CN111632094B

CN111632094B - Application of composition in preventing or treating diseases caused by escherichia coli of broiler chicken

Info

Publication number: CN111632094B
Application number: CN201910890785.7A
Authority: CN
Inventors: 杨鹰; 武振龙; 王梦梦
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2019-09-20
Filing date: 2019-09-20
Publication date: 2021-09-21
Anticipated expiration: 2039-09-20
Also published as: CN111632094A

Abstract

The present invention proposes a composition and its use in the manufacture of a medicament, the composition comprising: 12-18 parts by weight of scutellaria baicalensis; 8-12 parts by weight of phellodendron amurense; 2-8 parts by weight of patrinia; 2-8 parts by weight of agrimony; 2-8 parts by weight of fructus forsythiae; 2-8 parts by weight of licorice; 2-8 parts by weight of sanguisorba officinalis. The composition can effectively prevent or treat diseases caused by the infection of escherichia coli in organisms, and has theoretical research and clinical application values.

Description

Application of composition in preventing or treating diseases caused by escherichia coli of broiler chicken

Technical Field

The present invention relates to the field of medicine. In particular to application of the composition in preventing or treating diseases caused by escherichia coli of broiler chickens.

Background

After the pathogenic escherichia coli of the poultry invades the organism, acute colibacillosis can be caused. Broiler colibacillosis is an explosive disease frequently occurring in the breeding process, the disease is initially expressed as septicemia, then sudden death occurs or inflammatory lesions occur in a plurality of important organs of an organism, such as liver, heart, air sac and the like, the disease is a disease with high morbidity and mortality, and serious economic burden is caused to poultry producers in the world. Although the use of antibiotics and chemical antibacterial drugs can effectively relieve the invasion of pathogenic microorganisms to organisms, the occurrence of antibiotic residues and bacterial drug resistance causes food safety hidden dangers and public health problems, so that the development of safe and effective antibiotic substitutes for preventing and treating colibacillosis is needed.

Chinese herbal medicines are widely applied clinically in China, and valuable and rich experience is accumulated. The traditional Chinese medicine can directly inhibit and kill various infection sources such as bacteria, viruses and the like, remove toxin generated by the infection sources, eliminate inflammation caused by infection of an organism, resist invasion and damage of foreign matters by improving the immune function of the organism, has higher safety than antibiotics, has small toxic and side effects on the organism, and has great potential in replacing antibiotics.

The Chinese herbal medicines play roles mainly by depending on the effective components, different Chinese herbal medicines are combined according to pharmacological properties, the maximum effect of various Chinese herbal medicines can be fully played, and the effect of treating both principal and secondary aspects of diseases is achieved.

However, there is still a need for a Chinese herbal medicine suitable for preventing or treating diseases caused by E.coli infection.

Disclosure of Invention

The present invention aims to solve at least to some extent at least one of the technical problems of the prior art.

To this end, the invention proposes a composition. According to an embodiment of the invention, the composition comprises: 12-18 parts by weight of scutellaria baicalensis; 8-12 parts by weight of phellodendron amurense; 2-8 parts by weight of patrinia; 2-8 parts by weight of agrimony; 2-8 parts by weight of fructus forsythiae; 2-8 parts by weight of licorice; 2-8 parts by weight of sanguisorba officinalis. The scutellaria baicalensis is used as a main drug, and the phellodendron is used as a ministerial drug, and is combined with the main drug to enhance the functions of clearing heat and drying dampness, and purging intense heat and removing toxicity. Herba Patriniae, fructus forsythiae, garden burnet and herba et Gemma Agrimoniae are adjuvant drugs, and the main effects of herba Patriniae and fructus forsythiae are similar to those of main drugs, and mainly have the effects of clearing heat and detoxicating, reducing swelling and expelling pus, removing blood stasis and relieving pain, dispelling wind and heat and the like. The medicines are supplemented with sanguisorba officinalis for cooling blood and stopping bleeding, detoxifying and healing sore, agrimony for astringing and stopping bleeding, stopping dysentery and detoxifying, and liquorice as a guiding medicine to harmonize the properties of the medicines in the composition. The composition is mainly prepared from cold and cool medicines, and the cold and cool medicines have the effects of clearing heat and removing toxicity, resisting bacteria and diminishing inflammation, are synergistic mutually, can effectively prevent or treat diseases of organisms caused by infecting escherichia coli, and have theoretical research and clinical application values.

According to an embodiment of the invention, the above composition may also have the following additional technical features:

according to an embodiment of the invention, the composition comprises: the method comprises the following steps: 15 parts by weight of scutellaria baicalensis; 10 parts by weight of phellodendron; 5 parts by weight of herba patriniae; 5 parts by weight of hairyvein agrimony; 5 parts by weight of forsythia suspense; 5 parts by weight of licorice; 5 parts of sanguisorba officinalis. This can further improve the prophylactic and therapeutic effects.

In another aspect of the invention, the invention proposes the use of a composition for the manufacture of a medicament. According to an embodiment of the present invention, the medicament is used for preventing or treating diseases caused by infection with Escherichia coli. As mentioned above, the medicine can effectively prevent or treat diseases caused by the infection of Escherichia coli in organisms, and has theoretical research and clinical application values.

According to an embodiment of the invention, the infected subject is an animal comprising: chicken, duck, rabbit, mouse, pig, cattle, sheep, and human.

According to embodiments of the invention, the composition is used for decreasing crypt depth, increasing lipase activity in jejunum, increasing lactobacillus and bifidobacterium numbers. Therefore, the function of protecting the intestinal structure can be effectively achieved, the intestinal injury is reduced, and the diseases caused by the infection of escherichia coli are effectively prevented and treated.

According to an embodiment of the invention, the composition is for reducing inflammatory cell infiltration of the liver. Thereby, the recovery ability of the liver injury is improved.

According to an embodiment of the present invention, the composition is used for enhancing immunity of the body. Thereby, the purpose of preventing and treating diseases is achieved.

According to embodiments of the invention, the composition is used to increase spleen index, increase serum lysozyme activity, and decrease serum newcastle disease antibody (NDV antibody) titers. Therefore, the immunity of the organism is improved, and the purposes of preventing and treating diseases are achieved.

According to an embodiment of the invention, the composition is used for reducing serum glutamic-oxaloacetic transaminase activity and reducing inflammatory cell infiltration of the liver. Thereby, the damage degree of Escherichia coli to liver is reduced.

According to an embodiment of the invention, the composition is administered in a dose of 0.2-1.4% daily. Thereby, the purpose of prevention and treatment can be achieved. In some embodiments, a corn-soybean meal type basal diet is employed, as standardized by chinese chicken feeding standards (2004).

According to the embodiment of the invention, the administration dosage of the composition is 0.2-0.6% of daily ration, and the composition is used for preventing diseases caused by the infection of escherichia coli. Therefore, the mortality rate of infected broiler chickens can be reduced, the systemic immunity is improved, the intestinal injury and liver injury can be prevented and protected, the number of intestinal probiotics is increased, and a better prevention purpose is achieved.

According to the embodiment of the invention, the administration dose of the composition is 0.8-1.2% of daily ration, and the composition is used for treating diseases caused by Escherichia coli infection. Therefore, the death rate of infected broiler chickens can be reduced, the intestinal injury and liver injury can be treated, the activity of intestinal digestive enzymes is improved, the intestinal microbial balance is restored, and a better treatment purpose is achieved.

According to an embodiment of the invention, the medicament comprises one or more pharmaceutically or dietetically acceptable excipients. For example, magnesium carbonate, magnesium stearate, talc, sugar or lactose. Therefore, the instant food is convenient to eat and can achieve a better treatment purpose. The dosage form of the medicine can be selected from one of mixture, injection, tablet, granule, syrup, capsule, oral liquid, aerosol and spray. Thus, different dosage forms can be adopted conveniently according to different administration objects. For example, tablets, granules, syrups, capsules and oral liquids may be used for convenience of administration, and the dosage form of the drug may be adjusted according to the absorption site of the drug and the release requirement of the drug, thereby improving the bioavailability of the drug and prolonging the release time of the drug. For severe patients, injection can be adopted, so that the requirement of large-dose administration is met, and the influence of gastrointestinal circulation on the effective components of the medicine is avoided.

The term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects resulting from the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) preventing disease (e.g., preventing disease caused by E.coli) or the development of a disorder in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting a disease, e.g., arresting disease progression; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce, or inhibit a disease in the individual, including, but not limited to, administering a drug containing a composition described herein to an individual in need thereof.

It will be appreciated by those skilled in the art that the features and advantages previously described for the compositions apply equally to this use and will not be described in further detail herein.

Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.

Drawings

The above and/or additional aspects and advantages of the present invention will become apparent and readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings of which:

FIG. 1 is a schematic diagram showing the therapeutic effect of a composition according to one embodiment of the present invention on E.coli infected broiler chickens;

fig. 2 shows a schematic diagram of the effect of a composition of 1% ration on the jejunum morphology of broilers according to an embodiment of the present invention, wherein a represents a blank control group, B represents an escherichia coli O78 infected control group, C represents a composition group added after infection, 21d represents the age of 21 days of broilers, 25d represents the age of 25 days of broilers, and the magnification is 50 times;

fig. 3 shows a schematic diagram of the effect of a composition of 1% ration on liver of broilers according to an embodiment of the present invention, wherein a represents a blank control group, B represents an escherichia coli O78 infected control group, C represents a composition group added after infection, 21d represents the age of the broilers at 21 days, 25d represents the age of the broilers at 25 days, and the magnification is 400 times;

FIG. 4 is a graph showing the protective effect of a 0.5% ration composition on E.coli O78 infected broiler chickens according to one embodiment of the present invention;

FIG. 5 is a schematic diagram showing the effect of a composition of 0.5% ration on the liver of broilers according to an embodiment of the present invention, wherein A represents a blank control group, B represents an Escherichia coli O78 infected control group, C represents an infected group after the composition is added, 21d represents the age of the broilers at 21 days, 25d represents the age of the broilers at 25 days, and the magnification is 400 times;

fig. 6 shows a schematic diagram of the effect of the composition of 0.5% ration on the jejunum morphology of broilers according to an embodiment of the invention, wherein a represents a blank control group, B represents an escherichia coli O78 infected control group, C represents an infected group after the composition is added, 21d represents the age of 21 days of broilers, 25d represents the age of 25 days of broilers, and the magnification is 50 times.

Detailed Description

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Examples

Determination of diameter of inhibition zone

The diameter of the inhibition zone of the composition on Escherichia coli O78 is determined by plate agar perforation method. Taking 0.1mL of 10⁸Placing the CFU/mL bacterial solution in the prepared sterile Mackanka culture plate, uniformly coating, stopping coating the plate when the coater and the plate have certain viscosity, punching the plate by using a puncher with the diameter of 6mm, and filling the holes with 1g/mL composition liquid medicine and physiological saline. The flat plate is placed in a constant temperature incubator at 37 ℃, the flat plate is taken out after being cultured for 24 hours, and the diameter of the inhibition zone is measured by a vernier caliper.

Determination of minimum inhibitory concentration

The minimum inhibitory concentration of the composition on avian pathogenic escherichia coli O78 is determined by a test tube double dilution-plate method. Taking 9 sterilized test tubes, marking numbers 1-9 on the test tubes, respectively adding 1mL of sterilized LB broth into each test tube, adding 1mL of liquid medicine into the test tube with the number 1, gradually diluting twice to the test tube with the number 9, uniformly mixing the last tube, and discarding 1mL, wherein the concentrations of the medicines in the test tubes with the numbers 1-9 are respectively 500, 250, 125, 62.5, 31.25, 15.625, 7.81, 3.9 and 1.95mg/m L. And setting a negative control and a positive control at the same time, wherein only the bacteria liquid and the LB broth are added into a positive control tube, and the negative control tube does not contain any substances except the LB broth. The concentration of the additive added into each of the 11 tubes is 10⁶And (3) 100 mu L of CFU/ml bacterial liquid, uniformly mixing, and horizontally placing in a constant-temperature incubator at 37 ℃ for 24 hours. The dark color of the compound Chinese medicinal liquid can affect the eye sightObservation of the results requires further validation of the results in conjunction with the plating method. And sucking 100 mul of liquid from each test tube, uniformly smearing the liquid on a prepared sterile ordinary agar culture plate, culturing the liquid at the constant temperature of 37 ℃ for 24 hours, and taking the corresponding concentration of the plate without bacterial growth as the minimum bacteriostatic concentration of the medicine.

Prevention experiment and treatment experiment

And (3) treatment test: the experiment adopts single-factor random design, 210 AA chicks of 1 day age are randomly divided into 3 groups, each group has 7 repetitions, each repetition comprises 10 chickens, namely a blank control group, an escherichia coli treatment group and a composition and escherichia coli treatment group, the additive amount of the composition is 10 g (namely 1% for replacing the amount of zeolite powder in basic daily ration) added in each kilogram of daily ration, and the adding time is 19-25 d. The whole breeding period is 25 days. In the other groups except the blank control, the broiler chickens are subjected to intraperitoneal injection for virus attack at the age of 19 days, each broiler chicken in the test group is subjected to intraperitoneal injection of 1mL of escherichia coli O78 bacterial liquid for later use, and the blank control group is injected with the same amount of physiological saline.

Prevention test: the test adopts single-factor random design, 210 AA chicks of 1 day age are randomly divided into 3 groups, each group has 7 repetitions, each repetition has 10 chickens, and the groups are a blank control group, an escherichia coli treatment group and a composition and escherichia coli treatment group respectively, the additive amount of the composition is 5g (namely 0.5% for replacing the amount of zeolite powder in basic ration) added in each kilogram of ration, and the adding time is 15-21 d. The whole breeding period is 25 days. In the other groups except the blank control, the broiler chickens are subjected to intraperitoneal injection for virus attack at the age of 19 days, each broiler chicken in the test group is subjected to intraperitoneal injection of 1mL of escherichia coli O78 bacterial liquid for later use, and the blank control group is injected with the same amount of physiological saline.

Sample collection was performed at 21d and 25 d.

Determination of mortality

After Escherichia coli is attacked, the death number of the broiler chickens is recorded, and the ratio of the death number in the whole period to the total number of the chickens is used as the death rate.

Determination of intestinal morphology

Selecting 1 test chicken repeatedly, placing the middle part of jejunum tissue of the same part in formaldehyde solution for fixation, embedding the jejunum fixed tissue sample with paraffin after fixation, and staining with hematoxylin-eosin (HE) to obtain tissue slices. Observing the damaged state of the intestinal tract by using a living cell workstation under a microscope and taking a picture, randomly selecting 10 complete intestinal villi under an observation visual field, respectively measuring the height (VH) of the villi and the depth (CD) of the crypt by using Image pro plus 6.0 processing software, and calculating the ratio of the VH to the CD.

Determination of digestive enzymes in the gut

The jejunum chyme collected at 21d and 25d is made into 10% homogenate by using physiological saline to be used for measuring the activity of jejunum amylase (cat # C016-1-1), trypsin (cat # A080-2-2) and lipase (cat # A054-2-1), and the measuring steps are referred to the instruction of establishing the kit by Nanjing. Total protein concentration in the homogenate was determined using the BCA kit (cat # CW0014) available from Bio-technology Inc., of century. The final digestive enzyme activity is expressed as activity per mg protein.

Determination of intestinal microorganisms

Weighing 0.5g of ileum chyme, adding sterile physiological saline to prepare 10% homogenate, continuously diluting by 10 times, after pre-culturing to determine three continuous concentration gradients which can be counted, sucking 100 mu l of sample to MRS and BL selective culture medium and uniformly coating the sample by using a sterile coater, stopping coating when a coating rod and the culture medium have certain viscosity, putting a flat plate into a 37 ℃ incubator for culturing, taking out the flat plate after 24-36 hours, counting grown colonies, and expressing the number of the bacterial colonies contained in each gram of intestinal contents (lg CFU/g) by logarithm.

Detection of liver pathological section

Selecting 1 test chicken repeatedly, taking liver tissue of the same part, fixing in 4% paraformaldehyde solution, and making into liver pathological section. The operation steps are as follows: after the liver tissue is fixed, the liver is embedded and sliced by paraffin, tissue slices are prepared after HE staining, and the liver slices are observed by a microscope of a living cell workstation to see whether pathological changes appear.

Statistical analysis

Data are presented as mean ± SEMs and analyzed by one-way analysis of variance. Duncan-type multiple comparisons were performed using a spot 17.0 to determine differences, with P <0.05 considered significant.

As shown in tables 1 and 2, the bacteriostatic diameter of the composition against Escherichia coli O78 was 8mm, and the minimum inhibitory concentration was 31.25 mg/ml. The results show that the composition has an inhibiting effect on in vitro cultured Escherichia coli O78.

TABLE 1 in vitro zone diameter of inhibition for compositions of E.coli O78 (n ═ 3)

Treatment of	Average zone diameter (mm)
		Physiological saline	0
Composition comprising a metal oxide and a metal oxide	8

Table 2 in vitro MIC of compositions against e.coli O78 (n ═ 3)

Treatment of	MIC(mg/ml)
		Qinbaiqingdu powder	31.25

In a treatment test, the mortality rate can reflect the treatment and prevention effects of the composition on broiler chickens infected by Escherichia coli O78. The results are shown in fig. 1A, the composition with 1% ration added reduced mortality of broiler chickens. The results show that the 1% ration composition has therapeutic effect on E.coli infection.

The intestinal canal has complete morphological structure, normal digestion capability and intestinal canal flora balance, which are important indexes reflecting the intestinal canal health. As shown in fig. 2, the composition added with 1% of the ration can protect intestinal structures, remarkably reduce the depth of crypts (P <0.05) of broilers (fig. 1B), remarkably improve the activity of jejunal lipase (P <0.05) (fig. 1C), and remarkably improve the number of lactobacilli and bifidobacteria (P <0.05) (fig. 1D).

The Escherichia coli can damage the liver when being attacked by toxin, and the recovery of the liver damage can show that the composition has a treatment effect. The results are shown in figure 3, the composition is able to reduce inflammatory cell infiltration of the liver.

In the prevention experiment, the result is shown in fig. 4A, and the composition added with 0.5% of daily ration reduces the death rate of the broiler chickens. The results show that the composition of 0.5% of the daily ration has a preventive and protective effect on escherichia coli infection.

Immune organ index and serum NDV antibody titer can reflect the body's resistance to e.coli O78. Results as shown in fig. 4B and 4C, the composition significantly increased the spleen index of broiler chickens (P <0.05), significantly decreased serum NDV antibody titers (P < 0.05). The results show that the 0.5 percent daily ration composition can improve the immune function of an organism and effectively prevent the infection of Escherichia coli O78.

Escherichia coli can damage the liver, so that the liver is compensated and swollen, the liver structure is damaged, and the low or no damage of the liver damage degree can indicate that the composition has a prevention effect. The results are shown in the figure, the 0.5% ration composition significantly reduced liver index (P <0.05) (4D), significantly reduced serum glutamic-oxaloacetic transaminase activity (P <0.05) (4E), and reduced inflammatory cell infiltration of the liver (fig. 5).

The intestinal canal has complete morphological structure, normal digestion capability and intestinal canal flora balance, which are important indexes reflecting the intestinal canal health. The results are shown in fig. 6, the 0.5% ration composition was able to preserve intestinal architecture, significantly reduce crypt depth (fig. 4F), significantly increase villus height (P <0.05) (fig. 4G), significantly increase the number of intestinal bifidobacteria (P <0.05) (fig. 4H).

In conclusion, the composition with 0.5% of daily ration added into the daily ration of the broiler chickens can effectively prevent the infection of the escherichia coli on the broiler chickens, and the composition with 1% of daily ration added has a treatment effect on the injury of the escherichia coli infected broiler chickens. The results of the invention can be used not only in production to prevent colibacillosis, but also in veterinary clinical after outbreak of colibacillosis.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. A composition for preventing or treating a disease caused by infection of avian animals with e.coli, comprising:

12-18 parts by weight of scutellaria baicalensis;

8-12 parts by weight of phellodendron amurense;

2-8 parts by weight of patrinia;

2-8 parts by weight of agrimony;

2-8 parts by weight of fructus forsythiae;

2-8 parts by weight of licorice;

2-8 parts by weight of sanguisorba officinalis.

2. The composition for preventing or treating diseases caused by the infection of avian animals with E.coli according to claim 1, which is composed of:

15 parts by weight of scutellaria baicalensis;

10 parts by weight of phellodendron;

5 parts by weight of herba patriniae;

5 parts by weight of hairyvein agrimony;

5 parts by weight of forsythia suspense;

5 parts by weight of licorice;

5 parts of sanguisorba officinalis.

3. Use of a composition according to claim 1 or 2 for the manufacture of a medicament for the prevention or treatment of a disease caused by e.

4. Use according to claim 3, wherein the avian animal comprises: chicken and duck.

5. Use according to claim 3, wherein the composition is for reducing crypt depth, increasing lipase activity in the jejunum, increasing the number of lactobacilli and bifidobacteria.

6. Use according to claim 3, wherein the composition is for improving the immunity of the body;

the composition is used for increasing spleen index and reducing serum newcastle disease antibody titer.

7. The use according to claim 3, wherein the composition is used for reducing serum aspartate aminotransferase activity and reducing inflammatory cell infiltration of the liver.

8. Use according to claim 3, wherein the composition is administered in a dose of 0.2 to 1.4% of the daily ration.

9. The use according to claim 3, wherein the composition is administered in a dose of 0.2 to 0.6% of a daily ration for the prevention of diseases caused by the infection with E.

10. The use according to claim 3, wherein the composition is administered at a dose of 0.8 to 1.2% of the daily ration for the treatment of diseases caused by E.