CN111760032A

CN111760032A - Composition for preventing and treating drug-resistant coccidiosis of chicken and preparation method and application thereof

Info

Publication number: CN111760032A
Application number: CN202010710169.1A
Authority: CN
Inventors: 廖海标; 李城丰
Original assignee: Guangzhou Baiyunshan Baoshen Animal Health Products Co ltd
Current assignee: Guangzhou Baiyunshan Baoshen Animal Health Products Co ltd
Priority date: 2020-07-22
Filing date: 2020-07-22
Publication date: 2020-10-13
Anticipated expiration: 2040-07-22
Also published as: CN111760032B

Abstract

The composition for preventing and treating the chicken drug-resistant coccidiosis comprises an isothiazolinone compound and plant tannin or disodium ethylene diamine tetraacetate, or comprises the isothiazolinone compound, the disodium ethylene diamine tetraacetate and the plant tannin, and provides the composition for preventing and treating the chicken drug-resistant coccidiosis and the preparation method thereof aiming at the problem that the chicken coccidiosis is easy to generate drug-resistant drug resistance. The composition for preventing and treating the drug-resistant coccidiosis of the chicken can be used for preventing and treating coccidiosis infection of the chicken, and can also be used for treating mixed infection of coccidia of livestock and poultry and virus, bacteria, mould and other protozoa diseases. The invention also relates to the application of the isothiazolinone compounds in the treatment of preventing and treating the drug-resistant coccidiosis of the chicken.

Description

Composition for preventing and treating drug-resistant coccidiosis of chicken and preparation method and application thereof

The technical field is as follows:

the invention relates to the technical field of chicken coccidiosis treatment, in particular to a composition for preventing and treating chicken drug-resistant coccidiosis and a preparation method and application thereof.

Background art:

coccidiosis in chickens is caused by one or more Eimeria unicellular coccidiosis parasitizing in mucosal epithelial cells of different parts of the intestinal tract of chickens, and the clinical manifestations of the coccidiosis are parasitic diseases characterized by diarrhea, bloody stool, growth retardation, reduced feed conversion rate, higher morbidity and mortality. The Eimeria necatrix and Eimeria tenella are most pathogenic. The diseases are common and easy to occur under the high-temperature and humid summer climate conditions, and the diseases are visible throughout the year by intensive culture. The coccidiosis mainly damages chicks within 3 months of age, the morbidity and mortality of the chicks within 10-60 days of age are high, the disease mortality can reach more than 30-80 percent at most, and the coccidiosis is distributed worldwide. Losses due to the disease are reported to be as high as billions of dollars each year worldwide. Because of the complexity and the particularity of the coccidian in the chicken, the immunity of the coccidian is more complex than that of other organisms, and the coccidian vaccine is limited due to poor stability, weak toxicity, strong reversion and the like in the chicken production, so that the prevention and the treatment of the chicken coccidiosis are mainly chemical drugs at present, but the generation of drug-resistant insect strains is inevitably caused due to the long-term use of the anti-coccidian drugs. To date, several classes of anticoccidial drugs have developed resistant strains of coccidial species and have shown some degree of cross-resistance to other drugs that are the same species as the drug or that have the same mechanism of action. According to statistics, the medicine cost for resisting coccidiosis of chicken in China is up to 6-8 billion yuan every year, which is undoubtedly a great loss in the chicken industry. Therefore, the development of a drug resistant to coccidiosis in chicken is urgently needed, and the drug is extremely necessary and significant for preventing and treating coccidiosis in chicken production.

The invention content is as follows:

the invention aims to overcome the defects, and provides a composition for preventing and treating the drug-resistant coccidiosis of chicken and a preparation method thereof aiming at the problem that the coccidiosis of chicken is easy to generate drug-resistant drug resistance, so that the composition has important practical significance for preventing and treating the coccidiosis in chicken production. The composition for preventing and treating the drug-resistant coccidiosis of the chicken can be used for preventing and treating coccidiosis of the chicken and can also be applied to the mixed infection treatment of coccidiosis of livestock and poultry and virus, bacteria, mould and other protozoa diseases.

In order to solve the technical problems, the invention is realized by the following technical scheme:

the composition for preventing and treating the drug-resistant coccidiosis of the chicken comprises 100-900 parts of isothiazolinone compounds and at least one of 100-900 parts of disodium ethylenediamine tetraacetic acid and 100-900 parts of plant tannin; the content of isothiazolinone compounds in the product is as follows: the content of the isothiazolinone is 10-90% of the isothiazolinone as an active ingredient.

The components can be prepared according to parts by weight, parts by mass or parts by volume.

The above formula can be prepared without adding or adding proper pharmaceutical adjuvants according to the process.

The main effective component content of the composition for preventing and treating the drug-resistant coccidiosis of the chicken is calculated by isothiazolinone. Wherein:

(1) isothiazolinone compounds are generic terms referring to a series of derivatives containing an isothiazolinone ring. Isothiazolinone belongs to heterocyclic organic compounds, and the basic structural formula of the isothiazolinone is as follows:

the isothiazolinone derivatives refer to a series of compounds formed by substituting other substances at the 2-position, the 4-position and the 5-position.

(2) The plant tannin (including plant tannin, plant tannin and plant tannin extract of tannin) is a polyphenol mixed tannin extracted from various tannin plant resources and Chinese medicinal materials (such as Galla chinensis, fructus Chebulae, mume fructus, etc.) by various technical methods.

(3) Disodium ethylenediaminetetraacetate (EDTA-2Na), also known as edetate disodium, has a molecular formula of C₁₀H₁₄N₂NaO₃·2H₂O, molecular weight 336.1. The product is white crystal granule or powder, and has no odor and taste. Is easy to be dissolved in water and is very insoluble in ethanol. The structural formula is as follows:

the composition for preventing and treating the drug-resistant coccidiosis of the chicken is further optimized as follows: the isothiazolinone compound is 2-methyl-4-isothiazolin-3-one (MIT), 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), Benzisothiazolinone (BIT), 4, 5-dichloro-2-methyl-4-isothiazolin-3-one (DCMIT), 2-methyl-4-isothiazolin-3-one (MIT), Octylisothiazolinone (OIT), 4, 5-dichloro-N-octyl-4-isothiazolin-3-one (DCOIT), butylbenzoisothiazolinone (BBIT), N-methyl-1, 2-benzisothiazolin-3-one (MBIT), N-N-octyl-1, at least one of 10 derivative compounds of 2-benzisothiazolin-3-One (OBIT).

The composition for preventing and treating the drug-resistant coccidiosis of the chicken is further optimized as follows: the dosage is as follows: the composition is calculated by the effective component isothiazolinone, and simultaneously considers the comprehensive effect of the pharmacological action of the composition, and the weight dosage is as follows through research: the usage amount of the composition for preventing and treating the drug-resistant coccidiosis of the chicken is 5mg optimally per kg of the weight of the chicken; adding water or mixing with appropriate amount of feed.

The composition for preventing and treating the drug-resistant coccidiosis of the chicken is further optimized as follows: the dosage form of the composition for preventing and treating the drug-resistant coccidiosis of the chicken comprises: solid powders and liquid formulations 2, where liquid formulations comprise solutions, mixtures, emulsifiers and suspensions. The content of isothiazolinone compounds in the product is as follows: the content of the isothiazolinone is 10-90% of the isothiazolinone as an active ingredient.

The invention also relates to a preparation method of the composition or the optimized composition for preventing and treating the drug-resistant coccidiosis of the chicken, which comprises the following steps:

preparing materials according to a formula, adding no or proper auxiliary materials, stirring and mixing uniformly, filling and packaging, and labeling to obtain solid powder; or

Preparing materials according to a formula, adding no or proper auxiliary materials, homogenizing under certain conditions, stirring and mixing uniformly, and finally filling, bottling, sealing and labeling to obtain a liquid preparation;

the proper auxiliary materials are proper necessary auxiliary materials selected according to the production requirements of product preparations, and comprise one or more of a carrier, an adsorbent, a diluent, a solvent, a dispersant, a wetting agent, an emulsifier, a cosolvent, an antifreezing agent and a surfactant.

The invention also relates to the application of the composition for preventing and treating the drug-resistant coccidiosis of the chicken in the mixed infection treatment of coccidia of livestock and poultry and virus, bacteria, mould and other protozoa diseases.

The invention also relates to application of the isothiazolinone compound in treatment of preventing and treating the drug-resistant coccidiosis of the chicken, which is prepared by adding proper pharmaceutical excipients into the isothiazolinone compound.

Coccidiosis is a unicellular organism with complex structure and development process. According to the study, each chicken Eimeria tenella infectious oocyst contains four sporangia, and each sporangia contains two sporozoites. Each sporozoite generally has a structure including a nucleus, a nucleolus, a mitochondrion, an endoplasmic reticulum, a ribosome, a Golgi apparatus, a starch granule, a liposome, a protein granule, a vacuole and the like, and a special structure including a top recombiner (polar ring, rod-shaped body, micro-wire, micropore, microtubule, cone-like body and the like), a refractile body, a surface membrane and the like, wherein the surface membrane of the sporozoite (including merozoite) consists of three layers (an inner membrane is compounded into two layers and an outer membrane is compounded into one layer) of unit membrane and is a continuous structure wrapping the whole polypide. Therefore, the coccidian epiphysis plays an important role in keeping the body stable and participating in the interaction of the host, so that sporozoites can resist the digestion of intestinal pancreatin, and schizonts can resist hyaluronidase. According to the research, the whole growth and development process of the chicken coccidian is basically as follows: after the infectious oocysts enter the digestive tract of the chicken, under the action of various factors, sporozoites firstly de-capsulate and then enter intestinal mucosal epithelial cells of a host, the sporozoites develop into trophozoites, schizonts are generated through schizogenesis, the schizonts develop into merozoites, and the merozoites are released after maturation. When the schizont develops and multiplies to some extent over several generations, a large number of merozoites are released. According to the research, 1 sporulated oocyst of the chicken coccidia can develop into 250 ten thousand merozoites. At the moment, the merozoites can destroy epithelial cells of intestinal mucosa of chickens when released, and a series of pathological changes of mucosal necrosis and enteritis such as rupture and bleeding of intestinal mucosa blood vessels, mucosa necrosis, diarrhea, feed feces and the like are caused. After a certain number of generations of asexual reproduction, the merozoites can not develop into schizonts any more, but into large and small gametocytes. And finally, fusing the large gamete and the small gamete to form a zygote, wherein the worm body starts to form an oocyst wall, and the oocyst is discharged out of the body along with excrement after being formed to form infectious oocysts again.

Because of the structural characteristics of the coccidian of the chicken, the resistance of the oocysts is very strong, the oocysts can survive for 4-9 months in soil and 15-18 months in tree shade, and are not easy to be killed by a common disinfectant, so that the oocysts are greatly favorable for forming infectious sporulation structures in a warm and humid environment, and the chicken can be repeatedly infected with coccidiosis.

According to the research of the inventor, the isothiazolinone compound is thought to kill unicellular coccidium by the following mechanism: the isothiazolinone derivative penetrates through the coccidian sporozoite surface membrane, and the active part on the heterocyclic ring reacts with the coccidian intracellular protein to break the bond containing sulfhydryl in the molecule, such as the reaction of the isothiazolinone compound and Glutathione (GSH), so that the protein amino acid chain is broken, thereby destroying the coccidian cells;

on the other hand, isothiazolinone compounds can also bind with DNA to block the replication of nucleic acid, thereby inhibiting the multiplication of coccidian cells. The isothiazolinone compounds have strong penetrating power with the largest anti-coccidian effect, can inhibit nucleic acid replication and can destroy the protein structure of the isothiazolinone compounds. Therefore, coccidia is not easy to generate the problem of drug resistance. According to the data, the isothiazolinone compound has good environment-friendly factors, can be rapidly and naturally degraded into low-toxicity or non-toxicity in the environment, and cannot pollute the environmentAnd (5) dyeing the environment. Toxicity: the rat acute oral semi-Lethal Dose (LD) varies from species to species₅₀)110-2000 mg/kg. Acute transdermal semi-Lethal Dose (LD) for rabbits₅₀)600-2000 mg/kg. The stock solution has strong irritation and sensitization to skin and mucous membrane. Aquatic organisms: semi-Lethal Concentration (LC) of rainbow trout₅₀)0.19mg/L (96 h). half-Lethal Concentration (LC) of blue gill fish₅₀)10mg/L (96 h). The isothiazolinone compounds have the main advantages of broad spectrum, high efficiency, low toxicity, easy degradation and the like.

According to the research of the inventor, because the disodium ethylene diamine tetraacetate is a compound with strong polarity, a molecule and divalent and trivalent metal ions form a chelate in a ratio of 1:1, the disodium ethylene diamine tetraacetate is very easy to combine with metal ions such as magnesium, manganese, calcium, potassium and the like of a coccidian cell membrane in a solution to form a stable chelate, so that the permeability of the coccidian cell membrane is changed, the concentration of an anticoccidial drug entering a cell is improved, the synergistic enhancement effect of the anticoccidial drug is exerted, and particularly, the effect on the anticoccidial coccidian strain is more obvious.

Plant tannin refers to tannin substances (also called tannin, tannic acid) contained in plant body. Is a polyphenol compound which is widely present in the root, stem, leaf, fruit, bark and other parts of plants and has a relative molecular mass of 500-3000 u. Tannins in plants tend to be complex mixtures of various polyphenol derivatives. The natural tannin is generally colored amorphous solid, bitter and astringent, and is soluble in water and organic solution such as ethanol, acetone, ethyl acetate, etc.; the tannin can react with protein and enzyme to affect the conformation of the protein and the enzyme, thereby inhibiting the activity of the enzyme; tannin can also complex with ions such as copper, zinc, iron, etc., so that the activity of the metalloenzyme containing the ions is inhibited, and the bioactivity of oxidation resistance, ultraviolet absorption, etc. is achieved.

Tannins are classified into two categories according to their chemical composition and the characteristics of their chemical bonds: hydrolyzed tannins and condensed tannins.

(1) Hydrolyzing the tannin. Also known as hydrolysable tannins, have an ester bond within the molecule, usually centered on a carbohydrate (or polyol related substance), which is linked to a polyphenolic carboxylic acid via an ester bond, and are thus hydrolysed by the action of an acid, a base or an enzyme to produce a sugar or a polyol and a polyphenolic carboxylic acid. Depending on the polyphenol carboxylic acid obtained, the hydrolyzed tannins can be further subdivided into gallotannins (hydrolysis to produce gallic acid) and ellagitannins (hydrolysis to produce ellagic acid or derivatives thereof). Examples of the hydrolysable tannin include Galla chinensis tannin, Erighur tannin, and HUAXIANGGUO tannin.

(2) Condensed tannins. Also known as condensed tannins or non-hydrolysable tannins. Aromatic rings in such tannin molecules are all connected by carbon and carbon, and sugar does not form the whole structure of the molecules, so that hydrolysis is difficult. Most condensed tannins are condensates of flavanols. Mixed tannins have both the characteristics of hydrolyzed tannins and condensed tannins.

The plant tannin (including plant tannin, plant tannin and plant tannin extract) refers to a polyphenol mixed tannin substance extracted and purified from various tannin plant resources and Chinese medicinal materials (such as gallnut, myrobalan, dark plum and the like) by adopting various technical methods. The current methods for extracting and purifying the plant tannin comprise the following steps: soaking and extracting with solvent; ② ultrasonic auxiliary extraction method; thirdly, microwave-assisted extraction; enzyme conversion and extraction; a supercritical fluid CO2 extraction technology; extracting by membrane technique; seventhly, ultrahigh pressure extraction; eighthly, semi-bionic extraction; ninthly, fermentation and extraction; the crude extract of koc tannin contains a large amount of impurities such as sugar, protein, lipid, etc., and needs to be further separated and purified. The preliminary separation may be carried out by a hide powder method, a lead acetate precipitation method, a sodium chloride salting-out method, a dialysis method, an ultrafiltration method, a crystallization method, or the like.

According to the research of the inventor, the plant tannin has the following pharmacological and toxicological effects: the convergence action. The tannin can be used for treating gastrointestinal hemorrhage by oral administration, gastrointestinal hemorrhage by external application, and wound surface of wound and burn by external application, and can solidify protein in surface exudate to form crust membrane, protect wound surface, and prevent infection; ② antibacterial and antiviral effects. Tannin can solidify protoplasm in a microorganism body, so that the tannin has a certain bacteriostatic action; ③ the function of expelling and killing protozoa. According to the research, the tannin can penetrate through cell membranes of protozoa and solidify protoplasm in the protozoa, so that the tannin has a certain effect of inhibiting and killing the protozoa; in addition, the plant tannin also has the function of inhibiting the formation of a biofilm, and is beneficial to the reduction of drug resistance of the antiprotozoal drug which directly acts on protozoa; fourthly, the detoxification function. Because tannin can generate insoluble precipitate with heavy metal salt and alkaloid, some toxic heavy metals or alkaloids can be used as antidote by tannin after being absorbed by animal body, thereby reducing the absorption of toxic substances by the body; fifthly, other functions. Pharmacological test research shows that some tannins also have the effects of clearing free radicals in vivo, inhibiting nervous system, reducing the content of urea hydrogen in serum, resisting allergic reaction, resisting inflammation and the like. Sixthly, the action of toxicity. The white rat is not abnormal when the decoction is orally taken at 25g/kg of body weight.

In conclusion, the composition for preventing and treating the drug-resistant coccidiosis of the chicken is prepared by combining the isothiazolinone compound and the plant tannin or combining the isothiazolinone compound, the disodium ethylenediamine tetraacetic acid and the plant tannin. Wherein isothiazolinone is the main component effective component of the anticoccidial drug of the invention; disodium edetate is a secondary active ingredient of the anticoccidial drug of the invention; the plant tannin has the functions of astringing, protecting mucous membrane, resisting allergic reaction, stopping bleeding, diminishing inflammation and the like, and is an auxiliary component of the anticoccidial drug. Pharmacological action analysis shows that on one hand, isothiazolinone compounds can enter coccidian cells to inhibit nucleic acid replication and can damage protein structures of the coccidian cells; and the other side of the membrane can be combined with metal ions such as magnesium, manganese, calcium, potassium and the like of the coccidian cell membrane to form a stable chelate so as to change the permeability of the coccidian cell membrane. Therefore, the coccidia is not easy to generate the problem of drug resistance. Therefore, the isothiazolinone compound and the plant tannin are mutually compatible for preventing and treating the chicken coccidiosis, and particularly, the isothiazolinone compound, the disodium ethylenediamine tetraacetic acid and the plant tannin are mutually compatible for preventing and treating the chicken coccidiosis, so that the isothiazolinone compound and the plant tannin have obvious superposition and synergistic enhancement effects.

The composition for preventing and treating the drug-resistant coccidiosis of the chicken has the following characteristics:

1. the composition for preventing and treating the drug-resistant coccidiosis of the chicken is prepared by adding an isothiazolinone compound, disodium ethylene diamine tetraacetate and a proper pharmaceutical adjuvant; or is prepared from isothiazolinone compounds, vegetable tannin and appropriate pharmaceutical adjuvants; or isothiazolinone compounds, disodium edetate and vegetable tannin, and no adjuvants. It has strong penetrating power and unique anticoccidial action mechanism.

2. The composition for preventing and treating the drug-resistant coccidiosis of the chicken is used for preventing and treating the coccidiosis of the chicken, does not generate drug resistance, and is an important breakthrough in the production technology of the anti-coccidiosis drug.

3. The composition for preventing and treating the drug-resistant coccidiosis of the chicken has simple production process and easy operation. But the product has wide range of inhibiting and killing effects on pathogens (including viruses, bacteria, moulds and protozoa), and has certain curative effect on the infection of the viruses, the bacteria, the moulds and the protozoa except for preventing and treating coccidian infection.

4. The product has stable quality and small toxic and side effects.

5. The plant tannin is a plant extract and is very environment-friendly; the isothiazolinone compounds are easy to degrade in the environment, and cannot cause ecological environment pollution.

The specific implementation mode is as follows:

the present invention will be described in further detail with reference to examples.

Test one: preparation method and application of composition for preventing and treating drug-resistant coccidiosis of chicken

Examples 1 to 10: taking the production and preparation of solid powder containing 10% isothiazolinone compounds as an example, the preparation method of the composition for preventing and treating the drug-resistant coccidiosis of the chicken is concretely illustrated as follows:

firstly, determining the product formula.

Remarking: if the above is liquid raw material, proper adsorbent auxiliary material is added.

Secondly, preparing materials. Accurately weighing 100 parts of isothiazolinone compounds and 900 parts of plant tannin respectively.

And thirdly, mixing, stirring, filling and packaging.

Pouring the above two raw materials into a stirrer, stirring, packaging, sealing, and labeling.

The using method comprises the following steps: can be used for preventing and treating chicken coccidiosis. The dosage is as follows: 5mg per kg body weight of chicken calculated by main effective component isothiazolinone. The use method comprises the following steps: mixed drinking or mixed feeding: adding water or mixing with appropriate amount of materials, stirring, and feeding for 3-5 days.

Examples 11 to 20: taking the production and preparation of solid powder containing 20% of isothiazolinone compounds as an example, the preparation method of the composition for preventing and treating the drug-resistant coccidiosis of the chicken is concretely illustrated as follows:

firstly, determining the product formula.

Secondly, preparing materials. 200 parts of isothiazolinone compounds and 200 parts of ethylene diamine tetraacetic acid disodium are accurately weighed respectively, and 600 parts of anhydrous glucose added with proper auxiliary materials is also accurately weighed.

And thirdly, mixing, stirring, filling and packaging.

Examples 21 to 30: taking the production and preparation of solid powder containing 30% isothiazolinone compounds as an example, the preparation method of the composition for preventing and treating the drug-resistant coccidiosis of the chicken is concretely illustrated as follows:

firstly, determining the product formula.

And thirdly, mixing, stirring, filling and packaging.

Examples 31 to 37: taking the production and preparation of solid powder containing 40-90% of isothiazolinone compounds as an example, the preparation method of the composition for preventing and treating the drug-resistant coccidiosis of the chicken is concretely illustrated as follows:

firstly, determining the product formula.

Secondly, preparing materials. And accurately weighing the related components according to the components.

And thirdly, mixing, stirring, filling and packaging.

Example 38: taking the production of a liquid preparation containing 30 percent of isothiazolinone compounds as an example, the preparation method of the composition for preventing and treating the drug-resistant coccidiosis of the chicken is concretely illustrated as follows:

firstly, determining the product formula. Namely 300 parts of isothiazolinone compounds, 300 parts of ethylene diamine tetraacetic acid disodium and 400 parts of vegetable tannin.

Secondly, preparing materials. Respectively and accurately weighing 300kg of isothiazolinone compounds, 300kg of disodium ethylene diamine tetraacetate and 400kg of plant tannin.

And thirdly, processing and preparing the product. During the processing, suitable auxiliary materials such as a diluent, a solvent, an emulsifier, a cosolvent, an antifreezing agent, a surfactant, pharmaceutical water and the like are added until the total volume is 1000 liters. Then processing, homogenizing, emulsifying under certain temperature and pressure, stirring, mixing, filling, bottling, sealing, and labeling.

The using method comprises the following steps: can be used for preventing and treating chicken coccidiosis. The dosage is as follows: 5mg per kg body weight of chicken calculated by main effective component isothiazolinone. The use method comprises the following steps: the mixed drink is added with a proper amount of water, stirred uniformly and fed for 3-5 days continuously.

Example 39: taking the production and preparation of solid powder containing 50% of isothiazolinone compounds as an example, the preparation method of the composition for preventing and treating the drug-resistant coccidiosis of the chicken is concretely illustrated as follows:

firstly, determining the product formula. 500 parts of Benzisothiazolinone (BIT) and 500 parts of auxiliary materials.

Secondly, preparing materials. 500 parts of Benzisothiazolinone (BIT) and 500 parts of auxiliary materials are accurately weighed respectively.

And thirdly, mixing, stirring, filling and packaging.

The pharmacological and toxicological effects of the anti-drug resistant coccidian composition prepared according to the above examples are studied as follows:

(1) acute toxicological test. The toxicity of isothiazolinone compounds varies with species, and the rat acute oral LD50110-2000 mg/kg. Rabbit acute percutaneous LD50600-2000 mg/kg. The stock solution has strong irritation and sensitization to skin and mucous membrane.

(2) Other toxicological data.

Skin irritation: it is corrosive to rabbit.

Eye stimulation: it is corrosive to rabbit.

Sensitization: guinea pigs cause allergies.

Carcinogenicity: both percutaneous in mice and oral in rats, were found to be non-carcinogenic.

Carcinogenicity: the product is not a harmful product for reproduction. No distortion effect was observed in animal experiments.

Mutagenic property: is a substance which does not induce organism mutation.

(3) Acute toxicity test of poultry. Through the research of the inventor: such as Benzisothiazolinone (BIT), chicken semilethal dose (LD50), chicken 500mg/kg body weight.

And (2) test II: research on action efficacy of the drug of the invention on Eimeria tenella in chick embryos

The purpose and content research and the determination of the action effect of the isothiazolinone compound, the disodium ethylene diamine tetraacetate and the vegetable tannin on the Eimeria tenella in chicken embryos independently or in a compatible combination with each other. So as to provide basis for product combination design and optimization.

Materials and methods

1. An oocyst. The purified Eimeria tenella strain was isolated from the sick chicken in the chicken farm, and the test strain was propagated by the chick embryo in advance, and then stored in a refrigerator at 4 ℃.

2. Preparation of a sporozoite suspension. Taking the Eimeria tenella oocysts, preparing sporozoite suspension according to the technical requirements, counting sporozoite number by using a blood counting plate, and diluting by using ringer's solution according to the required inoculation dose when in use.

3. Eggs for receiving seminal eggs are provided by breeding hens, groups are arranged according to different concentrations and combinations of test drugs, each group comprises 20 eggs, and a control group 2 which is not infected, not administered and not administered is arranged additionally, the eggs are injected with the drugs when the eggs are incubated to 10 days old, and after the drugs are inoculated for 24 hours, 0.1ml of the test group containing 1 × 10 per embryo allantois is inoculated into each embryo allantois cavity⁴And (3) continuously placing the sporozoite suspension in an incubator for incubation after the pinhole is wax-sealed, checking the chick embryos once a day after inoculation, and observing and recording the death condition of the chick embryos. The chick embryos which die within 4 days are considered to be caused by operation pollution or drug poisoning; coccidia were considered to be lethal in 5-6 days with death accompanied by bleeding symptoms.

4. Protocol for the test. In the test, 3 groups are respectively set according to a single substance, namely isothiazolinone compounds, EDTA-2Na and plant tannin; and two substance combinations, namely isothiazolinone compounds + plant tannin, EDTA-2Na + isothiazolinone compounds set 2 groups; the three substance compositions, namely isothiazolinone compounds + EDTA-2Na + plant tannin, are arranged in 1 group, and each group is provided with 3 administration dose groups. In addition, a healthy negative control group without infection and administration and a positive control group without infection and administration are also provided. The administration mode is that the dosage of each chick embryo is diluted by sterilized normal saline for injection, and 0.2ml of each chick embryo is injected. The specific design is shown in table 1.

Table 1: the invention relates to a design scheme for determination and optimization test of efficacy of anticoccidial drugs

5. And (5) judging the anti-coccidian efficacy. Oocysts were collected from the chick embryos and counted on day 7 after infection, and the oocyst reduction rate was used as a criterion for the efficacy against coccidia.

And thirdly, testing results. As shown in table 2 below.

Table 2: effect of the different combinations of the invention on Eimeria tenella in chick embryos

From the in vitro test results of table 2, the preliminary analysis shows that:

(1) three substances of isothiazolinone compounds, disodium ethylene diamine tetraacetate and vegetable tannin are used for inhibiting and killing coccidia in chick embryos respectively, and the average reduction rate of oocysts is 86.7%, 34.47% and 5.72% respectively. The three compounds have the best effect as isothiazolinone compounds, and the next is disodium ethylene diamine tetraacetate. The vegetable tannin has the worst coccidian inhibition effect, but the traditional Chinese medicine physical effects of protecting intestinal mucosa, stopping bleeding and eliminating isothiazolinone compounds easily cause mucosa allergic reaction, and is one of essential components for resisting the drug-resistant coccidia.

(2) Isothiazolone and EDTA were combined with isothiazolone and vegetable tannin to kill coccidia in chick embryos with average oocyst reduction of 94.74% and 83.47%, respectively. The former combination was 11.27% higher than the latter combination.

(3) The isothiazolinone compound, the disodium ethylene diamine tetraacetate and the vegetable tannin are combined to inhibit and kill coccidian oocysts in the chick embryos, the average reduction rate is 99.67%, and the effect is optimal.

And (3) test III: study on influence of the drug on development of Eimeria tenella oocysts in chick embryos

First, purpose and content to study the effect of different dosages of the drug of the invention on the development of Eimeria tenella oocysts in chicken embryos, so as to provide a basis for determining the dosage of the drug of the invention.

Materials and methods

1. Test drugs the composition for preventing and treating drug-resistant coccidiosis in chickens of the present invention was prepared according to the preparation method of example 3.

2. The test materials and the equipment are selected from 80 hatching eggs with the weight of 50g, and are purchased from a certain chicken farm in Heizhou, Guangdong. The Eimeria tenella oocysts are automatically collected, purified and stored at 4 ℃ for later use. 1 small incubator.

3. Test methods the test was divided into 8 groups of 10 chick embryos each. The 6 groups inoculated with oocysts were injected with 0.1mg, 0.2mg, 0.4mg, 0.6mg, 0.8mg, 1mg of the drug (based on the active ingredient isothiazolinone compound, diluted to 0.2ml with physiological saline) respectively; the other two groups: group 1 is a control group of a coccidian-inoculated oocyst group, and group 1 is a control group of a drug only injection. The method comprises the following steps: selecting normally hatched chick embryos, inoculating 1 ten thousand coccidian oocysts in allantoic cavities of the chick embryos at the age of 10 days, and respectively injecting 0.2ml of the medicine with different doses into 6 groups of chick embryos on the day 2 after inoculation. Subsequently, chick embryo death was observed daily and oocyst examination was performed by taking sediment from allantoic fluid on day 8 after inoculation.

And thirdly, testing results. As shown in table 3 below.

Table 3: the influence of the drug of the invention on the development of Eimeria tenella oocysts in chicken embryos

The test result shows that:

(1) in the drug control chick embryos, only 10 chick embryos injected with the drug develop normally until the chick shells, which indicates that no toxic reaction is seen on the chick embryos.

(2) Control chick embryos inoculated with oocysts alone, on day 8 post inoculation, showed a large number of oocysts in allantoic fluid, indicating that Eimeria tenella regenerated oocysts in chick embryos to complete their life history through the asexual and sexual stages.

(3) The results of both the inoculation of oocysts and the injection of each dose of the drug group showed that no oocysts were detected from the 0.1mg to 1mg group, indicating that the drug had a killing effect on oocysts, with the best of the 0.4mg and 0.6mg groups, and also on gametophytes. When the allantoic fluid is detected with iodine, it is assumed that the surface membrane of coccidia schizont, merozoite, etc. is destroyed and the amylopectin granules are released to react with iodine.

Therefore, the anti-coccidium action mechanism of the product is supposed to be that the physiological functions of the substance metabolism, the energy metabolism and the like of the insect body are inhibited by destroying the structural integrity of the insect body, so that the coccidium is killed or the growth and the reproduction of the coccidium are inhibited.

And (4) testing: research on treatment test effect of the medicine and different dosages of the medicine for resisting coccidiosis

First, the purpose and content are to study the effect of the anti-coccidiosis treatment test of the invention in various combinations and different dosages in practical clinical application.

Materials and methods

1. Inoculating insect strains: the Eimeria tenella strain is derived from a chicken farm isolate from Guangdong.

2. Experimental animals: newly hatched broiler chicks are raised in a strictly disinfected chicken house and are grouped and caged into 11 groups, each group contains 10 chicks, feed and drinking water are strictly disinfected, and experimental medicines are mixed into the drinking water and fed to the chicks.

3. Experimental drugs and administration modes: the various compositions (prepared according to the embodiment) for preventing and treating the drug-resistant coccidiosis of the chicken and the three drugs of the qiuxiang, diclazuril and sulfachloropyrazine sodium are mixed into drinking water for feeding.

4. The virus inoculation method comprises the following steps: 5 million E.tenella sporulated oocysts were administered orally to each chicken. The administration method is adopted according to the dosage per kg body weight, namely, the medicine of the invention is mixed into the drinking water according to 6 dosages of 10mg, 8mg, 6mg, 4mg, 2mg and 1mg per kg body weight. The reference medicament is administered according to the recommended dosage of the instruction, namely 1m/L drinking water of diclazuril, 2.5g/L drinking water of qiuxin and lg/L drinking water of sulfachloropyrazine sodium. Diclazuril was administered 2 days before infection and the remaining drugs were administered the third day after infection.

5. Grouping tests: the medicine is provided with three combined treatments, namely an isothiazolinone compound and plant tannin, an isothiazolinone compound and disodium ethylene diamine tetraacetate and plant tannin, wherein the dosage of each treatment is 1 group of 10mg/kgW, 8mg/kgW, 6mg/kgW, 4mg/kgW, 2mg/kgW and 1mg/kgW according to 10 times of the dosage of chicken embryos; comparing 1 group of anticoccidial drugs including carbendazim, diclazuril and sulfaclozine sodium with 1 group of drugs which are not administered for infection and are not administered for infection.

6. The evaluation standard of the drug resistance is as follows: (1) the anticoccidial index ACI is more than or equal to 180, and the medicine is judged to be sensitive (effective medicine); (2) ACI is 160 to 179, and is judged as a partial drug resistance (partial effective drug); (3) ACI less than 160, it is considered to be drug-resistant (ineffective drug).

7. Detection indexes are as follows:

(1) ACI ═ relative rate of weight gain + survival) - (lesion score + oocyst score)

Wherein, the relative weight gain ratio (%) is the average weight gain of the infection administration medicine group/the average weight gain of the non-infection administration medicine group multiplied by 100;

(2) the survival rate (%) (number of live chickens in group/total number of chickens in group) is multiplied by 100;

(3) lesion score-mean lesion score × 10.

Table 4: evaluation criteria for lesion scores

(4) Oocyst ratio and oocyst value: after the chickens are killed, the cecum is homogenized, the oocyst fluid is sucked, the number of oocysts is counted by using a blood cell counting plate, and the ratio of the oocysts is calculated.

Oocyst ratio (%). healthy or experimental oocyst count/number of oocysts infected without administration of drug group ×%

Table 5: corresponding value table of oocyst ratio and oocyst value

And thirdly, testing results. See tables 6 and 7.

Table 6: ACI results of the inventive drugs

Remarking: INC is a positive control group and UNC is a negative control group.

② the combination I is isothiazolinone compounds and plant tannin. The combination II is isothiazolinone compounds and disodium ethylene diamine tetraacetate. The composition III is isothiazolinone compounds, disodium ethylene diamine tetraacetate and plant tannin.

Table 7: comparison of ACI values for the inventive drug and the control drug

Remarking: an ACI value greater than 180 indicates that the drug is effective; the ACI value is between 160 and 180, indicating that the drug moiety is effective; an ACI value of less than 160 indicates that the drug is not effective.

As can be seen from tables 6 and 7, the three compositions of combination I, combination II and combination III all have significant effects within the dosage range of 4-8mg/kgW, and the anticoccidial ACI values are not significantly different from the three anticoccidial drugs of sulfachloropyrazine sodium, diclazuril and carbendazim.

And (5) testing: experimental study on drug-resistant strain for preventing and treating chicken coccidiosis

The purpose and content are used for preventing and treating coccidian resistant strains, and the actual effect of the drug for preventing and treating chicken coccidian resistant strains is verified.

Materials and methods

1. And (4) experimental animals. Feeding 1 day old chicks in an coccidiosis-free environment to 12 days old, grouping, replacing padding every day in the feeding process, keeping the environment clean, and enabling experimental animals to freely eat drinking water.

2. Test insect strains. A field insect strain is separated from a certain chicken farm in Guangdong province, is identified as an Eimeria tenella strain, namely a Guangdong strain, and is stored in a laboratory of the company.

3. And (4) testing the medicine. The medicament of the invention is prepared by the method of example 6; other anticoccidial drugs, maduramicin ammonium, dinitramine, diclazuril, sulfachloropyrazine, and clopyralid are all qualified products produced by the company according to the standard of veterinary drug code.

4. And (5) detecting the drug resistance of the coccidia.

(1) Grouping and administration of the test. The average body mass of 12-day-old chicks is divided into 7 groups of 14 chicks, and the groups are maduramicin ammonium, dinitramine, diclazuril, sulfachloropyrazine, clopyralid, positive (INC) and negative (UNC) control groups respectively. Except for the control group, each administration group was given with the dressing 24h before infection. Each infected group was orally infected with sporulated oocysts 5 ten thousand per feather at 14 days old, except for the negative control group. After infection, the chickens were observed every day for mental status and ingestion status, weighed 8 days after infection and killed, and cecum was taken for observation and lesion scoring and oocyst count of cecum contents were performed. Cross contamination is avoided in the test process, and the drug resistance detection of 3 coccidia strains is respectively completed.

(2) Drug resistance determination

Anticoccidial index (ACI): calculated according to the following method:

ACI ═ (relative rate of weight gain + survival) - (lesion value + oocyst value). Wherein, the anti-coccidium efficacy standard is as follows: ACI >180, high efficiency; 160-180, medium effect; 120-; <120, invalid.

Percent gain ═ final average weight-initial average weight)/initial average weight × 100;

relative percent weight gain is the weight gain of the test group/the weight gain of the negative control group multiplied by 100;

the survival rate is the number of the live chickens in each test group/the number of the chickens in each test group is multiplied by 100;

mean lesion score for each group x 10. See table 8.

Table 8: evaluation criteria for lesion scores

Oocyst value: scaled according to the corresponding data listed in table 9 below.

Table 9: eimeria tenella oocyst value correspondence table

5. The drug of the invention is used for testing the curative effect of drug-resistant insect plants.

The test is divided into groups and administered, wherein 12-day-old chicks are divided into 4 groups according to the average body mass, each group comprises 18 chicks, and 1 group of the medicament is administered as an infection mixed material, 2 groups of the medicament are administered as infection drinking water, 3 groups of the medicament are administered as infection-free medicaments, and a negative control group is additionally arranged. Each administration group is administrated 24h before infection by mixing with feed or drinking water, and 14 days old orally infect sporulated oocysts 5 ten thousand per feather in each region.

6. And (6) analyzing the data. The test data is expressed by x-shy S, the test results are analyzed for the writability by adopting mathematical statistics software, and the differences among groups are compared.

Third, test results

1. Drug resistance of Eimeria tenella strains

Table 10: drug resistance detection result of Eimeria tenella strain

Remarking: INC is a positive control group and UNC is a negative control group.

As shown in Table 10, the ACI values of the administered groups were all below 160, and the drug resistance was positive. Wherein the ACI value of the dinitolmide group is 131, the dinitolmintic is low-efficiency anticoccidial drug, and the ACI values of the other groups are all lower than 120, and the dinitolmintic is completely resistant to drugs.

2. Determination result of ACI

Table 11: the anticoccidial index (ACI) of the medicine of the invention

Note: group 1 is administered with the infection dressing; group 2 was water-administered for infection: the group 3 is free food and water intake for infection-free drug-free group and negative control group.

As can be seen from table 11, ACI values of 197 and 182, which are greater than 180, in the compositions of the present invention for controlling drug-resistant coccidiosis in chicken of test 1 and 2 show that the composition prepared by the method of example 5 shows high efficacy against 5 coccidian-resistant coccidia strains. Therefore, the medicine has definite curative effect and obvious effect when being used for preventing and treating the drug-resistant coccidiosis of the chicken.

In conclusion, the product of the invention has different action mechanism with other anticoccidial drugs, has good anticoccidial effect, is effective after being continuously used for 3-5 days, is particularly suitable for drug-resistant strains, has the effects of promoting growth and improving feed conversion rate, and can be vigorously popularized and applied to stocking chicken households and intensive large-scale chicken farms.

And (6) test six: clinical application case of the medicine of the invention

Case 1: mixed infection of newcastle disease and coccidiosis in chicken

[ PROPERTIES OF THE PROBLEM ] A Guangdong Qingyuan Daiwan chicken breeder 7000 feathers of more than 40 days old. Because the mountain stocking mode is considered to be adopted and is far away from villages, there is a lucky psychology, the batch of chickens do not have any vaccine immunization, and the ground stocking sanitary condition is also poor. 24 days in 2019, 10 months and 24 days, the chicken flock suddenly attacks, and 170 feathers die in one night; 120 feathers die again in 25 days, and 90 feathers die again in the next day. Within 3 days of disease occurrence, 380 feathers die in sequence, the disease incidence reaches 60 percent, and the disease death rate is over 36 percent.

[ PROPERTIES ] sudden onset of disease, depressed spirit of sick chickens, reversed feather, no movement of crouched on the ground, discharge of white water-like feces or green dilute feces, and discharge of blood-containing feces of about 1/5 sick chickens.

[ Caesarean examination pathological changes ] 20 feathers of sick and dangerous chickens and chickens died of illness are successively subjected to cesarean examination, the pathological changes are basically consistent, and the conclusion is mainly as follows:

1. cockscomb of dead chicken is purple black, yellow green chicken manure is adhered to feather around anus, and fishy smell is given off.

2. The digestive tract. The glandular stomach of the chicken died of illness is swollen, the nipple has bleeding spot obviously, there is a band of obvious hemorrhage at the juncture of muscular stomach and glandular stomach, duodenum, caecum tonsil and rectum have hemorrhagic lesion, the caecum is full of blood.

3. A solid organ. The livers of the dead chickens are swollen and are scattered with bleeding points in different numbers; the spleen was enlarged and marbled. There are varying amounts of punctate bleeding in the pericardium, myocardium, coronary sulcus fat, apex, etc.

[ DIAGNOSIS ]

1. And (5) smear microscopy. Aseptic operation, taking small intestinal mucosa smear of dead chicken, adding 1-2 drops of normal saline, mixing, covering with glass slide, and observing under microscope to find coccidian schizont and coccidian oocyst.

2. And (5) virus separation. Aseptic operation, taking the brain, lung, spleen and trachea of the dead chick, grinding the brain, lung, spleen and trachea to prepare 1:10 suspension, adding penicillin and streptomycin to treat the suspension, inoculating the suspension into the allantoic cavity of a chick embryo of 10-embryo-old chick, placing 0.2ml of chick embryo in a 37 ℃ incubator to incubate for 120 hours, discarding 3 dead chick embryos within 24 hours after inoculation, collecting allantoic fluid of the remaining 7 chick embryos, and determining the titer of hemagglutination reaction (HA) to be (1:64) - (1: 256). The dead embryos are engorged systemically with varying numbers of bleeding spots on the head, chest and back.

3. Detecting Newcastle Disease Virus (NDV) by a real-time fluorescent RT-PCR method, and judging that the NDV is positive according to the result. Indicating that the chicken flock has newcastle disease.

According to on-site investigation, clinical manifestations of sick chicken, necropsy lesions of the sick chicken and laboratory test results, the chicken diseases of the chicken farmers are diagnosed as mixed infection of newcastle disease and coccidiosis.

[ MEANS FOR PREVENTING AND CONTROLLING ]

1. The composition prepared in example 6 was fed with the feed for group control in an amount of 5mg/kg body weight for 3 days to rapidly control coccidiosis in chicken groups, and also has prophylactic and therapeutic effects on newcastle disease and reduce the number of dead chicken.

2. Can be used in combination with therapy. The chicken is drunk with liquid supplementing salt, glucose and vitamin C to relieve dehydration and enhance disease resistance.

[ EFFECT ] after the chicken farm has epidemic situation and is preliminarily determined, the comprehensive emergency prevention and control measures are immediately taken, the effect is obvious, the sick chicken flocks are gradually recovered in less than 3 days, the sick chicken flocks are not dead again, and the production of the chicken farm is recovered to be normal in less than 1 week. During the treatment period, the accumulated death number is less than 100 feathers, and the cure rate is 98.57%.

Case 2: colibacillosis and coccidiosis mixed infection of spotted-brown chicken

[ PROBIAL CONDITIONS ] the number of broilers raised in a certain chicken farm in Guangxi Nanning is 100 ten thousand feather. Wherein, when one 12000 feather is raised to 20 days old, epidemic situation appears, and the death number is more than 120 feathers.

[ PROPERTIES ] sick chickens have the symptoms of depressed spirit, decreased feed intake, pale cockscomb, loose and disorderly feathers, eye closure and neck constriction, and purple and black feces discharge; the sick chicken die due to loss of appetite in the later period.

The caesarean lesion is 20 feathers of sick dead chickens and sick dangerous chickens which are successively and jointly caesarean examined, the lesions are basically consistent, and the summary is as follows:

(1) intestinal tract: the duodenum of the dead chicken spreads with purulent and hemorrhagic inflammation, and cecal swelling and is full of dark red blood; the mesentery is accompanied by a cheese-like exudate.

(2) And others: the breast muscles and leg muscles of the dead chicken are pale and have no blood color; turbid thoracic cavity air sac; the pericardium is accompanied by a cellulosic exudate.

[ DIAGNOSIS ]

1. And (6) detecting the contact lens. Aseptic operation, adopting the liver of a dead chicken, performing gram staining and microscopic examination, and finding gram-negative bacilli with blunt and round ends and medium size.

2. And (6) performing tabletting microscopic examination. And (4) performing aseptic operation, taking 10 g of fresh chicken manure of sick chicken, diluting the chicken manure with saturated saline, filtering out manure residues by using a filter screen, taking the suspension, tabletting and performing microscopic examination to find a large amount of ovate coccidian oocysts.

3. And (5) separating bacteria. Aseptically processing, collecting the liver of dead chicken, inoculating in the following two culture media, culturing at 37 deg.C for 24 hr, and observing. (1) On a common nutrient agar medium: grow into round, convex, smooth, wet edge neat, gray colony; (2) on mecnkia agar medium: grow into red colony with diameter of about 2 mm and concave center. And (3) performing aseptic operation, selecting the colony smear, performing gram staining and microscopic examination, and finding that the form of the isolate is the same as that of a direct contact blade of the liver of a chicken died of the disease.

4. And (4) biochemical reaction. The results all conform to the main characteristics of the biochemical reaction of the escherichia coli.

According to on-site investigation, clinical manifestations of sick chickens, autopsy lesions of dead chickens and laboratory examination results, the spotted-brown chickens can be diagnosed as colibacillosis and coccidiosis mixed infection.

[ MEANS FOR PREVENTING AND CONTROLLING ]

1. The composition prepared in example 18 was fed with the feed for group control in an amount of 5mg/kg body weight for 5 days to rapidly control coccidiosis in chicken groups, and also has prophylactic and therapeutic effects on newcastle disease and decrease death of sick chickens.

2. The chicken is drunk with liquid supplementing salt and vitamin C to relieve dehydration and enhance disease resistance.

After the spotted-brown chickens suffer from diseases, the comprehensive emergency prevention and control measures are immediately adopted according to the preliminary clinical diagnosis, the effect is very obvious, and the chickens do not die the next day. In less than 5 days, the sick chicken group recovers gradually, and in less than 1 week, the whole chicken group recovers normally. The cure rate almost reaches 100 percent.

Case 3: mixed infection of poultry histomoniasis and chicken coccidiosis

[ PROBIAL CONDITIONS ] A family of Sichuan chickens bought 6000 feathers of 10-day old Roman chickens from the field in 6 months of 2017, and raised in a simple booth separated by wood boards to breed on the ground. The batch of chicks had been immunized with marek's disease vaccine, infectious bursal disease vaccine, and newcastle disease vaccine. In order to prevent bacterial diseases, a proper amount of terramycin is added into the feed. When the chickens are raised to 30 days old, the chickens have disease epidemic situation, and the chickens have 200 feathers in turn and die 70 feathers more in succession.

[ PROPERTIES ] the sick chicken have the symptoms of lassitude, anorexia, even abominable appetite, increased drinking water, dislike of sports, neck loss, eye closure, somnolence, two-wing drooping, lusterless feather, body curling and aversion to cold. Diarrhea often occurs, and faint yellow thin feces are discharged; some loose stools are bloody. Sick chicken rapidly get slim, fossil fragments are uplifted. In the later stage of disease, the head of some sick chickens becomes purple. Finally, death is due to exhaustion.

The 18 feather dead chickens are subjected to cesarean examination in sequence, the lesions are basically consistent, and are mainly concentrated in the liver and the caecum. The summary is as follows:

(1) liver: the liver of the dead chicken is swollen, circular or irregular ulcer necrotic foci with different numbers are scattered on the surface, the size is as large as that of mung beans to peas, the middle of the necrotic foci is slightly sunken, the edge is neat or in a zigzag shape and is raised like a volcano shape, and the raised part is grey white or yellow white; severe lesions fuse into larger necrotic areas.

(2) Cecum: caecum lesions of dead chickens occur on both sides, and a few occur on one side. The cecum is swollen, the finger pressure cecum is firm and elastic, the cecum is filled with a dry and hard cheese-like embolism, and the surface is grey white; the mucous membrane of the caecum is inflamed and thickened, the content is scraped off, and a plurality of bleeding and ulcer of the intestinal mucosa are caused.

[ DIAGNOSIS ]

(1) And (5) smear microscopy. And (4) performing aseptic operation, and directly performing smear microscopy on caecan contents of the dead chicken to find a small amount of coccidian oocysts and a large amount of oval polyps.

(2) Hanging drop microscopy. Aseptically operating, collecting cecal contents of the newly dead chicken, diluting with warm normal saline to obtain hanging drop specimen, observing at room temperature (25-30 deg.C) under 400 times microscope, and finding large amount of round or oval blackhead tissue drop worm body in rapid rotation or pendulum motion.

According to on-site investigation, clinical manifestations of sick chickens, cesarean lesions of the sick and dead chickens and laboratory test results, the disease property of the Roman chicken flock is diagnosed as coccidiosis and poultry histomoniasis mixed infection.

[ MEANS FOR PROPHYLACTIC PROBLEMS ] first, the composition prepared in example 37 was mixed with a feed in an amount of 5mg/kg by weight and an appropriate amount of vitamin B complex was added thereto, and the mixture was continuously fed for 5 days. Secondly, the whole flock is disinfected and the feed trough and the drinking fountain are washed and disinfected by hot water or hot alkaline water (2%). And thirdly, removing the excrement in the henhouse in time, and keeping the ground dry.

[ EFFECT ] the prevention and control measures are taken immediately after the Roman chicken group has an epidemic situation, and the effect is obvious. After the medicine is taken for 3 to 4 days, the epidemic situation is basically controlled, the overall situation of the chicken flock is good, and clinical symptoms such as diarrhea, lassitude, anorexia and the like do not appear any more; in less than 1 week, the sick chicken group no longer appears dead chicken and recovers gradually, and if the healthy chicken group no longer appears sick chicken, the production is recovered to be normal. The cure rate is as high as 99.7%.

Case 4: coccidiosis and mycosis mixed infection of chicken

[ PROBIAL CONDITIONS ] the southern China is humid in the high temperature in 4-9 months per year, and is suitable for mass growth and reproduction of various moulds and coccidian; when the chicken flocks are in an environment with poor sanitation, the mixed infection of mould and coccidiosis is most likely to occur, the mortality rate is high, and the growth of chicks is hindered. A chicken farm of Guangdong river origin is purchased with 4200 feather of spotted-brown chicken seedlings from abroad in 2018, 5 months and 20 days. When the chickens are raised to 17 days old, the chickens have chicken diseases mainly including diarrhea, dysentery, blood dung and the like, the morbidity reaches 30%, and the mortality rate is 17%.

[ PROPERTIES ] most of the diseases are in depression, basically normal in body temperature, inappetence, increased drinking water, loose and disordered villi, drooping wings, unwilling to walk, somnolence and fond of being crowded together. A few young birds make paroxysmal chirps. Most of the diseases cause diarrhea, diarrhea and white and thin feces, some of which have blood. The course of disease is usually 3-5 days, and a few are 5-7 days. Finally, the disease is dyspnea, convulsion or coma and death. A few of the most acute early-late or morning chicks eat the chicken normally, but the chicken frequently attacks the chicken in the next morning or afternoon, spits fresh blood in the mouth and rapidly falls to kill the chicken. Some of them become chronic, and mainly have diarrhea, and can be well tolerated to gain immunity, but grow and develop slowly.

The heart, liver, kidney and glandular stomach mucous membrane of the young birds died of the disease spread larger bleeding points, some pectoral muscles also have bleeding points or have several fresh bleeding small points and nodules in between. The livers of the dead chicks are swollen, and yellow-white necrotic foci and nodules are scattered on the surfaces of the dead chicks; small nodules, small intestine swelling, mucosal hemorrhage or diffuse hemorrhage are scattered on the serosa of the intestinal tract, and some small grey speckles exist among the small nodules; swollen kidneys, anemia, slightly swollen cecum, with cheese-like material left in the interior, mixed with a small amount of blood, and the cloaca filled with loose stool.

[ DIAGNOSIS ]

1. And (4) mycosis. And (3) performing aseptic operation, randomly extracting the mixture eaten by sick chicken flocks, quantitatively inoculating the mixture on 3 Sambulo agar plates, culturing at 37 ℃ for 72 hours, observing and recording calculation results, and averagely containing more than 5000 aspergillus flavus spores per gram of feed. According to data, if more than 100 toxigenic aspergillus flavus spores are contained in each gram of feed, the disease of young poultry can be caused.

2. Coccidiosis. Sterile operation, taking the content and mucous membrane of the damaged small intestine section of the diseased chick, performing high power microscopic examination, and detecting to find a large amount of small intestine coccidiosis, wherein each visual field has more than 5 coccidiosis.

According to the epidemic characteristics, the chicks are purchased from other places, the henhouse is poor in sanitation, the climate is humid, and the feed is moldy. And diarrhea and intestinal mucosa bleeding of the sick chicks are used as main clinical characteristics and microscopic examination; can be diagnosed as the chicken infected with coccidiosis and mycotic and mycotoxin toxinism.

Firstly, create a good feeding management environment for chicks, make the henhouse sanitary, strictly isolate sick chicks, disinfect the polluted ground with 10% lime water, replace fresh feed, and feed multidimensional and glucose water for detoxification once a day. Next, the composition prepared in example 38 was administered at a dose of 5mg/kg body weight for a group control and a mixed drinking for 3 days to rapidly control coccidiosis in chicken groups and simultaneously prevent and treat chicken mold and reduce the death of sick chickens.

After the mixed infection of the chick group occurs, the disease condition is basically controlled in less than 5 days according to the comprehensive prevention and control measures, the symptoms are obviously improved, sick chickens do not die any more, and the healthy chick group is supposed to not discover the sick chickens any more; by day 7, the production of the flock returned to normal. The cure rate reaches more than 98 percent.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way; those skilled in the art should also realize that such equivalent variations, modifications, and alterations are possible in light of the above teachings without departing from the spirit and scope of the present invention; meanwhile, any equivalent changes, modifications and evolution made to the above embodiments according to the essential technology of the present invention still fall within the protection scope of the technical solution of the present invention.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The experimental method of the present invention, in which the specific conditions are not specified, is generally carried out under the conventional conditions or the conditions recommended by the manufacturers.

The various optimized technical schemes in the invention can be combined with each other unless otherwise stated.

Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The experimental procedures described in the specification and examples, which do not specify specific conditions, are generally carried out under the conditions which are customary or recommended by the manufacturers.

The various starting materials, reagents, components used in the present invention are, unless otherwise indicated, the corresponding starting materials commonly used in the art.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention.

Claims

1. A composition for preventing and treating drug-resistant coccidiosis in chicken is characterized in that:

the isothiazolinone compound comprises 100-900 parts of isothiazolinone compounds and at least one of 100-900 parts of disodium ethylene diamine tetraacetate and 100-900 parts of plant tannin; the content of isothiazolinone compounds in the product is as follows: the content of the isothiazolinone is 10-90% of the isothiazolinone as an active ingredient.

2. The composition for preventing and treating chicken drug-resistant coccidiosis as claimed in claim 1, wherein:

the isothiazolinone compound is 2-methyl-4-isothiazolin-3-one (MIT), 5-chloro-2-methyl-4-isothiazolin-3-one (CMIT), Benzisothiazolinone (BIT), 4, 5-dichloro-2-methyl-4-isothiazolin-3-one (DCMIT), 2-methyl-4-isothiazolin-3-one (MIT), Octylisothiazolinone (OIT), 4, 5-dichloro-N-octyl-4-isothiazolin-3-one (DCOIT), butylbenzoisothiazolinone (BBIT), N-methyl-1, 2-benzisothiazolin-3-one (MBIT), N-N-octyl-1, at least one of 10 derivative compounds of 2-benzisothiazolin-3-One (OBIT).

3. The composition for preventing and treating chicken drug-resistant coccidiosis as claimed in claim 1, wherein:

the dosage is as follows: the effective component isothiazolinone is calculated according to the weight ratio: the dosage of the composition for preventing and treating the drug-resistant coccidiosis of the chicken per kg of the weight of the chicken is 5 mg; adding water or mixing with appropriate amount of feed.

4. The composition for preventing and treating chicken drug-resistant coccidiosis as claimed in claim 1, wherein:

the dosage form of the composition for preventing and treating the drug-resistant coccidiosis of the chicken comprises: solid powders and liquid formulations 2, where liquid formulations comprise solutions, mixtures, emulsifiers and suspensions.

5. The preparation method of the composition for preventing and treating the drug-resistant coccidiosis in the chicken according to claim 1, wherein the composition comprises the following steps:

according to the ingredients, no proper auxiliary materials are added or proper auxiliary materials are added, then the mixture is stirred and mixed uniformly, and the mixture is filled, packaged and labeled to obtain solid powder; or

According to the component ingredients, no proper auxiliary materials are added or proper auxiliary materials are added, then the homogenization is carried out under certain conditions, the stirring and the mixing are carried out uniformly, and finally the liquid preparation is obtained by filling, bottling, sealing and labeling;

6. The use of the composition for preventing and treating chicken drug-resistant coccidiosis of any one of claims 1-5 in the treatment of mixed infection of coccidia and virus, bacteria, mold and other protozoa diseases of livestock and poultry.

7. The use of isothiazolinone compounds in the composition for preventing and treating chicken drug-resistant coccidiosis according to any one of claims 1 to 5 in the treatment of preventing and treating chicken drug-resistant coccidiosis.