CN117379500A

CN117379500A - Traditional Chinese medicine composition for treating chicken eimeria tenella and application thereof

Info

Publication number: CN117379500A
Application number: CN202311374611.8A
Authority: CN
Inventors: 刘明江; 陈慧敏; 田伟; 李金贵; 槐玉英; 王惠文; 郑淑媚; 伯若楠
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2023-10-23
Filing date: 2023-10-23
Publication date: 2024-01-12

Abstract

The invention belongs to the field of veterinary drug preparation, and discloses a traditional Chinese medicine composition for treating chicken eimeria tenella and application thereof, wherein the traditional Chinese medicine composition comprises the following components in parts by mass: 14-16 parts of mulberry leaf, 14-16 parts of rhizoma atractylodis, 8-12 parts of wrinkled gianthyssop, 18-22 parts of red halloysite, 8-12 parts of betel nut, 18-22 parts of milk root, 8-12 parts of amur corktree bark and 5-7 parts of liquorice. The traditional Chinese medicine composition has the effects of clearing heat and drying dampness, cooling blood and stopping diarrhea, promoting qi circulation and removing stagnation, has a good treatment effect on chicken coccidiosis, can improve the growth performance of chickens, and has no toxic or side effect.

Description

Traditional Chinese medicine composition for treating chicken eimeria tenella and application thereof

Technical Field

The invention belongs to the technical field of veterinary drug preparation, and particularly relates to a traditional Chinese medicine composition for treating chicken eimeria tenella and application thereof.

Background

Along with the continuous development of large-scale, intensive and industrialization of the livestock and poultry breeding industry, the growth speed or the production performance of the livestock and poultry are continuously improved, and more economic benefits are brought to the livestock and poultry breeding industry. However, as the raising density and raising amount are increased sharply, the living space and the environmental quality are limited, the resistance of the livestock is reduced, and the occurrence rate of diseases is increased, which becomes a troublesome problem for the development of animal husbandry. Chicken coccidiosis is a poultry parasitic disease that damages the breeding industry caused by one or more coccidia parasitized in chicken intestinal epithelial cells, and is one of the most serious diseases of the intensive farms. The disease is characterized by listlessness, slow growth, bloody dysentery, reduced growth performance, can also cause immunosuppression, cause failed vaccination or induce secondary infection, and seriously cause death of chickens, and is considered to be the parasitic disease with the greatest influence on the production economy of poultry. Currently, 7 commonly recognized species of coccidia that can infect chickens are the most common and most pathogenic.

At present, the prevention and treatment of chicken coccidiosis mainly based on ionophore drugs and chemical synthesis drugs. But the problem of coccidian resistance is continuously emerging due to the long-term massive use or abuse of drugs. A large number of documents report that almost all commercially used anticoccidial chemicals have developed corresponding resistant strains, and that the trend of multiple resistance and cross resistance is increasingly severe. The common drug resistance of anticoccidial drugs often causes failure in prevention and control or poor effect of chicken coccidiosis, so that once coccidiosis erupts, the anticoccidial drug faces the embarrassment of no drug availability, and huge economic loss is caused. Although researchers are working on developing novel anticoccidial drugs, the development speed of drugs often lags behind the speed of drug resistance, and many drugs have been reported separately about one year on the market. In addition, the vaccine seems to be an optional countermeasure, but at present, the traditional live vaccine or the emerging genetic engineering vaccine (nucleic acid vaccine, subunit vaccine and transgenic vaccine) has certain limitations, and is difficult to popularize and apply in a short period.

Therefore, in clinical practice, the mode of alternate medication, shuttle medication and combined medication is mainly adopted to cope with and slow down the occurrence of drug-resistant insect strains. However, in the actual production process, it is difficult to predict in time and accurately what degree of drug resistance is generated by coccidian to what kind of drug, so that some drugs with generated drug resistance are still used, and conditions are created for the generation of drug resistance of anticoccidial drugs; meanwhile, some breeders adopt a mode of administering a plurality of anticoccidial drugs or overdose to achieve the purpose of preventing and controlling coccidiosis, which can accelerate the generation of drug resistance, increase the drug residue risk of egg products and meat products and influence the health of people and animals.

Therefore, the development of alternative anticoccidial drugs meeting the requirements of 'new type, less residue, safety and reliability' and the like is urgent. Many alternatives are also proposed, wherein natural medicines, particularly traditional Chinese medicines and formulas thereof, are widely focused, and the traditional Chinese medicine preparation contains various effective components, has the advantages of multiple targets and multiple action ways, and is not easy to generate drug resistance; in addition, the traditional Chinese medicine preparation can improve the production performance or enhance the immunity of organisms and has the advantage of small toxic and side effects while the efficacy of preventing and treating diseases is exerted (under the guidance of the correct (veterinary) medical theory). Nowadays, under the large background of food safety and resistance limiting and forbidden, the traditional Chinese medicine preparation has become a large break for solving the drug resistance of coccidium.

Disclosure of Invention

The invention aims to provide a traditional Chinese medicine composition for effectively treating chicken eimeria tenella and application thereof, and aims to provide a new choice for treating chicken coccidiosis, in particular to relieve the dilemma of 'no medicine available' faced by drug-resistant insect strain outbreak. The invention is hopeful to reduce or replace the addition of anticoccidial drugs in chicken feed, relieve the drug resistance of chicken coccidiosis and make a contribution to the industry of 'reducing and forbidding' of livestock and poultry cultivation.

The technical scheme provided by the invention is as follows:

a traditional Chinese medicine composition for treating chicken eimeria tenella comprises the following components in parts by mass: 14-16 parts of mulberry leaf, 14-16 parts of rhizoma atractylodis, 8-12 parts of wrinkled gianthyssop, 18-22 parts of red halloysite, 8-12 parts of betel nut, 18-22 parts of milk root, 8-12 parts of amur corktree bark and 5-7 parts of liquorice.

Further, the coating comprises the following components in parts by weight: 15 parts of mulberry leaf, 15 parts of rhizoma atractylodis, 10 parts of wrinkled gianthyssop herb, 20 parts of red halloysite, 10 parts of betel nut, 20 parts of milk root, 10 parts of amur corktree bark and 6 parts of liquorice.

The invention also provides a preparation method of the traditional Chinese medicine composition for treating chicken eimeria tenella, which comprises the following steps:

adding distilled water with the volume of 3 times into the raw materials for soaking; boiling, decocting with slow fire for 2 hr, and filtering to obtain medicinal liquid; adding distilled water with volume 3 times of that of the residues, decocting with slow fire for 2 hr, filtering with 80 mesh filter screen, mixing the decoctions, concentrating with slow fire, and filtering to obtain water decoction.

The invention also provides application of the traditional Chinese medicine composition for treating the chicken eimeria tenella in preparing medicines for improving the survival rate and/or the relative weight gain rate of the chicken eimeria tenella infected chicken;

relative weight gain (%) = (final weight of each chicken in the group-initial weight of the chicken)/initial weight of the chicken x 100%.

The invention also provides application of the traditional Chinese medicine composition for treating the chicken eimeria tenella in preparing a medicine for reducing the infection of the chicken caecum lesions by the chicken eimeria tenella.

The invention also provides application of the traditional Chinese medicine composition for treating the chicken eimeria tenella in preparing a medicine for reducing the cecum tissue injury of the chicken infected by the chicken eimeria tenella.

The invention also provides application of the traditional Chinese medicine composition for treating the chicken eimeria tenella in preparing a medicine for reducing the inflammation of the cecum tissue of the chicken infected by the chicken eimeria tenella.

The invention also provides application of the traditional Chinese medicine composition for treating the chicken eimeria tenella in preparing a medicine for reducing the oxidative stress reaction of the chicken cecum tissues infected by the chicken eimeria tenella.

The invention also provides application of the traditional Chinese medicine composition for treating chicken eimeria tenella in preparing medicines for improving the anti-coccidium index of chicken eimeria tenella infected chicken;

anticoccidial index= (relative rate of weight gain + survival) - (lesion + oocyst value).

The invention also provides application of the traditional Chinese medicine composition for treating the chicken eimeria tenella in preparing medicines for reducing the indices of viscera of chicken infected by the chicken eimeria tenella; the viscera include liver and spleen;

organ index (%) = (organ weight/living body weight) ×100%.

The traditional Chinese medicine composition for treating chicken coccidiosis caused by infection of multiple drug-resistant strains of Eimeria tenella is prepared by proportionally grabbing 1000g of the traditional Chinese medicine composition, and adding distilled water with volume of 3 times to soak for 1.5 hours; decocting with slow fire for 2h after boiling, and filtering the liquid medicine with a 80-mesh filter screen; adding distilled water with volume 3 times to the residue, decocting with slow fire for 2 hr, filtering with 80 mesh filter screen, mixing the decoctions, concentrating with slow fire to 1000mL, filtering to obtain 1g/mL water decoction, adding drinking water (concentration 6 g/L), and administering with free drinking water.

The traditional Chinese medicine composition for treating chicken coccidiosis consists of 15g of mulberry leaf, 15g of rhizoma atractylodis, 10g of wrinkled gianthyssop, 20g of red halloysite, 10g of betel nut, 20g of milk root, 10g of amur corktree bark and 6g of liquorice. Wherein the mulberry leaf has the effects of dispelling wind and heat, cooling blood and stopping bleeding; rhizoma Atractylodis has effects of invigorating spleen, eliminating dampness, dispelling pathogenic wind and dispelling cold; herba Pogostemonis has effects of eliminating dampness with aromatics; halloysitum rubrum has effects of astringing intestine, stopping bleeding, promoting granulation and healing sore; betel nut has the effects of killing parasites, removing food retention, reducing qi and promoting diuresis. The milk root has the effects of stopping bleeding, removing food stagnation, dispelling wind and promoting diuresis; cortex Phellodendri has effects of clearing heat, eliminating dampness, purging pathogenic fire and removing toxic substances; licorice has the effects of invigorating spleen and replenishing qi, clearing heat and detoxicating, and harmonizing various medicines; the mulberry leaf, the rhizoma atractylodis, the wrinkled gianthyssop herb, the milk root, the amur corktree bark and the liquorice can strengthen the spleen of the sick chicken, remove dampness, clear heat and detoxify, and inhibit the growth and the reproduction of pathogenic microorganisms; betel nut and phellodendron bark have the functions of directly killing or inhibiting growth and reproduction of various parasites; halloysitum rubrum and radix Lac bovis seu Bubali can inhibit exudation, stop bleeding, and promote intestinal mucosa repair; the liquorice can harmonize various medicines and relieve spasm and pain; the combination of the medicines is beneficial to the recovery of intestinal functions of sick chickens. The traditional Chinese medicine composition has the effects of clearing heat and drying dampness, cooling blood and stopping diarrhea, and promoting qi circulation and removing stagnation, can act on drug-resistant coccidium strains, can obviously inhibit the propagation of coccidium in vivo, lighten intestinal tract injury of infected chickens, improve survival rate of the infected chickens and effectively restore growth performance of the infected chickens.

Advantageous effects

The traditional Chinese medicine composition can obviously reduce the discharge amount of E.tenella infected chicken oocysts, inhibit the inflammation of the chicken cecum tissue caused by E.tenella infection, reflect oxidative stress, reduce the damage of the structure of the infected chicken cecum tissue, and improve the survival rate and the growth performance of the infected chicken. The traditional Chinese medicine composition can reach the level of the medium-effect anticoccidial drug.

The traditional Chinese medicine composition for effectively treating chicken eimeria tenella provides a new choice for treating chicken coccidiosis, is hopeful to reduce or replace the addition of anticoccidial medicines in chicken feeds, relieves the drug resistance of chicken coccidiosis, and contributes to the industry of 'resistance reduction and resistance forbidding' of livestock and poultry cultivation.

Drawings

FIG. 1 is a graph showing survival and weight gain of each test group;

note that: a: survival B of chickens for each test group: the chickens of each test group had a body weight of 192 h. The letters are the same, indicating that the difference is not significant (P > 0.05), and the letters are different, indicating that the difference is significant (P < 0.05).

FIG. 2 is cecal eye lesions and lesion scores for chickens of each experimental group;

note that: a: cecum gross lesions B cecum score; the letters are the same, indicating that the difference is not significant (P > 0.05), and the letters are different, indicating that the difference is significant (P < 0.05).

FIG. 3 shows spleen index and liver index of each test group chicken;

note that: a: liver index; b: spleen index; the letters are the same, indicating that the difference is not significant (P > 0.05), and the letters are different, indicating that the difference is significant (P < 0.05).

FIG. 4 shows the results of HE staining of ceca from chickens of each test group;

note that: black arrows indicate pile breaks; red arrows indicate oocysts; blue arrows indicate the voids left by coccidian shedding.

FIG. 5 is the results of the cecal scanning electron microscope observation of the chickens of each experimental group;

note that: red arrows indicate oocysts; blue arrows indicate the voids left by coccidian shedding.

FIG. 6 is the content of the cecum tissue inflammation-related factor of each test group;

note that: a: IL-1 beta, B: IL-10, C: TNF-alpha; the letters are the same, indicating that the difference is not significant (P > 0.05), and the letters are different, indicating that the difference is significant (P < 0.05).

FIG. 7 is a graph showing the levels of indices related to the oxidative stress of the cecum tissue of chickens in each experimental group;

note that: a: SOD, B: GSH-Px, C: MDA; the letters are the same, indicating that the difference is not significant (P > 0.05), and the letters are different, indicating that the difference is significant (P < 0.05).

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

The embodiment of the invention provides a health care tea which is composed of 15g of mulberry leaf, 15g of rhizoma atractylodis, 10g of wrinkled gianthyssop, 20g of red halloysite, 10g of betel nut, 20g of milk root, 10g of amur corktree bark and 6g of liquorice. The traditional Chinese medicine preparation has the effects of clearing heat and drying dampness, cooling blood and stopping diarrhea, promoting qi circulation and removing stagnation, has a good treatment effect on chicken eimeria tenella infection, and has small toxic and side effects.

Example 1

Prevention effect of different traditional Chinese medicine compositions on chicken eimeria tenella infection

1. Method of

1.1 Experimental animal

Selecting 1-day-old male yellow-feather broilers, and feeding experimental animals in experimental animal houses of a veterinary medicine department of Yangzhou university; after the raising environment is cleaned, ammonia water is used for spraying, potassium permanganate is used for fumigation and disinfection, and alcohol flame is used for heat treatment after ventilation. The cages, the trough and the water tank for raising adopt the same heat treatment method, so that the chicken raising environment is ensured to be free of coccidium, and the test environment is ensured to be free of other influencing factors. The chickens can provide sufficient water and basic feed in the feeding process, can eat freely, and can drink water by double steaming. Feed sampling film-making microscope observation, no coccidian oocyst. The breeding is carried out by uniformly adopting a 40-watt bulb for illumination until 14 days of age for test. The feces of the chicken flock was collected for 3 consecutive days for examination of coccidian-free oocysts.

The feed formulation was formulated according to table 1 with reference to chicken nutrition standards.

Table 1 basic diet composition and nutrient level (air-dried basis)

Note that: per 1kg of premix, it is possible to provide: VA 1.25X105 IU, VD ₃ 5×104IU，VE 625IU，VK ₃ 50mg，VB ₁ 50mg，

VB ₂ 100mg，VB ₆ 75mg，VB ₁₂ 0.25mg of 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 water content less than or equal to 10%.

1.2 Experimental insect strains

The chicken Eimeria tenella strain is Nantong isolate NT6-7 which is isolated and stored in a veterinary teaching and research room in the university of Yangzhou animal doctor. Early researches show that the insect strain is completely resistant to diclazuril, chlorohydroxy pyridine and decoquinate and is a multi-resistant strain.

The insect strain is 5×10 before application ⁴ The 14-day old chicks were vaccinated with sporulated oocysts and were rejuvenated, fresh faeces were collected on day 7 post infection, the faeces were diluted with 5 times of tap water, filtered 2 times, first with 80 mesh screen and second with 120 mesh screen. Standing at normal temperature for precipitating filtrate for 3 hr, discarding supernatant, placing in a culture dish containing 2.5% potassium dichromate solution, ventilating and culturing at 29 deg.C, and preserving at 4deg.C for 30 days.

1.3 Chinese medicinal composition and preparation of decoction

The traditional Chinese medicine composition A comprises 15g of rhizoma atractylodis, 10g of wrinkled gianthyssop herb, 10g of betel nut and 6g of liquorice, and has the effects of strengthening spleen, eliminating dampness, promoting qi and promoting diuresis; the traditional Chinese medicine composition B consists of 15g of mulberry leaf, 10g of amur corktree bark, 20g of milk root, 20g of red halloysite and 6g of liquorice, and has the effects of clearing heat and promoting diuresis, astringing and stopping bleeding and relieving diarrhea with astringents. The traditional Chinese medicine composition A+B consists of all the medicines in the prescription, and has the effects of clearing heat and drying dampness, cooling blood and stopping diarrhea, and promoting qi circulation and removing stagnation.

Respectively weighing 1000g of each of the Chinese medicinal compositions A, B, A +B which are grabbed in proportion, adding distilled water with the volume being 3 times of that of the Chinese medicinal compositions, soaking for 1.5h, boiling, decocting with slow fire for 2h, and filtering the liquid medicine by using a 80-mesh filter screen; adding distilled water with volume 3 times of that of the residues, decocting with slow fire for 2 hr, filtering with 80 mesh filter screen, mixing the decoctions, concentrating with slow fire to 1000ml, and filtering to obtain 1g/ml water decoction.

1.4 Experimental grouping

60yellow-featherbroilersof14daysofsimilarweightwereselectedandrandomlydividedintoaControlgroup(Control),aModelgroup(Model),atraditionalChinesemedicinecompositionAgroup(TCM-A),atraditionalChinesemedicinecompositionBgroup(TCM-B),andatraditionalChinesemedicinecompositionA+Bgroup(TCM-A+B),eachgroupbeing12. Test chick flocks were weighed 15 days old as initial body weight and sporulated oocysts were orally taken on the day 5X 10 ⁴ Feces 120-192 h post infection were collected for oocyst count, weighed at 23 days of age as terminal body weight and cecal recorded intestinal lesions scored. The administration modes are shown in Table 2; the administration dosage is 3g/kg body weight, and the administration modes are free drinking water.

Table 2 experimental animal grouping and treatment

1.5 evaluation of anticoccidial efficacy of Chinese medicinal composition

The relative weight gain rate, survival rate, lesion value and oocyst value were calculated according to the merck company method in the united states after infection of eimeria tenella for 192 hours; the anticoccidial effect of the drug was comprehensively evaluated by the number of fecal oocysts per gram, weight gain, lesion score and mortality of the chickens of the test group and the control group.

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

According to the ACI value of the anticoccidial index, when ACI is more than or equal to 180 and is high, ACI is more than or equal to 160 and less than or equal to 180 and is medium, ACI is more than or equal to 120 and less than or equal to 160 and is low, and ACI is less than 120 and is invalid.

2 experimental results

2.1 anticoccidial index for each experimental group

As shown in Table 3, the oocyst value and the cecal lesion value of the Model group are obviously increased compared with those of the Control group, and the survival rate and the relative weight gain rate are obviously reduced; TCM-A, TCM-B, TCM-A+B showed significantly higher anticoccidial index than Model group and significantly lower oocyst and lesion values than Model group; therelativeweightgainrateandanticoccidialindexoftheTCM-A+BgrouparehigherthanthoseoftheTCM-A,TCM-Bgroup,thedeathrate,theoocystvalueandthelesionvaluearelowerthanthoseoftheTCM-A,TCM-Bgroup,andthetraditionalChinesemedicinecompositionA+Bgrouphastheoptimalanticoccidialeffectandisusedforsubsequentexperiments.

Table 3 anticoccidial index for each test group

3 knots

The anti-coccidiosis index of the traditional Chinese medicine composition A+B is higher than that of the traditional Chinese medicine composition A and B, the traditional Chinese medicine composition A+B can improve the survival rate and the relative weight gain rate of the test chickens, reduce the clinical lesions of the test chickens, and the treatment effect is better than that of the traditional Chinese medicine composition A and B, and the traditional Chinese medicine composition A+B is adopted for subsequent experiments.

Example 2

1. Method of

1.1 Experimental animal

Male yellow feather broilers at 1 day old purchased from national institutes of agriculture poultry institute, production license: agricultural chicken threo K050901; use license: SYXK 2017-0044. The experimental animals are fed into an experimental animal house of a Yangzhou university veterinary medicine department; the raising environment is thoroughly cleaned, sprayed with ammonia water, fumigated and disinfected with formaldehyde and potassium permanganate, and heat treated with alcohol flame after ventilation. The cages, the trough and the water tank for raising adopt the same heat treatment method, so that the chicken raising environment is ensured to be free of coccidium, and the experimental environment is ensured to be free of other influencing factors. The chickens can provide enough water and basic feed in the feeding process, eat the chickens freely, and drink water by distilled water which is autoclaved at 121 ℃ for 20 min. Feed sampling film-making microscope observation, no coccidian oocyst. The light of 40W bulbs is uniformly adopted in the breeding process, and the breeding is carried out until 14 days old for experiment. The feces of the chicken flock was collected for 3 consecutive days for examination of coccidian-free oocysts. All operations in the experimental process accord with the ' guiding opinion about animals to be tested ' published in 2006 of the scientific and technical part of the people's republic of China, and animal welfare behaviors are not violated. All animal experiments were conducted under the direction of the university of Yangzhou animal protection and utilization committee. All experimental animals were ethically approved by the university of Yangzhou laboratory animal ethics committee (No. 202103-009). The feed formulation was formulated according to table 4 with reference to chicken nutrition standards.

Table 4 basic diet composition and nutrient level (air-dried basis)

1.2 Experimental insect strains

The experimental chicken Eimeria tenella strain is a Nantong separated insect strain NT6-7 which is separated and stored in a veterinary textroom in the clinic veterinary medicine of Yangzhou university. Through early experiments, the strain is completely resistant to multiple coccidiosis such as diclazuril, chlorodipyridyl, decoquinate and the like, and is a multi-resistant strain. The insect strain is 5×10 before application ⁴ The sporulated oocysts were inoculated into 14-day-old chicks for rejuvenation after infectionFresh feces were collected on day 7, diluted with 5 times the amount of tap water, filtered 2 times, first with 80 mesh screen and second with 120 mesh screen. Standing at normal temperature for precipitating filtrate for 3 hr, discarding supernatant, placing in a culture dish containing 2.5% potassium dichromate solution, ventilating and culturing at 29 deg.C, and preserving at 4deg.C for 30 days.

1.3 preparation of decoction of Chinese medicinal composition

Weighing 1000g of each Chinese medicinal composition, soaking in 3 times of distilled water for 1.5 hr, and soaking in root and fruit for 3 hr; decocting with slow fire for 2 hr after boiling, and filtering the medicinal liquid with 80 mesh filter sieve; adding distilled water with volume 3 times of that of the residues, decocting with slow fire for 2 hr, filtering with 80 mesh filter screen, mixing the decoctions, concentrating with slow fire to 1000ml, and filtering to obtain 1g/ml water decoction.

1.4 Experimental methods

48 experimental chickens of 10 days of age were divided 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 weight, and each group comprises 12 chickens. Experimental chick flocks were weighed as initial body weight 15 days old and sporulated oocysts were orally taken on the day 5X 10 ⁴ Feces 120-192 h post infection were collected for oocyst count, weighed at 23 days of age as terminal body weight and cecal recorded intestinal lesions scored. All medicines are administrated according to the requirements of Table 5, and the concentration of the traditional Chinese medicine composition is 6g/L and the medicine composition is administrated by free drinking water; the diclazuril concentration is 1mg/L for administration with free drinking water.

Table 5 experimental animal grouping and treatment

1.5 survival and weight gain conditions

The survival of the chickens in each experimental group was counted at 192h after infection. Survival (%) for each group=number of live chickens in the group/total number of experimental chickens in the group x 100%.

The weight gain index was calculated by weighing the chickens on an empty stomach at 15 days of the chicks and at the morning after the end of the experiment, taking the weight at the beginning of the experiment as the initial weight and the weight of the chickens at the end of the experiment (23 days of the age) as the final weight.

Relative weight gain (%) = (final weight of each chicken in the group-initial weight of the chicken)/initial weight of the chicken×100%

1.6 score and lesion value for cecum lesions of chickens

At 192h post-infection, each experimental group was sacrificed and cecum was collected and after appearance lesions were observed, the cecum was cut off to observe lesions in the intestinal tract. The severe side of the cecum was selected for lesion scoring and the scoring criteria are shown in table 6.

TABLE 6 cecum lesions score criteria

Note that: according to the severity of the cecum lesions after the chicken is dissected, when the cecum lesions on two sides are inconsistent, the severe side is taken as the reference.

1.7 evaluation of anticoccidial efficacy of Chinese medicinal composition

After infection with Eimeria tenella for 192 hours, each group of chickens was weighed on an empty stomach and weight gain was calculated. And recording death conditions of the chickens in the experimental process, and counting survival rate. Relative weight gain, survival, lesion values (lesion score x 10) and oocyst values were calculated as per Merck company method, usa, and anticoccidial index (Anticoccidial index, ACI) was calculated. The anticoccidial effect of the drug is comprehensively evaluated by the number of fecal oocysts per gram, the weight increment condition, the lesion score and the mortality of the experimental group and the control group of chickens.

1.8 histological examination of the cecum tissue of chickens

The chickens of each experimental group were sacrificed 192h after infection, ceca were collected and lesion scored. One side cecum was fixed in 4% neutral formaldehyde, 2.5% glutaraldehyde for preparing conventional HE-stained sections and scanning electron microscope samples, and the other side cecum was frozen in a-80 ℃ refrigerator.

1.9 chicken organ index determination

Each experimental group was infected with chicken for 192h, pre-slaughter liveness of each chicken was weighed, spleen and liver were rapidly separated after sacrifice, fat and connective tissue on the surface of organs were removed, and the like, and after the blood water was sucked by filter paper, the chicken was weighed, and organ indexes were calculated.

Organ index (%) = (organ weight/living body weight) ×100%

1.10 determination of inflammatory factor of cecum tissue of chickens

TNF-alpha, IL-1 beta, IL-10 content detection: according to the kit instructions. Washing the microplate for 3 times, centrifuging the tissue homogenate at 1000r/min for 10min, adding 100 μl of supernatant into the microplate, incubating at 37deg.C for 2h, and washing the plate for 3 times; adding 100 μl of biotinylated antibody working solution, incubating at 37deg.C for 1 hr, and washing the plate 3 times; adding 100 μl of streptavidin-HRP working solution, incubating at 37deg.C for 0.5h, and washing for 3 times; adding 100 μl of substrate solution and incubating at 37deg.C in dark for 20min; immediately after 50 μl of stop solution is added, the detection is carried out by an enzyme-labeled instrument, the detection is carried out at 450nm, and the correction wavelength is 630nm; and (5) carrying out corresponding calculation and statistics by using a standard curve.

1.11 measurement of the redox index of the cecum tissue of chickens

Cecum tissue homogenate preparation: taking 100mg of cecum tissue, rinsing with pre-cooled physiological saline, wiping the filter paper, weighing, and adding RIPA lysate to prepare 10% tissue homogenate.

And (3) detecting SOD activity: centrifuging 10% tissue homogenate at 3000r/min for 10min; adding 20 μl of supernatant into a microplate, adding the prepared corresponding reaction solution into the microplate according to the requirements of the kit, incubating at 37deg.C for 20min, zeroing at 450nm with distilled water, reading OD values of each tube by an ELISA reader, and performing corresponding calculation and statistics by using the recommended method of the specification.

GSH-Px Activity assay: centrifuging 10% tissue homogenate at 3000r/min for 10min; adding 100 μl of supernatant and prepared corresponding reaction solution into a 4ml centrifuge tube according to the requirements of the kit, standing at room temperature for 15min, zeroing at 412nm by distilled water, reading OD values of each tube by an enzyme-labeling instrument, and performing corresponding calculation and statistics by using a description recommendation method.

MDA content detection: adding 100 μl of 10% tissue homogenate and the prepared corresponding reaction solution into a 4ml centrifuge tube, vortex mixing, cooling with running water, centrifuging at 4000r/min for 10min, taking supernatant, zeroing at 532nm with distilled water, reading OD values of each tube by an enzyme-labeling instrument, and performing corresponding calculation and statistics by using a specification recommendation method.

1.12 data analysis

Statistical analysis was performed using IBM SPSS statistics17.0 software and experimental data was graphed using Graphpad prism 9.5.1. Data are presented as Mean ± standard error (Mean ± SEM) and comparison between groups uses one-way ANOVA. The standard of significant difference was P <0.05.

2 results

2.1A Chinese medicinal composition for improving survival rate and relative weight gain rate of Eimeria tenella infected chicken

As shown in fig. 1, the chickens in TCMF group and CON group did not die, and the survival rate of MOD group and DIC group was 75%; at 192h of infection, the weights of chickens in tcmf and DIC groups were significantly higher than those in MOD (P < 0.05).

2.2A Chinese medicinal composition for reducing the score of cecal lesions in Eimeria tenella infected chickens

As shown in fig. 2, the cecum of the MOD group chickens was significantly shortened compared to the CON group, and the cecum core appeared in the intestinal canal with scattered bleeding points on the surface. Compared with the MOD group, the lengths of cecum of the chickens in the TCMF group and the DIC group are obviously increased, no obvious bleeding point is found on the surface of the cecum, and no cecal core is found in the intestinal canal. The integral of cecal lesions of the chickens in the TCMF group and the DIC group is obviously lower than that in the MOD group (P is less than 0.05), and the integral of cecal lesions of the chickens in the TCMF group is obviously lower than that in the DIC group (P is less than 0.05).

2.3 anticoccidial index for each experimental group

As shown in Table 7, the ACI index of MOD group was 55.2, and the ACI indexes of TCMF group and DIC group were 164.8 and 122.6, respectively, wherein the TCMF group drug reached the moderate anticoccidial level, which was superior to DIC.

TABLE 7 anticoccidial index for each test group drug

2.4 the Chinese medicinal composition reduces the liver and spleen index of Eimeria tenella infected chickens

As shown in fig. 3, compared with the CON group, the liver index and spleen index of the MOD group chicken are significantly higher than those of the CON group (P < 0.05), and the liver index and spleen index of the TCMF group chicken and DIC group chicken are significantly lower than those of the MOD group (P < 0.05); wherein, spleen index of TCMF group chicken is significantly lower than MOD group (P < 0.05).

2.5 traditional Chinese medicine composition for weakening the tissue injury of the cecum of chickens infected with Eimeria tenella

2.5.1 results of HE staining of cecal tissue from each experimental group

As shown in fig. 4, the cecal villi of the MOD group chickens became shorter or broken, without normal glandular structure, compared to the CON group; there is massive inflammatory cell infiltration and erythrocyte exudation in the intestinal mucosa, and massive oocysts are seen. The intestinal villi and glandular structures of the TCMF group and DIC group chickens are relatively complete, and inflammatory cell infiltration and erythrocyte exudation phenomena in intestinal mucosa are obviously reduced; oocysts are not seen in the intestinal mucosa. The cecal structural integrity of TCMF group chickens was superior to DIC group.

2.5.2 results of the cecum tissue scanning electron microscope for each Experimental group of chickens

As shown in fig. 5, the mucosa surface of cecum tissue of CON group was smooth and flat, and the microvilli structure was not damaged. In the MOD group, a large amount of oocysts were attached to the intestinal mucosal surface, and the mucosal structure was severely damaged, compared with the CON group. Compared with the MOD group, the damage degree of intestinal mucosa structures of the TCMF group and the DIC group is obviously reduced; wherein, oocysts are not found on the intestinal mucosa surface of the TCMF group, and a small amount of coccidian oocysts are attached to the intestinal mucosa surface of the DIC group.

2.6 traditional Chinese medicine composition for weakening the inflammatory response of the cecum tissue of chickens infected with Eimeria tenella

As shown in FIG. 6, the IL-1 beta and TNF-alpha content in the cecum tissue of the MOD group was significantly increased (P < 0.05) and the IL-10 content was significantly decreased (P < 0.05) compared to the CON group. Compared with the MOD group, the contents of IL-1 beta and TNF-alpha in the cecum tissues of the chickens in the TCMF group and the DIC group are obviously reduced (P is less than 0.05), and the content of IL-10 is obviously higher than that in the MOD group (P is less than 0.05). IL-1 beta and TNF-alpha contents in the cecum tissues of the TCMF group chickens are significantly lower than those of the DIC group (P < 0.05), and IL-10 contents are significantly higher than those of the DIC group (P < 0.05).

2.7 traditional Chinese medicine composition for weakening the oxidative stress of the cecum tissue of chickens infected with Eimeria tenella

As shown in FIG. 7, compared with CON group, the activities of SOD and GSH-Px in the cecum tissue of MOD group chicken are extremely reduced (P < 0.05), and the MDA content is remarkably increased (P < 0.05); compared with the MOD group, the activities of SOD and GSH-Px in the cecum tissues of the chickens in the TCMF group and the DIC group are obviously increased (P is less than 0.05), and the MDA content is obviously reduced (P is less than 0.05). ISOD and GSH-Px activity in the cecum tissue of the TCMF group is obviously higher than that of the DIC group (P < 0.05), and MDA content is obviously lower than that of the DIC group (P < 0.05).

3 knots

The traditional Chinese medicine composition can obviously reduce the discharge amount of E.tenella infected chicken oocysts, inhibit the inflammation of the chicken cecum tissue caused by E.tenella infection, reflect oxidative stress, reduce the damage of the structure of the infected chicken cecum tissue, and improve the survival rate and the growth performance of the infected chicken. The traditional Chinese medicine composition reaches the level of the medium-effect anticoccidial drug.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.

Claims

1. The traditional Chinese medicine composition for treating chicken eimeria tenella is characterized by comprising the following components in parts by weight: 14-16 parts of mulberry leaf, 14-16 parts of rhizoma atractylodis, 8-12 parts of wrinkled gianthyssop, 18-22 parts of red halloysite, 8-12 parts of betel nut, 18-22 parts of milk root, 8-12 parts of amur corktree bark and 5-7 parts of liquorice.

2. The traditional Chinese medicine composition for treating chicken eimeria tenella according to claim 1, which is characterized by comprising the following components in parts by mass: 15 parts of mulberry leaf, 15 parts of rhizoma atractylodis, 10 parts of wrinkled gianthyssop herb, 20 parts of red halloysite, 10 parts of betel nut, 20 parts of milk root, 10 parts of amur corktree bark and 6 parts of liquorice.

3. The method for preparing the traditional Chinese medicine composition for treating chicken eimeria tenella according to claim 1 or 2, which comprises the following steps:

4. Use of the traditional Chinese medicine composition for treating chicken eimeria tenella according to claim 1 or 2 in the preparation of a medicament for improving survival rate and/or relative weight gain rate of chicken eimeria tenella infected chicken;

5. Use of the traditional Chinese medicine composition for treating chicken eimeria tenella according to claim 1 or 2 in preparing a medicine for reducing chicken eimeria tenella infection chicken cecum lesions.

6. Use of the traditional Chinese medicine composition for treating chicken eimeria tenella according to claim 1 or 2 in preparing a medicine for reducing the cecum tissue injury of chicken eimeria tenella infected chicken.

7. Use of the traditional Chinese medicine composition for treating chicken eimeria tenella according to claim 1 or 2 in preparing a medicine for reducing inflammation of cecum tissue of chicken infected with chicken eimeria tenella.

8. Use of the traditional Chinese medicine composition for treating chicken eimeria tenella according to claim 1 or 2 in preparing a medicine for reducing oxidation stress response of chicken cecum tissue infected by chicken eimeria tenella.

9. The use of the traditional Chinese medicine composition for treating chicken eimeria tenella according to claim 1 or 2 in preparing a medicine for improving anti-coccidium index of chicken eimeria tenella infected chicken;

10. The use of the traditional Chinese medicine composition for treating chicken eimeria tenella according to claim 1 or 2 for preparing a medicine for reducing organ indexes of chicken eimeria tenella infected chicken; the viscera include liver and spleen;

organ index (%) = (organ weight/living body weight) ×100%.