A traditional Chinese medicine composition for treating chicken eimeria tenella and application thereof

Abstract

The application belongs to the field of veterinary medicine preparation and discloses a traditional Chinese medicine composition for treating chicken Eimeria tenella and application thereof. The traditional Chinese medicine composition comprises the following components in parts by mass: mulberry leaves 14-16 parts, atractylodes 14-16 parts, agastache 8-12 parts, cinnabar 18-22 parts, areca nut 8-12 parts, milk root 18-22 parts, Chinese catalpa 8-12 parts, and licorice 5-7 parts. The traditional Chinese medicine composition has the effects of clearing heat and drying dampness, cooling blood and stopping diarrhea, and promoting qi and removing stagnation, has a good treatment effect on chicken coccidiosis, can improve the growth performance of chicks, and has no toxic side effects.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of veterinary medicine preparation, and particularly relates to a traditional Chinese medicine composition for treating chicken Eimeria tenella and application thereof. BACKGROUND

[0002] With the continuous development of scale, intensification and industrialization of livestock and poultry breeding industry, the growth rate or production performance of livestock and poultry is continuously improved, which brings more economic benefits to the livestock and poultry breeding industry. However, with the sharp increase of feeding density and feeding amount, the living space and the quality of the environment are limited, and the resistance of livestock and poultry is decreased, resulting in an upward trend of the incidence of diseases, which has become a thorny problem for the development of animal husbandry. Chicken coccidiosis is a kind of poultry parasitic disease caused by one or more kinds of coccidia parasitizing in the epithelial cells of chicken intestinal tract, which is one of the most serious diseases in intensive breeding farms. The disease is characterized by listlessness, slow growth, bloody diarrhea and reduced growth performance, and can also cause immune suppression, resulting in failure of immunization or inducing secondary infection, and even death of chickens, and is considered to be the most economically important parasitic disease in poultry production. At present, among the seven recognized coccidia species that can infect chickens, Eimeria tenella is the most common and the most pathogenic.

[0003] At present, the prevention and treatment of chicken coccidiosis mainly relies on ionophore drugs and chemically synthesized drugs. However, due to the long-term and large-scale use or abuse of drugs, coccidia drug resistance problems continue to emerge. A large number of literatures report that almost all commercially used anticoccidial chemicals have corresponding drug-resistant strains, and the trend of multiple drug resistance and cross drug resistance is becoming increasingly serious. The universal drug resistance of anticoccidial drugs often leads to the failure or poor effect of the prevention and control of chicken coccidiosis, so that the outbreak of coccidiosis faces the predicament of no available drugs, causing huge economic losses. Although researchers are committed to the development of new anticoccidial drugs, the development speed of drugs often lags behind the speed of drug resistance, and many drugs have drug-resistant strains isolated and reported within one year of marketing. In addition, vaccines also seem to be an optional strategy, but at present, neither traditional live vaccines nor emerging genetic engineering vaccines (nucleic acid vaccines, subunit vaccines, transgenic vaccines) have certain limitations, and it is difficult to promote and apply them in the short term.

[0004] Therefore, in clinical practice, the methods of drug rotation, drug shuttle and drug combination are mainly adopted to cope with and slow down the emergence of drug-resistant strains. However, in the actual production process, it is difficult to predict when coccidia will develop resistance to which drug and to what extent, resulting in the use of some drugs that have already developed resistance, which also creates conditions for the occurrence of anticoccidial drug resistance; at the same time, some breeders use multiple anticoccidial drugs or super-dose administration to achieve the purpose of preventing and controlling coccidiosis, which will accelerate the development of drug resistance and increase the risk of drug residues in egg products and meat products, and affect the health of humans and animals.

[0005] Therefore, the research and development of alternative anticoccidial drugs that meet the requirements of "new, less residue, safe and reliable" is imminent. Many alternative solutions have been proposed, among which natural drugs, especially traditional Chinese medicine and its formulations, have attracted widespread attention. Traditional Chinese medicine preparations contain multiple active ingredients and have the advantages of multiple targets and multiple action pathways, and are less likely to develop drug resistance. In addition, while playing a role in preventing and treating diseases (under the correct guidance of veterinary theory), traditional Chinese medicine preparations can improve production performance or enhance the body's immunity, and have the advantage of small toxic and side effects. Today, under the background of food safety and "limiting and banning antibiotics", traditional Chinese medicine preparations have become a major breakthrough in solving coccidial drug resistance. SUMMARY

[0006] The purpose of the present application is to provide a traditional Chinese medicine composition for effectively treating chicken Eimeria tenella and its application, aiming to provide a new choice for the treatment of chicken coccidiosis, especially to alleviate the dilemma of "no available drugs" faced by the outbreak of drug-resistant strains. The present application is expected to reduce or replace the addition of anticoccidial drugs in chicken feed, alleviate chicken coccidial drug resistance, and contribute to the "reduction and ban of antibiotics" in livestock and poultry breeding.

[0007] The technical solution provided by the present application is as follows:

[0008] A traditional Chinese medicine composition for treating chicken Eimeria tenella, comprising the following components in mass fraction: mulberry leaves 14-16 parts, atractylodes 14-16 parts, agastache 8-12 parts, cinnabar 18-22 parts, areca nut 8-12 parts, milk root 18-22 parts, yellow oak 8-12 parts, and licorice 5-7 parts.

[0009] Further, the composition comprises the following components in mass fraction: mulberry leaves 15 parts, atractylodes 15 parts, agastache 10 parts, cinnabar 20 parts, areca nut 10 parts, milk root 20 parts, yellow oak 10 parts, and licorice 6 parts.

[0010] The present application also provides a preparation method of the traditional Chinese medicine composition for treating chicken Eimeria tenella as described above, comprising the following steps:

[0011] The raw materials are soaked in 3 times the volume of distilled water, then boiled and simmered for 2 hours, the liquid is filtered, 3 times the volume of distilled water is added to the residue, and the simmering process is continued for another 2 hours, the liquid is filtered through an 80-mesh sieve, and the two times of simmered liquid are combined and concentrated on low heat, then filtered to obtain the water decoction.

[0012] The present application also provides the use of the traditional Chinese medicine composition for treating chicken Eimeria tenella as described above in the preparation of a drug for improving the survival rate and / or relative weight gain rate of chickens infected with chicken Eimeria tenella.

[0013] Relative weight gain rate (%) = (final body weight of each chicken in the group - initial body weight of the chicken) / initial body weight of the chicken x 100%.

[0014] The application also provides the use of the Chinese medicine composition for treating chicken Eimeria tenella in the preparation of a medicine for reducing pathological changes of the cecum of a chicken infected with chicken Eimeria tenella.

[0015] The application also provides the use of the Chinese medicine composition for treating chicken Eimeria tenella in the preparation of a medicine for reducing tissue damage of the cecum of a chicken infected with chicken Eimeria tenella.

[0016] The application also provides the use of the Chinese medicine composition for treating chicken Eimeria tenella in the preparation of a medicine for reducing inflammation of the cecum of a chicken infected with chicken Eimeria tenella.

[0017] The application also provides the use of the Chinese medicine composition for treating chicken Eimeria tenella in the preparation of a medicine for reducing oxidative stress of the cecum of a chicken infected with chicken Eimeria tenella.

[0018] The application also provides the use of the Chinese medicine composition for treating chicken Eimeria tenella in the preparation of a medicine for improving the anticoccidial index of a chicken infected with chicken Eimeria tenella.

[0019] The anticoccidial index = (relative weight gain rate + survival rate) - (pathological change value + oocyst value).

[0020] The application also provides the use of the Chinese medicine composition for treating chicken Eimeria tenella in the preparation of a medicine for reducing the organ index of a chicken infected with chicken Eimeria tenella; the organs include the liver and the spleen.

[0021] The organ index (%) = (organ weight / live body weight) x 100 %.

[0022] A Chinese medicine composition for treating chicken coccidiosis caused by infection of a multiple drug-resistant strain of chicken Eimeria tenella, 1000g of the Chinese medicine composition is taken by proportion, 3 times the volume of distilled water is added to soak for 1.5h; after boiling, the medicine liquid is filtered through a 80-mesh sieve after being decocted on a low fire for 2h; 3 times the volume of distilled water is added to the dregs, and the medicine liquid is filtered through a 80-mesh sieve after being decocted on a low fire for 2h; the two times of decocted liquids are combined, and concentrated to 1000mL on a low fire, and then filtered to prepare a water decoction of 1g / mL, which is mixed into drinking water (concentration 6g / L) for free drinking.

[0023] The traditional Chinese medicine composition for treating chicken coccidiosis is composed of mulberry leaves 15g, atractylodes 15g, agastache 10g, cinnabar 20g, areca nut 10g, milk root 20g, goldenrain tree 10g, and licorice 6g. The mulberry leaves have the effects of dispelling wind-heat and cooling blood to stop bleeding; the atractylodes have the effects of invigorating the spleen, drying dampness, dispelling wind and cold; the agastache has the effect of aromatizing dampness; the cinnabar has the effects of astringing the intestines, stopping bleeding, and promoting tissue repair; the areca nut has the effects of killing insects, eliminating accumulation, and reducing qi and water; the milk root has the effects of stopping bleeding, eliminating accumulation, and dispelling wind and dampness; the goldenrain tree has the effects of clearing heat and drying dampness, and purging fire and detoxifying; and the licorice has the effects of tonifying the spleen and qi, clearing heat and detoxifying, and regulating various drugs. The mulberry leaves, atractylodes, agastache, milk root, goldenrain tree, and licorice can invigorate the spleen, dry dampness, clear heat and detoxify, and inhibit the growth and reproduction of pathogenic microorganisms; the areca nut and the goldenrain tree have the effects of directly killing or inhibiting the growth and reproduction of various parasites; the cinnabar and the milk root can inhibit exudation, stop bleeding, and promote intestinal mucosa repair; and the licorice can regulate various drugs, relieve pain, and relieve acute and chronic conditions. The combined use of the drugs is beneficial to the recovery of the intestinal function of the sick chickens. The traditional Chinese medicine composition has the effects of clearing heat and drying dampness, cooling blood to stop diarrhea, and promoting qi circulation and removing stagnation, can act on drug-resistant coccidia strains, can significantly inhibit the reproduction of coccidia in the body, can reduce the intestinal damage of the infected chickens, and can improve the survival rate and growth performance of the infected chickens.

[0024] Advantages

[0025] The traditional Chinese medicine composition can significantly reduce the ovum capsule discharge of E. tenella infected chickens, inhibit the inflammation and oxidative stress of the cecum tissue of the chickens caused by E. tenella infection, reduce the damage to the cecum tissue structure of the infected chickens, and improve the survival rate and growth performance of the infected chickens. The traditional Chinese medicine composition can reach the level of a medium-efficiency anticoccidial drug.

[0026] The application provides a traditional Chinese medicine composition for effectively treating chicken E. tenella, provides a new choice for the treatment of chicken coccidiosis, and is expected to reduce or replace the addition of anticoccidial drugs in chicken feed, alleviate the drug resistance of chickens, and make contributions to the "reduction and prohibition of drugs" in livestock and poultry breeding. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 The survival rate and weight gain rate of the chickens in each test group;

[0028] Note: A: the survival rate of the chickens in each test group; B: the body weight of the chickens in each test group at the 192nd hour. The same letter indicates that there is no significant difference (P>0.05), and different letters indicate that there is a significant difference (P<0.05).

[0029] Figure 2 The cecal visual lesions and lesion scores of the chickens in each experimental group;

[0030] Note: A: Gross lesions of the cecum; B: Cecum score; the same letter indicates no significant difference (P>0.05), different letters indicate significant difference (P<0.05).

[0031] Figure 3 These are the spleen and liver indices of chickens in each experimental group;

[0032] Note: A: Liver index; B: Spleen index; same letters indicate no significant difference (P>0.05), different letters indicate significant difference (P<0.05).

[0033] Figure 4 These are the HE staining results of the cecum of chickens in each experimental group;

[0034] Note: Black arrows indicate where the villi broke; red arrows indicate egg sacs; blue arrows indicate cavities left by shed coccidia.

[0035] Figure 5 These are the scanning electron microscope observation results of the cecum of chickens in each experimental group;

[0036] Note: Red arrows indicate oocysts; blue arrows indicate cavities left by shed coccidia.

[0037] Figure 6 It refers to the content of inflammation-related factors in the cecal tissue of chickens in each experimental group;

[0038] Note: A: IL-1β, B: IL-10, C: TNF-α; the same letter indicates no significant difference (P>0.05), different letters indicate significant difference (P<0.05).

[0039] Figure 7 These are the levels of oxidative stress-related indicators in the cecal tissue of chickens in each experimental group;

[0040] Note: A: SOD, B: GSH-Px, C: MDA; the same letter indicates no significant difference (P>0.05), different letters indicate significant difference (P<0.05). Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0042] This invention provides a preparation composed of 15g mulberry leaves, 15g Atractylodes lancea, 10g Pogostemon cablin, 20g red ochre, 10g areca nut, 20g milkweed root, 10g Phellodendron chinense, and 6g licorice. This traditional Chinese medicine preparation has the effects of clearing heat and drying dampness, cooling blood and stopping dysentery, and promoting qi circulation and relieving stagnation. It has a good therapeutic effect on Eimeria tenella infection in chickens and has few toxic side effects.

[0043] Example 1

[0044] Preventive effects of different traditional Chinese medicine compositions on chicken Eimeria tenella infection

[0045] 1. Method

[0046] 1.1 Experimental animals

[0047] One-day-old male yellow-feathered broilers were selected, and the experimental animals were raised in the experimental animal room of the Veterinary College of Yangzhou University; after the feeding environment was cleaned, it was disinfected by spraying with ammonia water, fumigation with potassium permanganate, and then heat-treated with alcohol flame after ventilation. The cages, feeders, and waterers used for feeding were also heat-treated in the same way to ensure that there were no coccidia in the chicken feeding environment and no other influencing factors in the test environment. During the feeding process, the chickens were provided with sufficient water and basic feed, and they were free to eat and drink double-distilled water. The feed samples were observed under a microscope, and there were no coccidia oocysts. During feeding, 40-watt light bulbs were used for uniform illumination, and the chickens were raised to 14 days of age for testing. Feces from the chicken population were collected for 3 consecutive days to check for coccidia oocysts.

[0048] The feed formula was prepared according to the chicken nutrition standard according to Table 1.

[0049] Table 1 Composition and nutritional level of basic feed (dry basis)

[0050]

[0051] Note: Each 1 kg of premix can provide: VA 1.25 x 105 IU, VD3 5 x 104 IU, VE 625 IU, VK3 50 mg, VB1 50 mg,

[0052] VB2 100 mg, VB6 75 mg, VB 12 0.25 mg, nicotinamide 750 mg, pantothenic acid 250 mg, folic acid 15 mg, biotin 3.75 mg, choline 11 g, copper 0.03 g, iron 0.87 g, manganese 1 g, zinc 1 g, iodine 5 mg, selenium 5 mg, methionine 5%, calcium 15%, total phosphorus 5%, sodium chloride 8%, and moisture ≤10%.

[0053] 1.2 Experimental strain

[0054] The chicken Eimeria tenella strain used was the Nantong isolated strain NT6-7 isolated and preserved by the Department of Traditional Chinese Veterinary Medicine of the Veterinary College of Yangzhou University. Previous studies have shown that this strain is completely resistant to decoquinate, clopidol, and decoquinate, and is a multi-drug resistant strain.

[0055] Before use, the strain was diluted to 5 x 10 4Rejuvenation of 14-day-old chicks inoculated with each sporulated oocyst, fresh feces were collected on the 7th day after infection, diluted with 5 times the amount of tap water, filtered twice, first with an 80 mesh sieve and second with a 120 mesh sieve. The filtrate was allowed to stand at room temperature for 3 hours, the supernatant was discarded, and then placed in a petri dish containing 2.5% potassium dichromate solution, incubated at 29°C with aeration, and stored at 4°C after the sporulation rate reached more than 95%, with a maximum storage time of 30 days.

[0056] 1.3 Composition of Chinese medicine composition and preparation of water decoction

[0057] Chinese medicine composition A contains atractylodes 15g, pogostemon 10g, areca nut 10g, and licorice 6g, with the functions of invigorating the spleen, drying dampness, promoting qi and draining water; Chinese medicine composition B is composed of mulberry leaf 15g, golden cypress 10g, milk root 20g, cinnabar 20g, and licorice 6g, with the functions of clearing heat and dampness, astringing and stopping bleeding, and astringing the intestines to stop diarrhea. Chinese medicine composition A+B is composed of all the drugs in the above prescriptions, with the functions of clearing heat and drying dampness, cooling blood to stop diarrhea, promoting qi and removing stagnation.

[0058] Each 1000g of Chinese medicine composition A, B, A+B was taken according to the proportion, 3 times the volume of distilled water was added and soaked for 1.5h, then boiled and simmered for 2h, and the filtrate was filtered with an 80 mesh sieve; 3 times the volume of distilled water was added to the residue and continued to simmer for 2h, and filtered with an 80 mesh sieve. The two decoctions were combined and concentrated to 1000ml with gentle heat, and then filtered to prepare 1g / ml water decoction.

[0059] 1.4 Experimental grouping

[0060] 60 14-day-old yellow-feathered broiler chickens with similar body weight were selected and randomly divided into control group (Control), model group (Model), Chinese medicine composition A group (TCM-A), Chinese medicine composition B group (TCM-B), and Chinese medicine composition A+B group (TCM-A+B), with 12 chickens in each group. The chickens were weighed at 15 days of age as the initial body weight, and on the same day, each chicken was orally administered with 5x10 4 oocysts / each, and the feces collected 120-192h after infection were counted for oocysts. The chickens were weighed at 23 days of age as the final body weight and the intestinal lesions were scored. The administration method is shown in Table 2; the administration dose was 3g / kg body weight, and the administration method was free drinking water.

[0061] Table 2 Grouping and treatment of experimental animals

[0062]

[0063] 1.5 Evaluation of the anti-coccidial efficacy of Chinese medicine composition

[0064] The relative weight gain rate, survival rate, lesion value and oocyst value were calculated according to the method of Merck Company of USA 192 hours after the chicken was infected with Eimeria tenella; the anti-coccidiosis effect of the drug was evaluated by the number of oocysts per gram of feces, weight gain, lesion score and mortality rate of the test group and the control group.

[0065] Anti-coccidiosis index (ACI) = (relative weight gain rate + survival rate) - (lesion value + oocyst value)

[0066] According to the anti-coccidiosis index ACI value, when ACI≥180, it is high efficiency, 160≤ACI<180, it is medium efficiency, 120≤ACI<160, it is low efficiency, and ACI<120, it is invalid.

[0067] 2 Experimental results

[0068] 2.1 Anti-coccidiosis index of each experimental group

[0069] As shown in Table 3, the oocyst value and cecal lesion value of the Model group were significantly higher than those of the Control group, and the survival rate and relative weight gain rate were significantly lower; compared with the Model group, the anti-coccidiosis index of the TCM-A, TCM-B and TCM-A+B groups was significantly increased, and the oocyst value and lesion value were significantly decreased; the relative weight gain rate and anti-coccidiosis index of the TCM-A+B group were higher than those of the TCM-A and TCM-B groups, and the mortality rate, oocyst value and lesion value were lower than those of the TCM-A and TCM-B groups, indicating that the anti-coccidiosis effect of the Chinese medicine composition A+B group was the best, and the Chinese medicine composition was used for subsequent test.

[0070] Table 3 Anti-coccidiosis index of each test group

[0071]

[0072] 3 Summary

[0073] The anti-coccidiosis index of the Chinese medicine composition A+B was higher than that of the Chinese medicine composition A and B groups, and the Chinese medicine composition A+B could improve the survival rate and relative weight gain rate of the test chickens, reduce the clinical lesions of the test chickens, and the treatment effect was better than that of the Chinese medicine composition A and B groups, and the Chinese medicine composition A+B was used for subsequent experiments.

[0074] Example 2

[0075] 1 Method

[0076] 1.1 Experimental animals

[0077] 1-day-old male yellow-feathered broilers were purchased from the Poultry Institute of the Chinese Academy of Agricultural Sciences, production license: agricultural chicken Su K050901; use license: SYXK (Su) 2017-0044. Experimental animals were raised in the Experimental Animal Room of the Veterinary College of Yangzhou University; the feeding environment was thoroughly cleaned and disinfected with ammonia water, formaldehyde, and potassium permanganate, and then treated with alcohol flame after ventilation. The cages, feeders, and waterers used for feeding were also treated in the same way to ensure that there were no coccidia in the chicken feeding environment and no other influencing factors in the experimental environment. During the feeding process, the chickens were provided with sufficient water and basic feed, and they were free to eat and drink water treated with 121 °C for 20 min. The feed was sampled, sliced, and observed under a microscope to check for coccidian oocysts. A 40-watt bulb was used for lighting during feeding, and the chickens were fed until they were 14 days old for experimental use. Feces from the chicken population were collected for 3 consecutive days to check for coccidian oocysts. All operations during the experiment complied with the Guidelines for the Treatment of Laboratory Animals issued by the Ministry of Science and Technology of the People's Republic of China in 2006, and there were no violations of animal welfare. All animal experiments were conducted under the guidance of the Yangzhou University Animal Protection and Utilization Committee. All experimental animals were approved by the Yangzhou University Experimental Animal Ethics Committee (No. 202103-009). The feed formula was prepared according to the chicken nutrition standards in Table 4.

[0078] Table 4 Composition and nutritional levels of the basic feed ration (dry basis)

[0079]

[0080] Note: Each 1 kg of premix can provide: VA 1.25 x 105IU, VD3 5 x 104IU, VE 625IU, VK3 50mg, VB1 50mg,

[0081] VB2 100mg, VB6 75mg, VB 12 0.25mg, nicotinamide 750mg, pantothenic acid 250mg, folic acid 15mg, biotin 3.75mg, choline 11g, copper 0.03g, iron 0.87g, manganese 1g, zinc 1g, iodine 5mg, selenium 5mg, methionine 5%, calcium 15%, total phosphorus 5%, sodium chloride 8%, and moisture ≤10%.

[0082] 1.2 Experimental strain

[0083] The experimental chicken Eimeria tenella strain was the Nantong isolated strain NT6-7 isolated and preserved by the Clinical Veterinary Medicine Teaching and Research Office of Yangzhou University. After preliminary experiments, the strain was completely resistant to diclazuril, clopidol, and decoquinate, and was a multi-drug resistant strain. Before use, the strain was diluted to 5 x 10 4Rejuvenation of 14-day-old chicks inoculated with each sporulated oocyst, collect fresh feces on the 7th day after infection, dilute the feces with 5 times the amount of tap water, filter twice, first with an 80 mesh sieve and second with a 120 mesh sieve. Let the filtrate stand at room temperature for 3 hours, discard the supernatant, then place it in a petri dish containing 2.5% potassium dichromate solution, incubate at 29°C with aeration, and store at 4°C after the sporulation rate reaches more than 95%, with a maximum storage time of 30 days.

[0084] 1.3 Preparation of water decoction of traditional Chinese medicine composition

[0085] Weigh 1000 grams of each proportionally grabbed traditional Chinese medicine composition, add 3 times the volume of distilled water for soaking, soak leafy and stemmed medicines for 1.5 hours, and soak medicines mainly composed of roots and fruits for 3 hours; after boiling, use low heat to decoct for 2 hours, filter the medicinal liquid using an 80 mesh filter; add 3 times the volume of distilled water to the residue, continue to decoct using low heat for 2 hours, filter using an 80 mesh filter, and combine the two decocted liquids, then concentrate to 1000 ml using low heat, filter, and prepare 1 g / ml water decoction.

[0086] 1.4 Experimental method

[0087] Divide 48 10-day-old experimental chickens into a blank control group (CON), a model group (MOD), a traditional Chinese medicine composition treatment group (TCMF), and a diclazuril treatment group (DIC) according to the principle of consistent body weight, with 12 chickens in each group. Weigh the experimental chickens at 15 days of age as the initial body weight, and orally administer 5 x 10 4 spores per chicken on the same day. Collect the feces from 120 to 192 hours after infection for oocyst counting, weigh the chickens at 23 days of age as the final body weight, and record the intestinal lesion scores from the cecum. All drugs are administered according to Table 5, with the traditional Chinese medicine composition concentration being 6 g / L for free drinking water administration, and the diclazuril concentration being 1 mg / L for free drinking water administration.

[0088] Table 5 Experimental animal grouping and treatment

[0089]

[0090] 1.5 Survival rate and weight gain

[0091] At 192 hours after infection, count the survival of the chickens in each group. The survival rate (%) of each group = the number of surviving chickens in the group / the total number of experimental chickens in the group x 100%.

[0092] Each group of experiments weighs the chickens on an empty stomach at 15 days of age and in the morning after the experiment ends, with the body weight at the beginning of the experiment as the initial body weight, and the body weight of the chickens at 23 days of age as the final body weight, to calculate the weight gain index.

[0093] Relative weight gain rate (%) = (the final body weight of each chicken in the group - the initial body weight of the chicken) / the initial body weight of the chicken x 100%

[0094] 1.6 Lesion score of caecum of chicken

[0095] At the 192nd hour after infection, the chickens in each experimental group were sacrificed and the caecum was collected. After observing the external lesions, the caecum was cut open to observe the lesions in the intestinal tract. The caecum with the most severe lesions was selected for lesion scoring, and the scoring standard is shown in Table 6.

[0096] Table 6 Scoring criteria for caecum lesions

[0097]

[0098] Note: According to the severity of the caecum lesions after the chickens were dissected, when the lesions of the two sides of the caecum were inconsistent, the side with the most severe lesions was used as the reference.

[0099] 1.7 Evaluation of anticoccidial efficacy of the traditional Chinese medicine composition

[0100] After 192 hours of infection with E. tenella, the weight of each group of chickens was measured on an empty stomach, and the weight gain was calculated. During the experiment, the mortality of the chickens was recorded, and the survival rate was calculated. The relative weight gain rate, survival rate, lesion value (lesion score x 10), and oocyst value were calculated according to the method of the American Merck Company, and the anticoccidial index (ACI) was calculated. The anticoccidial effect of the drug was evaluated comprehensively by the number of oocysts per gram of feces, weight gain, lesion score, and mortality of the chickens in the experimental and control groups.

[0101] Anticoccidial index (ACI) = (relative weight gain rate + survival rate) - (lesion value + oocyst value)

[0102] According to the anticoccidial index ACI value, when ACI ≥ 180, it is highly effective, 160 ≤ ACI < 180 is moderately effective, 120 ≤ ACI < 160 is low effective, and ACI < 120 is ineffective.

[0103] 1.8 Histological observation of chicken caecum tissue

[0104] After 192 hours of infection, the chickens in each experimental group were sacrificed, and the caecum was collected and scored for lesions. One side of the caecum was fixed in 4% neutral formaldehyde and 2.5% glutaraldehyde for the preparation of conventional HE-stained sections and scanning electron microscope samples, and the other side of the caecum was stored in a -80°C freezer.

[0105] 1.9 Determination of organ index of chicken

[0106] At the 192nd hour of infection, the pre-slaughter live weight of each chicken in each experimental group was weighed, and after being sacrificed, the spleen and liver were quickly separated, the fat and connective tissue on the surface of the organs were removed, and after the blood water was absorbed with filter paper, the organs were weighed to calculate the organ index.

[0107] Visceral index (%) = (visceral weight / live weight) x 100%

[0108] 1.10 Determination of inflammation factors in chicken cecal tissue

[0109] Detection of TNF-α, IL-1β and IL-10: After washing the microplate for 3 times, centrifuge the tissue homogenate at 1000 r / min for 10 min, take 100 μl supernatant and add into the microplate, incubate at 37℃ for 2 h, then wash the plate for 3 times; add 100 μl biotinylated antibody working solution, incubate at 37℃ for 1 h, then wash the plate for 3 times; add 100 μl streptavidin-HRP working solution, incubate at 37℃ for 0.5 h, then wash for 3 times; add 100 μl substrate solution and incubate at 37℃ for 20 min in dark; add 50 μl stop solution, then immediately detect by microplate reader at 450 nm, and the correction wavelength is 630 nm; use the standard curve to make corresponding calculation and statistics.

[0110] 1.11 Determination of redox index in chicken cecal tissue

[0111] Preparation of cecal tissue homogenate: take 100 mg of cecal tissue, rinse with pre-cooled normal saline, then wipe dry with filter paper, weigh, and add RIPA lysis buffer to prepare 10% tissue homogenate.

[0112] Detection of SOD activity: centrifuge the 10% tissue homogenate at 3000 r / min for 10 min; take 20 μl supernatant and add into the microplate, add the corresponding reaction solution prepared according to the kit requirements into the microplate, incubate at 37℃ for 20 min, then read OD value of each tube at 450 nm after zero adjustment with distilled water, and make corresponding calculation and statistics by the method recommended in the instruction.

[0113] Detection of GSH-Px activity: centrifuge the 10% tissue homogenate at 3000 r / min for 10 min; take 100 μl supernatant and add into 4 ml centrifuge tube together with the corresponding reaction solution prepared according to the kit requirements, stand at room temperature for 15 min, then read OD value of each tube at 412 nm after zero adjustment with distilled water, and make corresponding calculation and statistics by the method recommended in the instruction.

[0114] Detection of MDA content: take 100 μl of 10% tissue homogenate and add into 4 ml centrifuge tube together with the corresponding reaction solution prepared, vortex to mix, then incubate at 95℃ water bath for 40 min, cool with running water, then centrifuge at 4000 r / min for 10 min, take the supernatant, read OD value of each tube at 532 nm after zero adjustment with distilled water, and make corresponding calculation and statistics by the method recommended in the instruction.

[0115] 1.12 Data analysis

[0116] Statistical analysis was performed using IBM SPSS Statistics 17.0 software, and graphs were created using Graphpad Prism 9.5.1. All data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used for comparisons between groups. A p-value < 0.05 was considered statistically significant.

[0117] 2 Results

[0118] 2.1 Traditional Chinese medicine composition improves the survival rate and relative weight gain rate of chickens infected with Eimeria tenella.

[0119] like Figure 1 As shown, no chickens in the TCMF and CON groups died, while the survival rate in the MOD and DIC groups was 75%. At 192 hours after infection, the body weight of chickens in the TCMF and DIC groups was significantly higher than that in the MOD group (P < 0.05).

[0120] 2.2 Traditional Chinese medicine composition reduces cecal lesion score in chickens infected with Eimeria tenella.

[0121] like Figure 2 As shown, compared with the CON group, the cecum of chickens in the MOD group was significantly shorter, with scattered hemorrhages on the surface and a cecal core in the intestinal tract. Compared with the MOD group, the cecum length of chickens in the TCMF and DIC groups was significantly increased, with no obvious hemorrhages on the cecum surface and no cecal core in the intestinal tract. The cecal lesion scores of chickens in the TCMF and DIC groups were significantly lower than those in the MOD group (P < 0.05), and the cecal lesion score of chickens in the TCMF group was significantly lower than that in the DIC group (P < 0.05).

[0122] 2.3 Anticoccidial index of each experimental group

[0123] As shown in Table 7, the ACI index of the MOD group was 55.2, while the ACI indices of the TCMF group and the DIC group were 164.8 and 122.6, respectively. The TCMF group's drug reached the level of a moderately effective anticoccidial drug, which was better than that of DIC.

[0124] Table 7. Anticoccidial index of drugs in each experimental group

[0125]

[0126] 2.4 Traditional Chinese medicine composition reduces liver and spleen indices in chickens infected with Eimeria tenella.

[0127] like Figure 3As shown in Table 2, compared with the CON group, the liver index and spleen index of the MOD group chickens were significantly higher (P<0.05), and the liver index and spleen index of the TCMF group and the DIC group chickens were significantly lower than those of the MOD group (P<0.05); among them, the spleen index of the TCMF group chickens was significantly lower than that of the MOD group (P<0.05).

[0128] 2.5 The Chinese medicine composition reduces the damage of the cecum tissue of the chicken infected with the Eimeria tenella

[0129] 2.5.1 HE staining results of the cecum tissue of each experimental group

[0130] As shown in Table 2, compared with the CON group, the liver index and spleen index of the MOD group chickens were significantly higher (P<0.05), and the liver index and spleen index of the TCMF group and the DIC group chickens were significantly lower than those of the MOD group (P<0.05); among them, the spleen index of the TCMF group chickens was significantly lower than that of the MOD group (P<0.05). Figure 4 As shown in Table 3, compared with the CON group, the intestinal villi of the MOD group chickens were short or broken, and there was no normal gland structure; a large number of inflammatory cell infiltration and red blood cell exudation existed in the intestinal mucosa, and a large number of oocysts could be seen. The intestinal villi and gland structure of the TCMF group and the DIC group chickens were relatively complete, and the inflammatory cell infiltration and red blood cell exudation in the intestinal mucosa were obviously reduced; no oocysts were found in the intestinal mucosa. The integrity of the cecum structure of the TCMF group chickens was better than that of the DIC group.

[0131] 2.5.2 Scanning electron microscope results of the cecum tissue of each experimental group

[0132] As shown in Table 3, compared with the CON group, the intestinal villi of the MOD group chickens were short or broken, and there was no normal gland structure; a large number of inflammatory cell infiltration and red blood cell exudation existed in the intestinal mucosa, and a large number of oocysts could be seen. The intestinal villi and gland structure of the TCMF group and the DIC group chickens were relatively complete, and the inflammatory cell infiltration and red blood cell exudation in the intestinal mucosa were obviously reduced; no oocysts were found in the intestinal mucosa. The integrity of the cecum structure of the TCMF group chickens was better than that of the DIC group. Figure 5 2.6 The Chinese medicine composition reduces the inflammatory response of the cecum tissue of the chicken infected with the Eimeria tenella

[0133] As shown in Table 4, compared with the CON group, the IL-1β and TNF-α contents in the cecum tissue of the MOD group chickens were significantly increased (P<0.05), and the IL-10 content was significantly reduced (P<0.05). Compared with the MOD group, the IL-1β and TNF-α contents in the cecum tissue of the TCMF group and the DIC group chickens were significantly reduced (P<0.05), and the IL-10 content was significantly higher than that of the MOD group (P<0.05). The IL-1β and TNF-α contents in the cecum tissue of the TCMF group chickens were significantly lower than those of the DIC group (P<0.05), and the IL-10 content was significantly higher than that of the DIC group (P<0.05).

[0134] Figure 6 2.7 The Chinese medicine composition reduces the oxidative stress response of the cecum tissue of the chicken infected with the Eimeria tenella

[0135] 2.7 The Chinese medicine composition reduces the oxidative stress response of the cecum tissue of the chicken infected with the Eimeria tenella ​

[0136] like Figure 7 As shown, compared with the CON group, the activities of SOD and GSH-Px in the cecal tissue of chickens in the MOD group were significantly decreased (P < 0.05), while the MDA content was significantly increased (P < 0.05). Compared with the MOD group, the activities of SOD and GSH-Px in the cecal tissue of chickens in the TCMF group and DIC group were significantly increased (P < 0.05), while the MDA content was significantly decreased (P < 0.05). The activities of ISOD and GSH-Px in the cecal tissue of chickens in the TCMF group were significantly higher than those in the DIC group (P < 0.05), while the MDA content was significantly lower than that in the DIC group (P < 0.05).

[0137] 3. Summary

[0138] The traditional Chinese medicine composition can significantly reduce the amount of oocysts excreted in E. tenella-infected chickens, inhibit inflammation and oxidative stress in the cecal tissue caused by E. tenella infection, alleviate structural damage to the cecal tissue of infected chickens, and improve the survival rate and growth performance of infected chickens. The traditional Chinese medicine composition reaches the level of a moderately effective anticoccidial drug.

[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A traditional Chinese medicine composition for treating Eimeria tenella in chickens, characterized in that, The traditional Chinese medicine composition is made of traditional Chinese medicine composition A and traditional Chinese medicine composition B, the traditional Chinese medicine composition A is made of 15 parts of atractylodes, 10 parts of agastache, 10 parts of areca nut and 6 parts of licorice, and the traditional Chinese medicine composition B is made of 15 parts of mulberry leaves, 10 parts of Chinese cork tree, 20 parts of milk root, 20 parts of cinnabar and 6 parts of licorice.

2. The preparation method of the traditional Chinese medicine composition for treating chicken Eimeria tenella according to claim 1, characterized in that, The method comprises the following steps: The raw materials are soaked in 3 times the volume of distilled water, and then boiled and decocted for 2 hours, and the decoction is filtered; 3 times the volume of distilled water is added to the residue, and then boiled and decocted for 2 hours, and the decoction is filtered through an 80-mesh sieve; the two decoctions are combined and concentrated on low heat, and then filtered to obtain the water decoction.

3. Use of the traditional Chinese medicine composition for treating chicken Eimeria tenella in claim 1 in the preparation of a medicine for increasing the survival rate and / or relative weight gain rate of chickens infected with chicken Eimeria tenella. Relative weight gain rate (%) = (final body weight of each chicken in the group - initial body weight of the chicken) / initial body weight of the chicken x 100%.

4. Use of the traditional Chinese medicine composition for treating chicken Eimeria tenella in claim 1 in the preparation of a medicine for reducing the cecal lesion of chickens infected with chicken Eimeria tenella.

5. Use of the traditional Chinese medicine composition for treating chicken Eimeria tenella in claim 1 in the preparation of a medicine for reducing the cecal tissue damage of chickens infected with chicken Eimeria tenella.

6. Use of the traditional Chinese medicine composition for treating chicken Eimeria tenella in claim 1 in the preparation of a medicine for reducing the cecal tissue inflammation of chickens infected with chicken Eimeria tenella.

7. Use of the traditional Chinese medicine composition for treating chicken Eimeria tenella in claim 1 in the preparation of a medicine for reducing the cecal tissue oxidative stress of chickens infected with chicken Eimeria tenella.

8. Use of the traditional Chinese medicine composition for treating chicken Eimeria tenella in claim 1 in the preparation of a medicine for increasing the anticoccidial index of chickens infected with chicken Eimeria tenella. Anticoccidial index = (relative weight gain rate + survival rate) - (lesion value + oocyst value).

9. Use of the traditional Chinese medicine composition for treating chicken Eimeria tenella in claim 1 in the preparation of a medicine for reducing the organ index of chickens infected with chicken Eimeria tenella; the organs include liver and spleen. Organ index (%) = (organ weight / live body weight) x 100%.

Images