effects, such as herbal extracts [11,12]. Compared with chemotherapeutic drugs, anticoccidial herbal medicines usually exhibit less drug residues and less drug resistance [13]. Cnidium monnieri (L.) Cuss. is a widely used traditional herbal medicine in China, Vietnam, and Japan, and its fruits have been used to treat a variety of diseases, including female vulva pain, male impotence, epilepsy, and ulcers [14]. Many phytochemicals, mainly coumarins, which are the dominant active constituents, have been identified from C. monnieri, and osthole is used as a quality marker in the Pharmacopoeia of China [15]. Taraxacum mongolicum Hand.-Mazz. is a Chinese medicine commonly used to treat infection, fever, upper respiratory tract infections, pneumonia, and other infectious diseases [16]. The phytochemical constituents of T. mongolicum can mainly be divided into sesquiterpenoid, triterpenoid, phytosterol, flavonoids, phenolic acids, and organic acids [17]. Caffeic acid, which is a kind of phenolic acid, is used as a quality marker in the Pharmacopoeia of China. Based on the traditional Chinese veterinary medicine theory, unlike other herbal extracts, this study prepared the herbal formula “Shi Ying Zi”, which is a powder consisting of a mixture of C. monnieri, T. mongolicum, and sodium chloride at a ratio of 55:40:5. The anticoccidial effects of “Shi Ying Zi” powder were evaluated in chickens infected with E. tenella through supplementation for 3 d prior to infection and administration after infection for determination of the preventive and therapeutic effects of “Shi Ying Zi” powder.

2. Materials and Methods

2.1. Preparation of “Shi Ying Zi” Powder

“Shi Ying Zi” powder consisted of C. monnieri, T. mongolicum, and sodium chloride at a ratio of 55:40:5. The two herbals were procured from Baicaotang pharmaceutical chain Co. LTD (Chengdu, China). The samples of the two plant materials were deposited at the herbarium of the Natural Medicine Research Center, College of Veterinary Medicine, Sichuan Agricultural University (Chengdu, China), with voucher numbers 2017-0168 and 2017-0205, respectively. The content of osthole in C. monnieri was 1.28% and the content of caffeic acid was 0.041% in T. mongolicum Hand-Mazz. The quality criteria of the two herbals were fitted for the standards of the Pharmacopoeia of China (Version 2015, China Pharmacopoeia Committee). The mixtures of C. monnieri, T. mongolicum, and sodium chloride were smashed into powder, followed by sieving through 150 μm ± 6.6 μm screen cloth, to give the “Shi Ying Zi” powder.

2.2. Oocyst

The sporulated oocysts of E. tenella (BJ strain) were provided by the College of Veterinary Medicine, China Agricultural University (Beijing, China) [18]. For the propagation of oocysts, twenty 14-day-old chicks free of coccidiosis were inoculated with 2 × 10⁴ sporulated oocysts of E. tenella by oral gavage. The cecum contents and feces from infected chicks were collected at 7 days post infection (dpi), and oocysts were separated by flotation in saturated saline, as described by Kumar (2014) [19]. The oocysts were then allowed to sporulate in 2.5% (w/v) potassium dichromate solution at 28 °C for three days, followed by washing thrice with normal saline. After the determination of the total number of oocysts by the McMaster technique, as previously described [20], the sporulated oocysts were diluted to 2 × 10⁴ oocysts/mL in normal saline.

2.3. Experimental Design

Two hundred 1-day-old broiler chicks free of coccidiosis were purchased from a commercial hatchery (Tianchi Poultry Co. Ltd., Mianyang, China) and reared in a sterilized poultry shed of the animal houses of Sichuan Agricultural University (Ya’an, China). The chicks were fed a ⁶⁰Co-sterilized diet free of anticoccidial drugs (Supplementary Table S1; Xietong Biotechnology Co., Ltd., Nanjing, China). The feces were collected daily to check for oocysts [21]. At the 11th day of age, the chicks were all free of coccidiosis and were then equally divided into 10 groups (10 males and 10 females per group and each with four replicates of five chickens/cage), including an uninfected-untreated group, an infected-untreated group, four prophylactic groups (positive control group and three “Shi Ying Zi” powder groups), and four therapeutic groups (positive control group and three “Shi Ying Zi” powder groups). The chicks in the three “Shi Ying Zi” powder protective groups were prophylactically supplemented with “Shi Ying Zi” powder at doses of 5 g/kg feed (ShiYingZi-PL), 10 g/kg feed (ShiYingZi-PM), and 15 g/kg feed (ShiYingZi-PH), respectively. In the positive control group, monensin was added (100 mg/kg feed). For obtaining homogeneous mixtures of feed with “Shi Ying Zi”, the powder was firstly mixed with an equal quantity of feed, and the feed was added to the mixture at an equal quantity. The mixing process continued until all of the feed had been added. The uninfected-untreated group and infected-untreated group were fed a regular diet. At the 14th day of age, chicks, except for those in the uninfected-untreated group, were inoculated with 2 × 10⁴ sporulated oocysts of E. tenella by oral gavage. At 1 dpi, the chicks in the three “Shi Ying Zi” powder therapeutic groups were supplemented with “Shi Ying Zi” powder at doses of 5 g/kg feed, (ShiYingZi-TL), 10 g/kg feed (ShiYingZi-TM), and 15 g/kg feed (ShiYingZi-TH), respectively. In the positive control group, sulfachloropyrazine sodium was added to water (1 g/L).

2.4. Ethical Approval

All procedures involving animals and their care in this study were approved by the Ethics Committee of Sichuan Agricultural University according to the Regulation of Experimental Animal Management (State Scientific and Technological Commission of the People’s Republic of China, No. 2, 1988) and The Interim Measures of Sichuan Province Experimental Animal Management (Science and Technology Bureau of Sichuan, China, No. 25, 2013). At the end of the study (8 dpi), the chicks were anesthetized through intravenous injection (underwing vein) with sodium pentobarbital (30 mg/kg body weight) within 2 min of the birds being removed from their home pen [22]. The wing venous blood samples and tissue samples (five chicks per group) were collected. The rest of the anesthetized chicks were euthanized by manual cervical dislocation. All efforts were made to minimize the suffering of birds.

2.5. Clinical Symptoms

The chicks were examined daily for recording clinical signs, such as anorexia, huddling together, disheveled feathers, bloody stools, and mortality [23].

2.6. Bloody Diarrheal Score

The bloody diarrheal score is a qualitative estimation of the deviation of the fecal appearance from a normal state. It was obtained by scoring the bloody feces each morning from 4 to 8 dpi. The bloody diarrheal score ranged from 0 to 4: A score of 0 indicated normal feces without hemorrhage; a score of 1 indicated 1–25% hemorrhage in the feces; a score of 2 indicated 25–50% hemorrhage in the feces; a score of 3 indicated 51–75% hemorrhage in the feces; and a score of 4 indicated 76–100% hemorrhage in the feces [11].

2.7. Lesion Score of the Cecum

The postmortem examination was conducted 8 dpi. The dead and slaughtered birds were incised, and gross lesions in the cecum were examined by recording lesion scoring as described by Johnson and Reid [24]. The lesions included hemorrhages, thickening of the cecum wall, and mucoid discharge. The lesion score ranged from 0 to 4: A score of 0 indicated no lesion; a score of 1 indicated a mild lesion; a score of 2 indicated a moderate lesion; a score of 3 indicated a severe lesion; and a score of 4 indicated a more severe lesion.

2.8. Oocyst Counts

The oocyst counts in feces were measured by the McMaster method [20]. Briefly, 2 g of feces collected from 0 to 8 dpi were uniformly dispersed in 60 mL saturated salt solution with vigorous stirring, followed by filtration. Then, the filtrate was loaded onto a McMaster Egg Slide (Chongqing DiShui Experimental apparatus Co., Ltd., Chongqing, China), which has two chambers with two identical 10 mm × 10 mm × 1.5 mm grids (0.15 mL), for oocyst counting. The total number of oocysts under both of the grids was recorded. The oocyst counts (oocysts per gram) were calculated by multiplying the total number of oocysts in the two chambers by 100. The cecum contents were collected 8 dpi after postmortem examination. The oocyst counts in the cecum contents were determined as described above. The score of the oocysts index at 8 dpi ranged from 0 to 40: A score of 0 indicated the oocyst counts were 0–0.1 × 10⁶; a score of 5 indicated the oocyst counts were 0.11 × 10⁶–1 × 10⁶; a score of 10 indicated the oocyst counts were 1.1 × 10⁶–1.9 × 10⁶; a score of 20 indicated the oocyst counts were 2.0 × 10⁶–5.9 × 10⁶; a score of 30 indicated the oocyst counts were 6.0 × 10⁶–10.9 ×10⁶; and a score of 40 indicated the oocyst counts were more than 1.1 × 10⁷.

2.9. Anti-Coccidiosis Index

The anti-coccidiosis index (ACI) = (Relative weight gain rate + Survival rate) − (Oocysts index in cecum + Gross lesions score) [25].

The relative weight gain rate (×100%) is the ratio of the average weight gain of the infected group to the average weight gain of the uninfected-untreated group. The survival rate (×100%) is the ratio of the number of alive chicks to the total number of chicks. The average initial weight is the average weight of the chicks at 0 dpi, and the average final weight is the average weight of the chicks at 8 dpi.

When ACI is larger than 180, it means that the drug has a high efficiency; when ACI is 160–180, it means that it has a medium efficiency; when ACI is 120–160, it means that it has a low efficiency; and when ACI is lower than 120, it means that the drug does not possess anti-coccidiosis effects.

2.10. Biochemical Indexes

For biochemical studies, 3–4 mL wing venous blood was collected from five randomly selected chicks of each group into tubes, without any anticoagulant, at intervals of 1, 4 and 8 dpi. After clotting, serum was separated and various parameters were estimated, including total serum protein, albumin, aspartate aminotransferase, alanine aminotransferase, creatinine, and triglyceride. The globulins were calculated by subtracting the values of albumin from total serum proteins.

2.11. Histopathology

After dissection, the cecum, liver, and kidney tissue samples were subjected to common histological preparation. A 5 μm section of each tissue was procured and stained with hematoxylin and eosin (HE) for histopathological examination.

2.12. Statistical Analysis

The statistical significance was compared for the uninfected-untreated control and experimental groups by one way analysis of variance (ANOVA), followed by the Student–Newman–Keuls test, using the IBM SPSS Statistics, Version 24 program (IBM Corporation. Somers, NY, USA). The differences between groups were considered significant when values of p < 0.05.

3. Results

3.1. Clinical Symptoms and Mortality

After being challenged with E. tenella, the clinical signs and symptoms were closely monitored throughout the trial. The clinical signs, including disheveled feathers, dullness, and anorexia, were observed in the infected-untreated group at 3 dpi. The symptoms in ShiYingZi protective groups were observed at 4 dpi, which were similar to the infected-untreated group, but much milder. The mortality of the infected-untreated group was highest in all groups. The appearance of hemorrhage in feces was noted at 4 dpi in the infected-untreated group and many oocysts were found in the cecum, suggesting that the model was successfully established. For pretreatment with “Shi Ying Zi” powder, bloody stools were detected at 5 dpi.

E. tenella infection induced a 25% mortality rate in the infected-untreated group, which is significantly higher than the uninfected-untreated control and other infected groups. Pretreatment with “Shi Ying Zi” powder only caused a 5% mortality rate in the ShiYingZi-PL group and ShiYingZi-PH group. There was no mortality in the ShiYingZi-PM group and monensin group (Table 1). When the drugs were supplemented after infection, the mortality rates were 10% in the ShiYingZi-TL group and 5% in the ShiYingZi-TM group. In the ShiYingZi-TH group and sulfachloropyrazine sodium group, there was no mortality (Table 1). These results suggested that “Shi Ying Zi” powder and positive drugs could significantly reduce the mortality rate of chicks infected with E. tenella.

Table 1.

Mortality in different groups from 4 to 8 days post infection (dpi).

Groups	4 dpi	5 dpi	6 dpi	Total Mortality
Uninfected-untreated control	0	0	0	0 ^a
Infected-untreated control	2	2	1	5 ^b
ShiYingZi-PL	0	1	0	1 ^ac
ShiYingZi-PM	0	0	0	0 ^a
ShiYingZi-PH	1	0	0	1 ^ac
Monensin	0	0	0	0 ^a
ShiYingZi-TL	1	1	0	2 ^ac
ShiYingZi-TM	0	1	0	1 ^ac
ShiYingZi-TH	0	0	0	0 ^a
Sulfachloropyrazine sodium	0	0	0	0 ^a

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^a,b,c Different letters for the total mortality indicate that a significant difference existed (p < 0.05).

3.2. Anticoccidial Efficacy in Chickens

The bloody diarrheal score from 4 to 8 dpi (Table 2) and the total number of oocysts per gram of feces from 0 to 8 dpi (Table 3) were measured. Oocyst shedding in feces could be observed from 0 to 3 dpi. When the hemorrhagic feces were observed in the infected groups at 4 dpi, the oocyst shedding in feces could be detected, except for the ShiYingZi-PH and monensin groups. The number of oocysts in feces was decreased by ShiYingZi treatment from 4 to 8 dpi. The ShiYingZi therapeutic group (10 g/kg) exhibited a significantly decreased bloody diarrheal score in comparison with the infected-untreated control at 5 dpi. Both the prophylactic and therapeutic administration of “Shi Ying Zi” powder could significantly decrease the bloody diarrheal score in comparison with the infected-untreated control at 6 and 7 dpi. “Shi Ying Zi” powder exhibited equal potency when compared with monensin and sulfachloropyrazine sodium. These results suggested that “Shi Ying Zi” powder could alleviate the hemorrhages in feces and accelerate recovery from bloody stools. The amount of oocyst shedding in feces was also reduced.

Table 2.

The bloody diarrheal score from 4 to 8 dpi.

Groups	4 dpi	5 dpi	6 dpi	7 dpi	8 dpi
Uninfected-untreated control	0 ^a	0 ^a	0 ^a	0 ^a	0 ^a
Infected-untreated control	1 ^a	2 ^b	3 ^b	3 ^b	1 ^a
ShiYingZi-PL	1 ^a	2 ^b	3 ^b	2 ^c	1 ^a
ShiYingZi-PM	0 ^a	2 ^b	2 ^c	2 ^c	0 ^a
ShiYingZi-PH	1 ^a	2 ^b	3 ^b	3 ^b	0 ^a
Monensin	0 ^a	2 ^b	2^c	2 ^c	0 ^a
ShiYingZi-TL	0 ^a	2 ^b	3 ^b	2 ^c	0 ^a
ShiYingZi-TM	0 ^a	1 ^a	2 ^c	1 ^a	1 ^a
ShiYingZi-TH	1 ^a	2 ^b	2 ^c	2 ^c	0 ^a
Sulfachloropyrazine sodium	0 ^a	1 ^a	2 ^c	1 ^a	0 ^a

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^a,b,c Different letters indicate that a significant difference existed (p < 0.05).

Table 3.

The number of oocysts per gram of feces (×10⁶).

Groups	4 dpi	5 dpi	6 dpi	7 dpi	8 dpi
Uninfected-untreated control	0	0	0	0	0
Infected-untreated control	0.27	2.78	1.93	1.54	1.15
ShiYingZi-PL	0.09	2	1.71	0.85	0.55
ShiYingZi-PM	0.14	2.13	1.24	1.03	0.60
ShiYingZi-PH	0	3.12	1.75	1.06	0.83
Monensin	0	1.56	1.69	0.75	0.42
ShiYingZi-TL	0.21	2.17	1.78	1.13	0.90
ShiYingZi-TM	0.33	2.33	1.51	1.23	0.95
ShiYingZi-TH	0.25	2.49	1.88	1.07	0.46
Sulfachloropyrazine sodium	0.19	2.12	1.25	0.99	0.78

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Coccidiosis induced decreased weight gain, and the relative weight gain rate was 53% compared with the uninfected-untreated group (Table 4). The prophylactic administration of “Shi Ying Zi” powder could recover the relative weight gain rate up to 86% compared with the uninfected-untreated group, which is better than monensin. The relative weight gain rate of the “Shi Ying Zi” powder therapeutic group could reach to 77% in comparison with the infected-untreated group. The gross lesions score and cecum oocyst index were decreased after the prophylactic and therapeutic administration of “Shi Ying Zi” powder and the positive drugs. The prophylactic administration of “Shi Ying Zi” powder at the dose of 10 g/kg showed the highest anticoccidial activity, with an ACI value of 165, which is higher than that obtained for monensin (159).

Table 4.

Anti-coccidiosis index.

Indicators	Uninfected-Untreated Control	Infected-Untreated Control	Protective Effect				Therapeutic Effect
Indicators	Uninfected-Untreated Control	Infected-Untreated Control	ShiYingZi-PL	ShiYingZi-PM	ShiYingZi-PH	Monensin	ShiYingZi-TL	ShiYingZi-TM	ShiYingZi-TH	Sulfachloropyrazine Sodium
Average initial weight/g	165.27 ± 20.5	157.07 ± 25.1	164.07 ± 20.72	155.13 ± 14.17	160.43 ± 14.98	146.33 ± 12.56	160.13 ± 18.12	152.4 ± 18.72	149.33 ± 19.29	159.2 ± 19.93
Average final weight/g	225 ± 26.06 ^b	189.14 ± 29.44 ^a	210.27 ± 22.29 ^ab	204.82 ± 18.07 ^ab	209.41 ± 16.57 ^ab	191.6 ± 14.9 ^a	204 ± 21.01 ^a	188.73 ± 23.08 ^a	189.91 ± 23.4 ^a	207.44 ± 17.9 ^ab
Relative weight gain rate (%)	100	53	78	86	83	78	74	71	77	80
Survival rate (%)	100	75	95	100	95	100	90	95	100	100
Gross lesions score	0 ± 0 ^a	35 ± 5.27 ^c	14 ± 11.74 ^b	11 ± 9.94 ^b	14 ± 11.74 ^b	9 ± 8.76 ^b	19 ± 13.7 ^b	15 ± 10.8 ^b	13 ± 4.83 ^b	11 ± 7.38 ^b
Cecum oocyst index	0	40	20	10	10	10	20	20	20	10
ACI	-	-	139	165	154	159	125	131	144	159
Therapeutic evaluation	-	-	low	medium	low	low	low	low	low	low

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^a,b,c Significant differences exist when there are different letters in each column (p < 0.05). n = 20. The initial weight was the weight on the day of infection and the final weight was the weight at 8 dpi.

3.3. Biochemical Indexes

The contents of total protein (Table 5), albumin (Table 6), and triglyceride (Table 7) were decreased after infection on 4 and 8 dpi (p < 0.05), but they were increased after pretreatment or treatment with “Shi Ying Zi” powder and positive drugs (p < 0.05). The serum globulin concentrations (Table 8) in the infected chicks with or without treatment were increased on 4 and 8 dpi (p < 0.05), and no significant difference was observed among all of the infected chicks. The contents of alanine aminotransferase (Table 9) and creatinine (Table 10) in the infected chicks were increased on 4 and 8 dpi (p < 0.05), and after the prophylactic and therapeutic administration of “Shi Ying Zi” powder, they were decreased (p < 0.05). Coccidiosis led to an increased (p < 0.05) content of aspartate aminotransferase on 4 and 8 dpi (Table 11), and after the prophylactic and therapeutic administration of “Shi Ying Zi” powder, it was decreased on 8 dpi (p < 0.05).