CN117547556A

CN117547556A - A kind of medicine for treating avian trichomoniasis and its preparation method and application

Info

Publication number: CN117547556A
Application number: CN202310158938.5A
Authority: CN
Inventors: 刘鼎阔; 刘芳; 董惠峰; 王俊菊; 杨晓伟; 何志霞; 李源; 胡鹏程; 李道稳; 王磊; 任连泉; 史燕燕; 张博; 张怀瑾; 刘宁; 高晶晶
Original assignee: Dingzheng Xinxing Biotechnology Tianjin Co ltd
Current assignee: Dingzheng Xinxing Biotechnology Tianjin Co ltd
Priority date: 2023-02-24
Filing date: 2023-02-24
Publication date: 2024-02-13

Abstract

The invention provides a medicine for treating trichomonas fowl, which is prepared by culturing enterobacter hewanensis; the enterobacter hewanensis is enterobacter hewanensis DZ04001. In the invention, the strain has the effect of killing trichomonas, and the medicinal preparation obtained after cultivation is fed to sick birds, so that the growth of trichomonas can be inhibited, the spirit of the sick birds is recovered, the appetite of the sick birds is normal, and the hair color is gradually recovered to be bright.

Description

Medicine for treating trichomonas fowl and its preparation method and application

Technical Field

The invention relates to the technical field of prevention and treatment of trichomonas avionica, in particular to a medicine for treating trichomonas avionica, a preparation method and application thereof.

Background

Avian trichomoniasis is a protozoal disease caused by trichomonas and characterized by ulcers and diarrhea of the upper digestive tract of the birds, which is transmitted through contaminated feed and drinking water, and birds such as turkeys, chickens, ducks, geese, pigeons, hawks and the like are susceptible and the onset of young birds is severe, and is characterized by a cheesy accumulation in the throat, often accompanied by weight loss. The sick birds infected by the upper digestive tract show poor appetite, mental wilt, crop collapse, frequent extension of the neck to swallow, water-supply secretion of eyes, difficult closing, light green to light yellow mucus flowing out of the mouth and foul smell; the sick birds infected by the lower digestive tract show mental depression, appetite decline or abolish, loose feathers, unstable gait, slow-lying and happy, thin feces of light yellow water sample are pulled, and the weight declines and takes a sleeping shape until death. Trichomonas avians attack mucosal surfaces of the oral cavity, nasal cavity, pharynx, esophagus and crop.

The farm needs to strictly execute environmental sanitation and disinfection systems, such as: the method uses fumigation, insolation and other methods to kill the environment, and the ground excrement is cleaned in time to cut off the transmission path and the infectious agent, and the method can also be used for raising in a leveling mode as cage culture. In addition to the physical means, medicine treatment is adopted in the prevention and treatment of trichomonas avionica, nitroimidazole medicines are favored by people since the advent of the prior art, and metronidazole is outstanding, but the problem of drug resistance is also outstanding along with the long-term application of the metronidazole. The relevant departments in China have specified that the application of the metronidazole in the production of animal-derived foods is forbidden. The other nitroimidazole drugs and the metronidazole drugs belong to the same class, and also have the risk of carcinogenesis and mutation. Therefore, searching for new safe and effective chemical drugs and plant extracts to replace nitroimidazoles has been delayed, some plant extracts have been reported to have good effects against trichomonas at present, and research on biological preparations for resisting trichomonas has not been reported.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a biological agent for trichomonas avionica. In the invention, the enterobacter hewanensis DZ04001 is screened, the strain has the effect of killing trichomonas, and the medicinal preparation obtained after culture is used for feeding sick birds, so that the growth of trichomonas can be inhibited, the spirit of the sick birds is recovered, the appetite of the sick birds is normal, and the hair color is gradually recovered.

The technical scheme adopted by the invention is as follows:

a medicine for treating trichomonas fowl, which is characterized in that: the therapeutic medicine is prepared by culturing enterobacter hewanensis;

the enterobacter is enterobacter freundii DZ04001, is classified and named as Lelliottia amnigena, has a preservation unit of China general microbiological culture Collection center (CGMCC) No.24403, and has a preservation address of North Chen Xiyu No. 1 and North Chen Xiyu No. 3 in the Chaoyang area of Beijing city.

The invention also aims to provide a preparation method of the drug for treating the trichomonas avionica, which comprises the steps of inoculating enterobacter hewanensis into a culture medium, and culturing for a period of time to obtain a biological preparation;

Further: the living bacteria in the biological agent is 6.25X10 ⁴ -6.25×10 ⁵ CFU. The culture medium comprises 2-8 parts of peptone, 1-5 parts of yeast powder, 2-8 parts of NaCl and 5-15 parts of glucose. The culture medium comprises 5 parts of peptone, 2.5 parts of yeast powder, 5 parts of NaCl and 10 parts of glucose.

Another object of the present invention is to provide a method for preparing a drug for treating trichomonas fowl, comprising the steps of:

weighing peptone, yeast powder, naCl and glucose, adding water to constant volume, and shaking;

sterilizing at high temperature, and standing in a sterile environment;

when the temperature is room temperature, absorbing bacterial liquid to perform inoculation amount, and culturing at constant temperature to obtain mother liquid;

and (5) carrying out industrial amplification through a three-stage fermentation tank, and diluting to obtain a finished product.

Further: the inoculum size was 5%. The conditions of the constant temperature culture are as follows: the constant temperature water bath shaking table is set to 30 ℃ and the rotating speed is 180r/min, and the constant temperature culture is carried out for 48 hours.

The invention also aims to provide an application of the enterobacter hewanensis DZ04001 in preparing medicines for treating trichomonas avians.

The invention has the advantages and positive effects that:

in the invention, the enterobacter hewanensis DZ04001 is screened, the strain has the effect of killing trichomonas, and the medicinal preparation obtained after culture is used for feeding sick birds, so that the growth of trichomonas can be inhibited, the spirit of the sick birds is recovered, the appetite of the sick birds is normal, and the hair color is gradually recovered.

Detailed Description

The invention will now be further illustrated by reference to the following examples, which are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.

A medicine for treating trichomonas fowl, which is characterized in that: the therapeutic medicine is prepared by culturing enterobacter hewanensis; the enterobacter is enterobacter freundii DZ04001, is classified and named as Lelliottia amnigena, has a preservation unit of China general microbiological culture Collection center (CGMCC) No.24403, and has a preservation address of North Chen Xiyu No. 1 and North Chen Xiyu No. 3 in the Chaoyang area of Beijing city.

The preparation method of the therapeutic drug comprises the following steps: inoculating enterobacter hewanensis into a culture medium, and culturing for a period of time to obtain a biological preparation; the living bacteria in the biological preparation is 6.25X10 ⁴ -6.25×10 ⁵ CFU. The culture medium comprises 2-8 parts of peptone, 1-5 parts of yeast powder, 2-8 parts of NaCl and 5-15 parts of glucose. The preferable scheme is as follows: the culture medium comprises 5 parts of peptone, 2.5 parts of yeast powder, 5 parts of NaCl and 10 parts of glucose.

Optimizing the conditions of a strain culture medium formula, a culture temperature, a culture time inoculation amount, a rotating speed and the like according to the insecticidal effect and the safety to obtain optimal culture conditions: preparation of a culture medium: 5.0g of peptone, 2.5g of yeast powder, 5.0g of NaCl and 10g of glucose are respectively weighed, purified water is added to a constant volume of 1L, the mixture is inverted and shaken uniformly, and the mixture is placed in a sterile environment after high-temperature sterilization. Cooling to room temperature, sucking bacterial liquid, inoculating 5%, setting the constant temperature water bath table to 30deg.C, rotating speed of 180r/min, and culturing at constant temperature for 48 hr to obtain 1.0X10 ⁹ CFU bacterial liquid. Industrial amplification is carried out through a three-stage fermentation tank to obtain fermentation liquor, and the fermentation liquor is introduced into a dilution tank for dilution to obtain a finished product.

Screening of strains: during the investigation of the cultivation environment, various bacteria were found from the soil in the vicinity of the collected farm, and microbial strains capable of inhibiting pathogens were selected according to the direction of study of "lilac in lilac". Through blood agar culture enrichment and single colony line drawing culture, a group of gram negative bacterial strains (DZ 04001) with smooth surfaces and regular edges and short and small rod shapes are screened out, and antibacterial, antiviral and antiparasitic tests are respectively carried out, so that the bacterial strains only have the effect of killing parasites. Then the strain is sent to China Committee for culture Collection of microorganisms, and the strain (DZ 04001) is identified as Enterobacter hewanensis (Lelliottia amnigena) with the collection number of 24403.

Minimum effective dose confirmation: taking 15g of fresh pork liver, cutting, adding a proper amount of pure water, placing in a refrigerator at 4 ℃ for overnight, decocting the pork liver into liver soaking soup, filtering, and taking supernatant. L-cysteine, peptone and sodium chloride (with the proportion shown in Table 1), the supernatant of the liver soaking soup is used for constant volume to 100mL, the pH is regulated to 6, and the mixture is preserved in a refrigerator at 4 ℃ for standby after high-temperature high-pressure sterilization. Before use, 20% sterilized calf serum, penicillin and streptomycin 100U/mL are added.

Table 1: trichomonas culture medium composition ratio

Composition of the components	Content of
		L-cysteine	0.2g
Peptone	2g
		Sodium chloride	0.5g

Collecting oral mucus and yellow and white secretion of nasal sinuses in oral cavity of pigeon suffering from trichomonas, inoculating into 5mL liver soaking broth (trichomonas culture medium is shown in table 1), and culturing in a constant temperature incubator at 37deg.C. Step-by-step centrifugation is carried out on the pigeon trichomonas which is stably cultured for 3 generations in vitro, dead and weak-activity insect bodies are screened out, and the pigeon trichomonas is mixed with liver soaking soup according to the following ratio of 1:50 proportions.

Preparing DZ04001 bacterial liquid into a liver soaking soup culture medium with volume fraction of 0.2% Tween-80 for standby. The test was divided into drug groups and blank groups, wherein the drug groups were 1.0X10 ⁶ Adding DZ04001 bacterial liquid for CFU as an initial value gradient; the blank group was not dosed, the remaining factors were identical to the drug group. Each test group was set with 3 parallel controls and the final concentration of trichomonas pigeon was 2X 10 ⁵ And each mL. The drug group and the blank group were placed in a 37℃incubator for cultivation. Incubation was performed at constant temperature for 6, 12, 24 and 36 hours, respectively, and samples were counted using a hemocytometer and mortality rates of trichomonas pigeon were calculated. The activity of trichomonas on the blood cell counting plate can be obviously seen under an optical microscope, and the minimum content of viable bacteria of the DZ04001 bacterial liquid for resisting the pigeon trichomonas is 6.25X10 ⁴ CFU。

Animal regressive tests determine safe dose ranges: the mice were observed 5 hours after injecting 30 Kunming mice intraperitoneally with the following doses (Table 2) of DZ04001 bacteria solution, and the results are shown in Table 2:

table 2: injection results

Sequence number

A

B

C

D

E

F

Bacterial liquid amount (CFU)

6.25×10 ⁴

6.25×10 ⁵

6.25×10 ⁶

6.25×10 ⁷

6.25×10 ⁸

0

Number of mice (Only)

6

A. Group B and F mice were not abnormal; group C mice showed poor appetite and did not die; group D mice showed listlessness and individually died; group E mice, with clear fluid flowing out of the eyes, die after 8 hours. Meanwhile, the mice are subjected to sectioning examination, and A, B and F groups of mice have no obvious pathological changes; C. group D mice developed intestinal tract ventilation; group E mice showed global hemorrhage in the intestinal tract, air blowing, and large-area white necrosis of the liver. Therefore, the safe bacterial liquid amount range is 6.25X10 ⁴ Up to 6.25X10 ⁵ CFU。

The drug sensitivity test results show that: the isolated strain is sensitive to drugs such as kanamycin, streptomycin, amikacin and neomycin, and resistant to drugs such as penicillin and gentamicin.

And (3) determining the optimal dosage of bacterial liquid:

the meat pigeons 42 feathers infected with trichomonas were diagnosed and randomly divided into 7 groups, and were dosed with free water according to table 3, and after 5 days, the survival rate was counted (number of surviving pigeons/number of animals per group x 100%) while checking oral cavity mucus parasites.

Table 3: test grouping situation

Grouping	Number of test animals	Whether or not to take medicine	Variety of medicine	Bacterial liquid amount (CFU)	Dosage of medication
						A	6 feather	Whether or not	/	/	/
B	6 feather	Is that	The bacterial liquid of the invention	6.25×10 ⁴	0.2mL/L
						C	6 feather	Is that	The bacterial liquid of the invention	1.0×10 ⁵	0.2mL/L
D	6 feather	Is that	The bacterial liquid of the invention	1.5×10 ⁵	0.2mL/L
						E	6 feather	Is that	The bacterial liquid of the invention	3.0×10 ⁵	0.2mL/L
F	6 feather	Is that	The bacterial liquid of the invention	4.5×10 ⁵	0.2mL/L
						G	6 feather	Is that	The bacterial liquid of the invention	6.25×10 ⁵	0.2mL/L

The test results are shown in Table 4, and the bacterial liquid dosage is 6.25X10 ⁴ -1.0×10 ⁵ When in CFU, the survival rate of the sick pigeons is 50% -67%, the mental state is obviously improved, the mucus on the oral cavity wall is collected, and the microscopic examination of trichomonas is positive; the bacterial liquid dosage is 1.5X10 ⁵ When CFU is carried out, the survival rate of the sick pigeons is 83%, and the mental state is obviously improvedRecovering appetite, collecting oral cavity wall mucus, and detecting that trichomonas is negative; the bacterial liquid dosage is 3.0X10 ⁵ CFU～6.285×10 ⁵ When in CFU, the survival rate of the sick pigeons is 100%, the mental state is obviously improved, appetite is recovered, feathers become glossy, oral cavity wall mucus is collected, and microscopic examination of trichomonas is negative. The above shows that the trichomonas tests of the groups D to G are negative, the active rate of the groups E to G is 100%, and the group E is the lowest curative bacterial liquid concentration.

Table 4: results of administration

Grouping

A

B

C

D

E

F

G

Survival rate of

0

50％

67％

83％

100％

Trichomonas

+

-

Example 1

The 90 feather pigeons infected with trichomonas and 30 feather checked parasite-free pigeons from the same farm were fed with free water according to table 5 in a house, and after 5 days, the survival rate (number of pigeons/number of animals per group x 100%) was counted and the oral cavity mucous parasites were checked.

Table 5: test grouping situation

Table 6: results of administration

Grouping

A

B

C

D

E

F

G

Survival rate of

100％

10％

90％

93％

97％

100％

Trichomonas

-

+

-

+

-

The test results are shown in Table 6, and after the metronidazole solution and the bacterial liquid are used, the death rate of the sick pigeons is obviously reduced, and meanwhile, the trichomonas growth and reproduction can be well inhibited. 0.2mL/L is 3.0X10 ⁵ The CFU bacterial liquid has the least effective dosage and can replace antibiotics.

Example 2

The 500 feather reserve pigeons raised by a certain raiser are checked and diagnosed as trichomonas by a laboratory. The mucous membranes of the parts of the pigeon mouth, the pharynx, the larynx and the like have yellow button-shaped deposits, the disease course is 9d, and the total death rate is 165 and reaches 33 percent. Meanwhile, the sick pigeons have dysphagia and dyspnea, depression, loose feathers, reduced appetite and yellow green thin feces discharge.

According to 3.0X10 ⁵ CFU dosage, 0.2mL/L water is added to continuously apply the bacterial liquid of the invention, and the chicken flock stops dying and mental state at the 5 th dayObviously improved, appetite is restored on the 8 th day, feathers become glossy, oral cavity wall mucus is collected on the 11 th day, and microscopic examination of trichomonas is negative.

Example 3

The young black-bone chicken of 12 days old raised by a certain raiser in the gallery starts to develop from 3 days old, suffers from mental depression, loose feathers, sagging wings, inappetence, discharges white rice soup-like feces, and makes individual chickens feel green and thin, and some chickens give a screaming when discharging. The disease progresses for 1-3 days, the disease young grows slowly, emaciation and failure death. After the chicken is dissected and examined, the intestinal nodule is scraped and placed on a glass slide, physiological saline and a cover slip are added dropwise, the trichomonas living body is visible under the lens, and the trichomonas disease is further diagnosed by combining clinical symptoms.

According to 3.0X10 ⁵ The bacterial liquid is continuously applied by adding water into 0.3mL/L of CFU dosage, the chicken flock is stopped dying on the 3 rd day, the mental state is obviously improved, the appetite is recovered on the 5 th day, the feather becomes glossy, the oral cavity wall mucus is collected on the 7 th day, and the microscopic examination of the trichomonas is negative.

Example 4

500 hen raised by a certain farmer in Xuyi county, jiangsu province, is checked and diagnosed as trichomonas disease by a laboratory, and the sick chicken has the phenomena of reduced feed intake, listlessness, emaciation, dyspnea, light yellow or light green thin feces in succession.

According to 3.0X10 ⁵ The bacterial liquid is continuously applied by adding water into 0.3mL/L of CFU dosage, the spirit of the chicken flock is obviously improved on the 4 th day, no death case can occur any more, appetite is restored on the 5 th day, oral cavity wall mucus is collected on the 7 th day, and microscopic examination of trichomonas is negative.

In addition to examples 1 to 4, the treatment of a plurality of sick chicken farms and sick pigeon farms infected with coccidiosis in different areas was performed according to 2.0X10 respectively ⁵ CFU、2.5×10 ⁵ CFU、3.5×10 ⁵ CFU、3.5×10 ⁵ CFU、4.0×10 ⁵ CFU、4.5×10 ⁵ The CFU dose was fed in the same manner as in examples 1 to 4, and the sick birds stopped dying on days 3 to 5, gradually recovered from mental and appetite on days 5 to 7, and after 7 days the trichomonas microscopic examination was negative.

Claims

1. A kind of medicine for the treatment of avian trichomoniasis, characterized in that: the medicine is prepared by culturing Enterobacter fluvium;

The Enterobacter fluvium is Enterobacter fluvium DZ04001, the classification name is Lelliottia amnigena, the depository unit is the General Microbiology Center of the Chinese Microbial Culture Collection Committee, the deposit number is CGMCC No. 24403, and the deposit address is Beichen West, Chaoyang District, Beijing No. 3, No. 1 Courtyard, Road.

2. A method for preparing a drug for treating avian trichomoniasis, which is characterized by: inoculating Enterobacter fluvium into a culture medium, and cultivating it for a period of time to obtain a biological preparation;

3. The preparation method of a drug for treating avian trichomoniasis according to claim 2, characterized in that the viable bacteria in the biological preparation are 6.25×10 ⁴ -6.25×10 ⁵ CFU.

4. The preparation method of a kind of avian trichomoniasis treatment drug according to claim 3, characterized in that: the culture medium is 2-8 parts of peptone, 1-5 parts of yeast powder, 2-8 parts of NaCl, and glucose Serves 5-15.

5. The preparation method of a kind of avian trichomoniasis treatment drug according to claim 2 or 3 or 4, characterized in that: the culture medium is 5 parts of peptone, 2.5 parts of yeast powder, 5 parts of NaCl, and 10 parts of glucose. .

6. The preparation method of a kind of avian trichomoniasis treatment drug according to claim 5, characterized in that: comprising the following steps:

Weigh peptone, yeast powder, NaCl and glucose, add water to make up to volume, and shake well;

After high temperature sterilization, let it stand in a sterile environment;

When the temperature is room temperature, draw the bacterial liquid to inoculate the amount, and obtain the mother liquid after constant temperature cultivation;

Industrial amplification is carried out through three-stage fermentation tanks, and the finished product is obtained after dilution.

7. The preparation method of a drug for treating avian trichomoniasis according to claim 6, characterized in that: the inoculation amount is 5%.

8. The preparation method of a kind of avian trichomoniasis treatment drug according to claim 6, characterized in that: the conditions of the constant temperature culture are: a constant temperature water bath shaker is set to 30°C, a rotation speed of 180r/min, and the constant temperature culture is 48 hours. .

9. Application of Enterobacter fluvium DZ04001 in the preparation of drugs for the treatment of avian trichomoniasis.