Anticoccidial effect of “Shi Ying Zi” Powder in chickens infected with Eimeria tenella

Background: Coccidiosis is one of the most economically important diseases affecting the poultry industry. Currently, anticoccidial drugs used in veterinary clinical showed many deficiencies, and new control measures are urgently needed. Many plant-derived drugs, such as herbal extracts, showed effects with less drug residues and resistance. Development of herbal medicine as the anticoccidial drug is one of the most prospective method for treating coccidiosis. Methods:“Shi Ying Zi” is the herbal powder that consisted of Cnidium monnieri (L.) Cuss, Taraxacum mongolicum Hand-Mazz and salt with the proportion of 55: 40: 5 (g/g/g). 200 1-day-old broiler chicks free of coccidiosis were equally divided into 10 groups: normal group, untreated group, 4 protective groups (positive control group and three “Shi Ying Zi” powder groups) and 4 therapeutical groups (positive control group and three “Shi Ying Zi” powder groups). At 11 th day of age, the chicks in the four protective groups were prophylactically supplemented with “Shi Ying Zi” powder at the doses of 5g/kg, 10g/kg and 15g/kg and monensin (100mg/kg), respectively. At 14 th day of age, chicks except those in blank group were inoculated with 2×10 4 sporulated oocysts of E. tenella . When the first hemorrhagic fecal was found, therapeutical drugs started administration. The low (5g/kg), medium (10g/kg) and high doses (15g/kg) of “Shi Ying Zi” powder were added into the diet, respectively. Sulfachloropyrazine sodium (positive control) was added into water (1g/L). The anti-coccidial index, biochemical parameters and tissue lesions were determined from 4 d post infection to 8 d post infection. Results: Supplement with “Shi Yin Zi” powder for 3d prior to infection or 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 contents of glutamic-pyruvic transaminase, reaches to The anti-coccidial effects were equal to positive controls (monensin and sulfamlopyrazine). Conclusions: Ying Zi” powder possesses potent anticoccidial effect and exhibits the potential to control E.tenella infection.


Introduction
Coccidiosis 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 diarrhoea and listlessness and can cause heavy mortality in affected flocks [2]. Eimeria oocysts are highly infectious and resilient, and easily transferred by animals, insects, dust, contaminated feed, water and equipment [3]. Eimeria oocysts typically invade intestinal epithelium cells and cause destruction of the infected cells, resulting in reduction of feed conversion, body weight gain, egg production, and increased morbidity and mortality [4].
Therefore, management of coccidiosis and maintenance of the immune functions for maximum performance, growth and production in poultry industry are fundamental requirements for profitable farming [5]. Thus far, chemoprophylaxis and anticoccidial feed additives are two typical methods for controlling the disease, but coccidiosis oocysts gradually show drug tolerance and eventually the phenomenon of survival and reproduction [6]. 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 the increased cost of poultry products. Furthermore, drug or antibiotic residue in the poultry products is potentially annoyance to consumer [6]. In recent years, the anticoccidial drug development is slower than the emergence of drug resistant Eimeria spp., which has stimulated the search for new and alternative control methods for avian coccidiosis [7].
There are many plant-derived drugs with anticoccidial effects, such as herbal extracts [8][9]. Compared with chemotherapeutic drugs, herbal anticoccidials usually show less drug residues and less drug resistance [10]. 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 ulcer [11]. Taraxacum mongolicum Hand-Mazz is a commonly used Chinese medicine to treat infection, fever, upper respiratory tract infection, pneumonia, and other infectious diseases [12]. These two herbals are recorded in the Pharmacopoeia of the People's Republic of China. Based on the traditional Chinese veterinary medicine theory, unlike other herbal extracts, this study prepares a herbal formula "Shi Ying Zi" which is the powder of the mixture of Cnidium monnieri (L.) Cuss, Taraxacum mongolicum Hand-Mazz. and salt in the ratio of 55:40:5 (g/g/g). The anticoccidial effects of "Shi Ying Zi" powder were evaluated in chickens infected with Eimeria tenella through supplement for 3d prior to infection or after infection for determination of the preventive effect and therapeutic effect of "Shi Ying Zi" powder. pharmacopoeia committee).The content of osthole in Cnidium monnieri (L.) Cuss is not less than 1% and the content of caffeic acid is not less than 0.02% in Taraxacum mongolicum Hand-Mazz. The mixture of Cnidium monnieri (L.) Cuss, Taraxacum mongolicum Hand-Mazz and salt were smashed into powder, followed by sieving through 150μm ± 6.6μm screen cloth to give the "Shi Ying Zi" powder.

Experiment design
200 1-day-old broiler chicks free of coccidiosis were purchased from a local hatchery and equally divided into 10 groups which contained normal group, untreated group, 4 protective groups (positive control group and three "Shi Ying Zi" powder groups) and 4 therapeutical groups (positive control group and three "Shi Ying Zi" powder groups).
Chicks were reared in poultry shed and maintained free from coccidian infection and anticoccidial drug At 11 th day of age, the chicks in the three "Shi Ying Zi" powder groups were prophylactically supplemented with "Shi Ying Zi" powder at the doses of 5g/kg (ShiYingZi-PL), 10g/kg (ShiYingZi-PM) and 15g/kg (ShiYingZi-PH), respectively. In the positive control group, monensin was added (100mg/kg). The normal group and untreated group were fed with normal diet. At 14 th day of age, chicks except those in blank group were inoculated with 2×10 4 sporulated oocysts of E. tenella. When the first hemorrhagic fecal was found, therapeutical drugs started administration. The low (5g/kg, ShiYingZi-TL), medium (10g/kg, ShiYingZi-TM) and high doses (15g/kg, ShiYingZi-TH) of "Shi Ying Zi" powder were added into the diet, respectively. In the positive control group, sulfachloropyrazine sodium was added into water (1g/L).

Clinical symptoms
The chicks were examined daily for recording clinical signs, such as ruffled feathers, anorexia, huddling together, ruffled feathers, loose dropping, hemorrhagicy feces, and mortality.

Hemorrhagic fecal score
Hemorrhagic fecal score is a qualitative estimation of the deviation of fecal appearance from normal. They were obtained by scoring the fecal each morning from 4dpi to 8dpi.

Lesion score of caecum
The postmortem examination was conducted on 8dpi. The dead and slaughtered birds were incised, and gross lesions in caecum were recorded. The pathogenicity and severity of the disease was studied by recording lesion scoring, which was evaluated based on extent of lesions in a particular organ as described by Johnson and Reid [13]. The lesions included petechial hemorrhages, thickening of caecum wall, hemorrhagic fecal contents, and mucoid discharge. Lesion score was made from 0 to 4: a score of 0 indicated no lesion; a score of 1 indicated mild lesion; a score of 2 indicated moderate lesion; a score of 3 indicated severe lesion; a score of 4 indicated more severe lesion.

Oocysts index in caecum
Caecum contents were collected on 8dpi after postmortem examination. The counting of oocysts was made by McMaster method [14]. The score was made from 0 to 40: a score of 0 indicated the oocysts in feces were 0-0.1×10 6 a score of 5 indicated the oocysts in content were 0.11×10 6 -1×10 6 a score of 10 indicated the oocysts in content were 1.1×10 6 -1.9×10 6 a score of 20 indicated the oocysts in content were 2.0×10 6 -5.9 ×10 6 a score of 30 indicated the oocysts in content were 6.0×10 6 -10.9×10 6 a score of 40 indicated the oocysts in content were more than 1.1×10 7
When ACI is larger than 180 it means the anti-coccidiosis drug is high efficiency; when ACI is 160-180, it means the anti-coccidiosis drug is medium efficiency; when ACI is 120-160, it means the anti-coccidiosis drug is low efficiency, and when ACI is lower than 120, it means the drug do not possess anti-coccidiosis efficiency.

Biochemical indexes
For biochemical studies, 3-4 mL blood was collected from randomly selected 5 chicks of each group into tubes without any anticoagulant at intervals 0, 4 and 8dpi. After clotting, serum was separated and various parameters were estimated, including total serum protein, albumin, aspartate aminotransferase and alanine aminotransferase, serum creatinine and serum triglyceride. The globulins were calculated by subtracting the values of albumin from total serum proteins.

Histopathology
After dissection, the caecum, liver, kidney tissue samples were excised, fixed with neutral buffered formalin (10%), and then embedded in paraffin. Approximately 4 μm thick cross sections were excised and stained with hematoxylin and eosin (HE) for histopathological examination.

Clinical symptoms and mortality
After challenged with E. tenella, the clinical signs and symptoms were closely monitored throughout the trial. The clinical signs, including dullness, anorexic and loose dropping, were first observed in the untreated group at 3dpi. The symptoms in ShiYingZi-treated groups of protective administration were observed at 4dpi, which is similar with untreated group, but much milder than untreated group. Mortality of untreated group was highest in all groups. Appearance of hemorrhage in feces was noted on 4dpi in untreated group and many oocysts were found in caecum, suggesting the model was successfully established.
E. tenella infection induced 25% mortality rate in the untreated group. Pretreatment only caused 5% mortality rate in the ShiYingZi-PL group and ShiYingZi-PH group. There were 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 were no mortality ( Table 1).

Anticoccidial efficacy
Hemorrhagic fecal score ( Table 2) and the total number of oocysts per gram of feces (Table 3) were measured from 4dpi to 8dpi. Pretreatment and therapeutical administration could alleviate the hemorrhage in feces and accelerate recover from bloody stool. The number of oocysts shedding in feces were also reduced by the two routes.
Coccidiosis induced decreased weight gain, and the relative weight gain rate is 53% compared with normal group (Table 4). ShiYingZi-pretreatment could recover the relative weight gain rate up to 86% compared with normal group, which is better than monensin (77%). With treatment with ShiYing Zi, the relative weight gain rate could reach to 77% in comparison with untreated group. The gross lesions score and caecum oocyst index were decreased after pretreatment or treatment with "Shi Ying Zi" powder and the positive controls. Pretreatment with "Shi Ying Zi" powder at the dose of 10g/kg showed highest anti-coccidiasis activity that the ACI was 165, which is higher than monensin (159)

Biochemical indexes
The contents of total protein (Table 5), albumin ( Table 6) and triglyceride (Table 11) were significantly decreased after infection on 4dpi and 8dpi (p 0.05), but they were significantly (p 0.05) increased after pretreatment or treatment with "Shi Ying Zi" powder and positive controls. Serum globulin concentrations ( Table 7) in the infected chicks with or without treatment were significantly (P<0.05) increased on 4dpi and 8dpi, no significant difference was observed among all the infected chicks. The contents of glutamic-pyruvic transaminase (Table 8), and creatinine (Table 10) in the infected chicks were significantly increased on 4dpi and 8dpi (P<0.05), after pretreatment or treatment, they were significantly decreased (P<0.05). Coccidiosis induced significantly (P<0.05) increased content of aspartate aminotransferase on 4dpi and 8dpi (Table 9), after pretreatment or treatment, it was significantly decreased on 8dpi (P<0.05).

Histopathological examination
In the caecum, oocysts invaded caecum mucosa and intestinal gland ( in the uninfected group (Fig.1A), caecum showed the normal structure.
In the kidney, serious granular and vacuolar degeneration and cell necrosis were observed in the untreated group (Fig.1N); few granular and vacuolar degeneration were observed in the ShiYingZi-PM group (Fig.1O), ShiYingZi-TH group (Fig.1Q) and positive control groupS (Fig.1P, monensin; Fig.1R , sulfachloropyrazine sodium); more inflammatory cell infiltration was observed in the sulfachloropyrazine sodium group (Fig.1R) than that in the ShiYingZi-TH group (Fig.1Q); without infection, kidney showed the normal structure (Fig.1I).

Discussion
Coccidiosis is one of most economically important diseases, but in common used anticoccidiosis drugs often cause drug residue in meat and eggs [16]. This study provides a new herbal drug candidate "Shi Ying Zi" for treating coccidiosis, and the potency is equal to monensin and sulfamlopyrazine. Unlike other herbal medicine, "Shi Ying Zi" don't need any extraction process and it is the powder of Cnidium monnieri (L.) Cuss, Taraxacum mongolicum Hand-Mazz and salt. Therefore, it is more financially acceptable, which is the one of the most important point for drugs used in poultry industry. This study revealed that "Shi Ying Zi" powder administrated through two routes, showed potent anticoccidial effect, suggesting that it could be used as prophylactic drug or therapeutic drug. For pretreatment, the recommended dosage of "Shi Ying Zi" powder is 10 g/kg; for treatment, it is 15g/kg. Coccidiosis usually reduces body weight gain in broiler chicks as a result of reduced feed intake, digestibility and absorption of macronutrients [17]. It is accepted that weight gain is the more sensitive variable to coccidiosis and anticoccidial treatments [18]. When infective sporozoites enter the caecum mucosa by penetrating villus epithelial cells, leading to extensive destruction of the caecum epithelium, hemorrhagic feces output and a large number of oocysts excretion [19][20]. In this study, infected chicks were dull and depressed with ruffled feather and less feed intake, which may be due to altered gut homeostasis that leads to poor feed intake, metabolism and thus decreased weight gains [21,22]. "Shi Ying Zi" powder can alleviate the histopathological changes of caecum, and the number of oocysts and mucosa cell necrocytosis in caecum has decreased. Therefore, the relative weight gain rate of "Shi Ying Z" groups were also improved. The anti-coccidial index is a common index to evaluate the anticoccidial activity of drugs. The ACI was 165 in the ShiYingZi-pretreated group at the dose of 10 g/kg, which is higher than positive control monensin. These results suggested that "Shi Ying Zi" powder is moderate-potency anticoccidial drug. Similarly, there was a statistically significant decrease in the contents of albumin, total protein and triglyceride and significant increase in the contents of glutamic-pyruvic transaminase and aspartate amino transferase after infection. Pretreatment or treatment with "Shi Ying Zi" powder, these changes was recovered to a degree. In histopathology findings, serious liver damage after E. tenella infection can be observed. While "Shi Ying Zi" powder can alleviate these symptoms. These results suggested that coccidiosis could affect the body's liver function, and "Shi Ying Zi" could protect and alleviate the liver damage.
Creatinine is a metabolite of muscle creatine and phosphate, which is not affected by the food protein content and protein metabolism. The content of creatinine in the body is relatively constant and only reflected by the renal clearance and renal function [27-28].
When the renal insufficiency, creatinine accumulates in the body and becomes a toxin that is harmful to the body, so that creatinine content can respond to the damage to renal . At the end of the study, blood samples were collected under anesthesia with sodium pentobarbital (30 mg/mL) within 2 min of the birds removal from its home pen, followed by euthanasia. Then, tissue samples were collected. All efforts were made to minimize suffering of birds.

Consent for publication
Not applicable

Availability of data and materials
All data generated or analysed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.