CN111621450B

CN111621450B - Duck-source gallibacterium and application thereof

Info

Publication number: CN111621450B
Application number: CN202010664292.4A
Authority: CN
Inventors: 张玉杰; 刘�东; 刘红祥; 郭玉广; 高天佐; 杜元钊; 赵鹏
Original assignee: Qingdao Yebio Bioengineering Co Ltd
Current assignee: Qingdao Yebio Bioengineering Co Ltd
Priority date: 2020-07-10
Filing date: 2020-07-10
Publication date: 2022-07-05
Anticipated expiration: 2040-07-10
Also published as: CN111621450A

Abstract

The invention provides duck-origin chicken bacillus and an inactivated vaccine prepared from the same, wherein the vaccine prepared from a duck-origin chicken bacillus YT-1 strain with the preservation number of CCTCC NO: M2020272 can be used for preventing diseases of egg laying reduction, fertilization rate reduction and salpingitis of breeding hens caused by the duck-origin chicken bacillus, so that the blank that NO duck-origin chicken bacillus vaccine exists in China is filled. According to the invention, pathogen separation, identification and purification are carried out on the cases of egg drop of the clinically laying hens and broiler breeders after artificial insemination to obtain a strain of duck-origin chicken bacilli, the screened bacteria are determined to be the duck-origin chicken bacilli through separation culture, specific PCR identification, 16s rRNA sequence analysis and animal regression tests, and the inactivated vaccine immune chicken flock prepared by the strain is used.

Description

Duck-source gallibacterium and application thereof

Technical Field

The invention belongs to the technical field of preparation of poultry vaccines, and particularly relates to duck-origin gallibacterium and an inactivated vaccine prepared from the duck-origin gallibacterium.

Background

Kjos-Hansen et al reported for the first time in 1950 that gallibacterium gallinarum, Christensen, et al established, in 2003, Duck pasteurella, salpingitis pasteurella and hemolytic pasteurella in the family Pasteurellaceae as gallibacterium species alone, and Bisgaard, et al, 2009, further identified gallibacterium gallinarum as a representative species of gallibacterium gallinarum. The bacillus gallinarum can be separated from various poultry such as chicken, turkey, duck, goose, parrot, partridge, guinea fowl and the like, and different bacillus gallinarum can also be separated from poultry with different clinical symptoms.

Studies have shown that gallinaceous bacilli can be isolated from healthy birds and it is believed that these bacteria may be part of the normal microflora of the upper and lower respiratory tracts; on the contrary, the bacillus can be isolated from the laying hens suffering from reproductive system diseases by kohlert, mirle and the like, and pathogenic bacteria are considered to be potential pathogenic bacteria of laying hen salpingitis, oviduct cyst and septicemia, so that the egg yield and the egg quality are reduced; another study showed that certain avian bacilli cause endocarditis, sinusitis, or upper respiratory tract infections in chickens. In addition, the pathogenic bacteria can also contribute to a higher mortality rate in chickens with immunosuppressive diseases. The domestic research on the strain is mainly limited to the strain isolation and culture characteristics, but no conclusion is made on the clinical hazard of the strain and relevant animal regression test data in a laboratory, Wang Chuanqing equal to 2008 reports the existence of duck-origin gallibacterium in China for the first time, Zheng deer equal to 2010 separates and identifies 45 duck-origin gallibacterium from different organs of 64 chickens suffering from salpingitis, Zhai equal to 2014 separates 1 duck-origin bacterium from glandular stomach of 10-day-old chickens suffering from glandular stomachache.

Research shows that the duck-origin chicken bacillus can cause the chicken oviduct cyst and the egg yield to be reduced, but has no influence on the fertilization rate. Since 2000 years, artificial insemination of each batch of chicken in some large-scale chicken breeding farms in China after development can cause the situation of double reduction of egg yield and hatchability, and the artificial insemination tends to be more and more severe, so that huge economic loss is caused for chicken raising enterprises, and due to numerous duck-origin gallibacterium serotypes, the disease is preliminarily guessed to be possibly related to duck-origin gallibacterium with a new serum type. In order to effectively prevent and control the disease, the screening of new serotype allium duarium is urgent.

Disclosure of Invention

The invention aims to provide duck-origin chicken bacillus and an inactivated vaccine prepared from the same, and the vaccine prepared from the duck-origin chicken bacillus can be used for preventing diseases of egg laying reduction, fertilization rate reduction and salpingitis of breeding hens caused by the duck-origin chicken bacillus, so that the blank that no duck-origin chicken bacillus vaccine exists in China is filled.

The invention firstly provides a duck source Gallibacterium (Gallibacterium anatis) YT-1 strain which is preserved in China center for type culture Collection in Wuhan in 7-3 months in 2020, with the preservation number of CCTCC NO: M2020272;

the duck source gallibacterium provided by the invention is YT-1 strain used for preparing vaccine;

the vaccine is an inactivated vaccine.

The invention also provides a duck-origin chicken bacillus inactivated vaccine, which comprises an inactivated duck-origin chicken bacillus strain antigen and a vaccine adjuvant, wherein the antigen is a duck-origin chicken bacillus (Gallibacterium anatis) YT-1 strain;

wherein the antigen content in the inactivated vaccine is about 12.5 hundred million/mL;

wherein the duck-origin chicken bacillus is inactivated by using formaldehyde solution.

The vaccine adjuvant is a water-phase adjuvant.

The preparation method of the inactivated vaccine of the invention comprises the following steps:

1) bacterial liquid culture: culturing a duck source gallibacterium YT-1 strain by using a fermentation tank to obtain a bacterial liquid;

2) bacterial liquid inactivation: adding the harvested bacterium liquid into 10% formaldehyde solution to enable the final concentration to be 0.3%, and inactivating the bacterium liquid for 96 hours at the temperature of 4 ℃;

3) antigen liquid preparation: according to the counting result of the thalli, the inactivated YT-1 strain is adjusted to 12.5 hundred million/mL by using sterile PBS buffer solution;

4) preparing seedlings: introducing 95 parts of antigen liquid into an emulsifying tank, starting a motor to rotate and stir at a slow speed, simultaneously adding 5 parts of MONTANIDE GEL 02 adjuvant, stirring fully and uniformly mixing after adding.

According to the invention, pathogen separation, identification and purification are carried out on the cases of egg drop of the clinically laying hens and broiler breeders after artificial insemination to obtain a strain of duck-origin chicken bacilli, the screened bacteria are determined to be the duck-origin chicken bacilli through separation culture, specific PCR identification, 16s rRNA sequence analysis and animal regression tests, and the inactivated vaccine immune chicken flock prepared by the strain is used.

Drawings

FIG. 1: clinical symptom chart of sick chicken;

FIG. 2 is a photograph of the colonies under light microscopy after TSA, blood plating and gram staining;

FIG. 3: electrophoresis chart of PCR amplification result;

FIG. 4: a genetic evolution analysis diagram based on the gene sequence of the duck-origin chicken bacillus 16s rRNA, wherein black circles in the diagram represent experimentally separated duck-origin chicken bacillus YT-1 strains;

FIG. 5: a genetic evolution analysis diagram based on the amino acid sequence of the duck-origin chicken bacillus OmpA gene, wherein a black circle in the diagram represents a duck-origin chicken bacillus YT-1 strain separated in a test;

FIG. 6: an OmpA gene amino acid comparison diagram of the duck-origin chicken bacillus YT-1 strain and a reference strain;

FIG. 7 is a schematic view of: animal regression test charts;

FIG. 8: and (3) a diagram of the reproductive system lesion and the eggshell quality change of the sick chicken after attacking the duck source bacillus, wherein the eggs laid by the test groups 2 and 4 are arranged in the dotted line frame on the right side of the diagram A, and the eggs laid by the test groups 5 are arranged on the left side of the diagram A.

Detailed Description

The invention collects suspected samples of the sick breeding hens from Shandong, Liaoning and Henan, and determines the pathogeny causing the reduction of the egg yield and the reduction of the hatchability of the breeding hens after artificial insemination as duck-origin bacillus gallinarum through the separation and identification of viruses and bacteria and animal regression test, thereby laying a foundation for the deep research of the disease and having important significance for the prevention and the control of the disease.

The present invention will be described in detail below with reference to examples and the accompanying drawings.

Example 1: isolation of bacterial strains

1. Basic conditions are as follows: from 10 months to the present in 2019, egg breeders and broiler breeder farms in Shandong, Liaoning, Henan and Guangxi areas have a new infectious disease which is characterized by egg drop and poor eggshell quality after artificial insemination of laying chicken flocks. After the chicken flocks are infected, no obvious clinical symptoms generally exist, the laying chicken flocks, particularly the chicken flocks with the upper peak, can have a continuous material reduction phenomenon after being infected, the weight is different from 10g to 20g, the egg laying rate is reduced, the quality of the eggshell is deteriorated, white shell eggs and soft preserved eggs are increased (shown in figure 1-C), the process generally lasts for about 2 weeks, the recovery is started after the lowest point is reached and the egg laying rate is stabilized for several days, the feed intake is increased again, the egg laying rate is increased, but the level can not be increased before the disease onset, the process also lasts for about 2 weeks, and the whole course of disease is 5 weeks to 7 weeks. During the period, some sick chickens are listless, the eyes are closed and constricted (figure 1-E), the water-drawing sample is taken or the milk yellow thick and thin stool (figure 1-D), some chickens lay eggs and generally have no obvious death phenomenon, and some chickens die slightly more than normal due to improper management. The necroses of dead and sick chickens are mainly characterized by stripe necrosis of the liver with red and yellow phases, follicular rupture, ovum congestion, cyst of oviduct, yellow and white cheese-like secretion in oviduct (figure 1-A, B), and no obvious change of other organs.

2. Virus separation: aseptically collecting tissues of fallopian tube, follicle and liver of sick chicken, removing yolk in the follicle, cutting the follicle membrane, fallopian tube and liver into pieces, adding appropriate amount of normal saline, homogenizing with homogenizer, adding 1000U of double antibody, repeatedly freezing and thawing for 3 times, centrifuging at 4 deg.C and 8000 r/min for 15min, sucking supernatant, and filtering with 0.22 μm filter for sterilization. The filtrate is inoculated with 20 SPF chick embryos of 10 days old, 20 SPF duck embryos of 12 days old and 0.2 mL/embryo through an allantoic cavity. After inoculation, the eggs are placed in a biochemical incubator at the temperature of 37 ℃ for constant-temperature incubation. Discarding the dead chick embryos and duck embryos within 24h, continuously observing for 5d, harvesting chick embryo allantoic fluid and duck embryo allantoic fluid inoculated for 24h, and freezing and storing at-80 ℃ for later use. Blind passage 3 generations according to the above method.

3. And (3) separating culture detection of mycoplasma: aseptically taking oviduct and follicle of sick chicken, removing egg yolk in the follicle, washing with aseptic normal saline for several times, grinding oviduct and follicle membrane with autoclave sterilized mortar, adding appropriate amount of aseptic normal saline, fully vortexing, standing at 4 deg.C for 1h, collecting supernatant, filtering with 0.45 μm aseptic filter, sterilizing, inoculating 6-day-old SPF chick embryo with yolk sac, and culturing at 37 deg.C for 7 d. The dead chick embryos within 24h are discarded, and the chick embryo vitelline membranes inoculated for 24h are harvested. And continuously blind-transferring for 3 generations.

The result shows that 66 samples inoculated with SPF chick embryos and SPF duck embryos have no death phenomenon after blind transmission for 3 generations, have no abnormal embryo bodies, have no hemagglutination activity of allantoic fluid, and are not suspected to be like viruses; mycoplasma was not detected separately after yolk sac inoculation.

4. Separation, purification and preliminary identification of bacteria: opening the abdominal cavity of a sick chicken, dissecting an oviduct, dipping the wall surface of the oviduct tube and the surface of a diseased follicle by using an autoclaved cotton stick, immersing the dipped oviduct tube and the surface of the diseased follicle in a 2ml centrifuge tube filled with a proper amount of normal saline, aseptically picking up a cock testis, placing the cock testis around an alcohol lamp of an ultra-clean workbench, longitudinally cutting the testis by using a reddened blade, dipping the content of the testis in the aseptic cotton stick, immersing the testis in the 2ml centrifuge tube filled with a proper amount of normal saline, fully whirling the above samples, sucking 0.2ml of liquid on a TSA (TSA) plate of 3% horse serum, uniformly smearing the liquid on the TSA plate by using a plate coater, and culturing the mixture at 37 ℃ for overnight; and picking suspected colonies on a sheep blood agar plate, culturing overnight at 37 ℃, performing gram staining and microscopic examination, and inoculating the suspected colonies on the sheep blood agar plate again for further purification. And respectively inoculating the purified colonies to a MacconKa culture medium and an eosin methylene blue culture medium, and observing the growth conditions of the colonies.

5. And (4) screening results: 66 strains with consistent morphological characteristics are separated from 66 samples, most of the separated strains are planted in the oviduct and ovary of the laying hen, the semen of the sexually mature cock, the testis and the cloaca, and other organs such as liver, trachea, lung and the like are few, as shown in table 1. The isolated bacteria are in a needle point shape on a TSA culture medium, grow vigorously after 3% horse serum is added, and are small colonies which are round, grey white, semitransparent and neat in edge and have the diameter of 1-2 mm, as shown in a figure 2-A, and are in beta hemolysis on a sheep blood agar plate, as shown in a figure 2-B. The obtained product is observed under an optical microscope to be in a shape of a fine rod, and gram stain is negative, as shown in FIG. 2-C. No growth was observed on mecnkia agar plates and eosin methylene blue agar plates.

Table 1: strain separated from different organs in 66 samples

Note: the 33 hens and the cock were bred in cages for artificial insemination, "-" indicates that there was no hen

1 suspected duck source gallibacterium isolated from a tobacco station area is named as YT-1 strain, which is preserved in China center for type culture Collection in Wuhan in 7 months and 3 days of 2020, with the preservation number of CCTCC NO: m2020272.

Example 2: identification of YT-1 Strain

First, specific PCR identification

Using chicken bacillus conserved genes rpoB, 16s rRNA and 23s rRNA as identification primers (as shown in Table 2), and carrying out specific PCR identification on the YT-1 strain; then using chicken bacillus 16s rRNA gene (1461bp) and OmpA gene (1076bp) full-length primers to carry out PCR amplification on the YT-1 strain, and sending the target fragment obtained by amplification to the biological engineering (Shanghai) company Limited for sequencing.

Table 2: primer sequence Listing

The results show that the YT-1 strains can obtain 3 items of bands after PCR amplification: 560bp, 790bp, 1030bp or 1080bp, and primarily determining the YT-1 strain as the chicken bacillus. The positive strain was PCR-amplified for the full length of 16s rRNA gene (FIG. 3-B) and OmpA gene (FIG. 3-C), and the obtained target fragment was sequenced.

Second, 16s rRNA Gene analysis for bacterial Classification

Comparing and analyzing a 16s rRNA gene full-length sequence (SEQ ID NO:1) obtained by PCR amplification with a 16s rRNA gene sequence of gallibacterium anatis disclosed in NCBI by using MegAlign software; the genetic evolution analysis was performed with MEGA 6.0 in 1000 replicates of bootstrap, a Neighbor-join method.

The results show that the nucleotide homology of the 16s rRNA gene of the YT-1 strain of the allium vulgare and the strain of the allium vulgare published in NCBI is between 99.0% and 99.9% (Table 3), the 16s rRNA gene is closest to the homology of the reference strains in Henan, Denmark and the United states of China, and the allium vulgare branches are all in the evolutionary tree (figure 4), and the newly isolated bacterium YT-1 strain is the allium vulgare.

Table 3: homology comparison of duck-origin gallibacterium YT-1 strain and reference strain 16s rRNA gene nucleotide

Thirdly, gene analysis of outer membrane protein A (OmpA) of main protective antigen gene

After the OmpA gene full-length sequence obtained by PCR amplification is translated into an amino acid sequence (SEQ ID NO:2 and SEQ ID NO:3), the OmpA gene amino acid sequence of the duck-origin gallibacterium published in NCBI is compared and analyzed by using MegAlign software; the genetic evolution analysis was performed with MEGA 6.0 in 1000 replicates of bootstrap, a Neighbor-join method.

The results show that the nucleotide homology of the OmpA gene of the YT-1 strain of the duck-origin chicken bacillus and the OmpA gene of the duck-origin chicken bacillus strain published in NCBI is between 84.9 and 86.2 percent, and the amino acid homology is between 46.0 and 46.2 percent (Table 4 and figure 6), which shows that the homology of the OmpA gene of different strains at the nucleotide and amino acid levels is lower, and obvious strain difference is shown, and the amino acid genetic evolution analysis of the OmpA gene shows that the YT-1 strain is in a single evolutionary branch (figure 5).

TABLE 4 comparison of homology between Duck-origin Chicken Bacillus YT-1 strain and reference strain OmpA Gene

Example 3 animal regression test-establishment of challenge animal model

24 healthy Hailan brown laying hens aged 15 weeks are averagely divided into 3 groups and 8 groups, the groups are raised in a closed room which is provided with a ventilating duct and can provide filtered air, and a Hailan laying hen raising management scheme is strictly executed by illumination, ventilation, drinking water, feed and the like. 1 week before challenge, cloaca swab and throat swab samples of each chicken were collected for bacterial isolation, and all tested chickens were negative for gallibacteroides detection.

Chickens at 135 days of age began the test according to the test design of table 5 below, with 1 group undergoing one challenge, 2 groups undergoing two challenges, and 3 groups serving as controls. The chickens were observed daily for clinical abnormalities only, and each group of eggs was collected and counted at 17:00 pm daily. Collecting cloaca swabs and genital tract swabs (the depth of the sterile cotton swab is at least 6cm) of each group of chickens on 3 rd, 5 th and 7 th days after bacteria attack respectively for bacteria separation, wherein the experimental process lasts for 31 days, and all the chickens are killed, subjected to autopsy, sampled and subjected to bacteria separation after the experiment is finished.

TABLE 5 test design Table

The experiment is divided intoGroup of	Number of	Treatment of	Bacteria attack and dosage	Bacteria attack mode
					1	8	1 time of bacteria attack	G.anatis 8.66×10⁷cfu/200ul	Simulation of artificial insemination
2	8	2 times of bacteria attack	G.anatis 8.66×10⁷cfu/200ul	Simulation of artificial insemination
					3	8	—	—	—

Note: the artificial insemination is to perform insemination on the hens every 5 days, so that the high fertilization rate is ensured, and the hens need to perform artificial insemination for N times in a lifetime until the hens are eliminated.

In test 1, the feed intake speed of the chicken flocks is reduced from 3 days after tapping bacteria, the feed intake is reduced to 5 g/day at most (Table 6), the daily egg laying rate is reduced from 87.5-100% to about 75% from 23 days after tapping bacteria, the level is maintained (figure 7), the course of disease is 3-4 w, and the quality of the egg shells of the produced eggs is normal. The situation of the chicken group in the test 2 after primary bacteria attack is consistent with that of the chicken group in the test 1, the feed loss reaches the maximum 10 g/day in the 2 nd day after secondary bacteria attack, the severe egg drop phenomenon occurs from the 5 th day after secondary bacteria attack, the daily laying rate is reduced from 87.5-100% to 0-25%, some chickens are even dead, the daily laying rate starts to recover gradually with the passage of time but can not recover to the level before the disease, the final daily laying rate is about 50% and loitering (figure 7), the disease course is 4-5 w, the quality of eggs and eggshells of individual chickens in the middle and later stages of egg laying is poor, the skin is thin and fragile, and the situation is completely consistent with the actual disease situation on site. During the whole test, the control group of chickens was normal.

Animal regression experiments show that the duck-origin gallibacterium can cause the egg laying reduction of the artificially inseminated hens, which is completely consistent with the clinical morbidity situation, and the animal model is established, thereby laying a foundation for the evaluation of the subsequent duck-origin gallibacterium vaccine.

Table 6: clinical manifestations of various chicken groups after bacterial attack

EXAMPLE 4 preparation of vaccine and bacterial neutralization assay

1 preparation of the vaccine

1.1 culture and inactivation of bacterial solution

(1) The strain culture adopts fermentation tank to culture duck-origin chicken bacillus YT-1 strain and F149 strain (duck-origin chicken bacillus F149 strain (deposition number NCTC11413) purchased from Beijing Baiohobowei biotechnology limited, and strain obtained from American strain collection center with deposition number

43329^TM) Respectively culturing, adding TSB culture medium according to 60-80% of the volume of the fermentation tank, simultaneously adding antifoaming agent, introducing high pressure steam for sterilization, cooling to 37 deg.C, adding horse serum and duck source chicken bacillus strain, the final concentration of each component is respectively2.5 percent and 2.5 percent of the total weight of the mixture, and carrying out fermentation culture. Culturing at 37 deg.C, stirring at 100r/min, pH of 7.2-7.5, culturing for 5-6 hr, and sampling for pure inspection. And (5) centrifuging to obtain bacterial sludge. The number of bacterial sludge is measured by a turbidimetric method. And (5) storing at 2-8 ℃ for later use.

(2) Bacterial liquid inactivation according to the counting result of bacterial sludge, bacterial sludge is re-dissolved by using a proper amount of sterilized PBS buffer solution until the number of bacteria is 50.0 hundred million/mL, 10 percent formaldehyde solution is added to ensure that the final concentration is 0.3 percent, and inactivation is carried out for 96 hours at the temperature of 2-8 ℃. Sampling for inactivation test. Diluting the inactivated bacteria liquid with a proper amount of sterilized PBS buffer solution, sampling, counting thalli, and storing at 2-8 ℃ for later use.

1.2 inspection of semi-finished products

(1) Purely checking and taking the bacterial liquid before inactivation, checking according to the method in the appendix of the current Chinese veterinary pharmacopoeia, and carrying out purely checking.

(2) Inactivation test bacteria liquid before inactivation is taken, and is tested according to the method in the appendix of the current Chinese veterinary pharmacopoeia, and the bacteria liquid is required to grow aseptically.

(3) And (3) counting the bacteria, namely taking diluted inactivated bacteria liquid, and counting the bacteria according to a turbidimetric method, wherein the bacteria number of the duck-origin gallibacterium YT-1 strain and the F149 strain is not less than 12.5 hundred million/mL.

1.3 preparation of inactivated vaccine: and (5) preparing the vaccine by using the semi-finished antigen after the semi-finished antigen is qualified through inspection.

(1) Preparation of antigen solution according to the counting result of thalli, adjusting qualified YT-1 strain and F149 strain to 12.5 hundred million/mL by using sterile PBS buffer solution;

(2) and (3) introducing 95 parts of antigen liquid into an emulsifying tank, starting a motor to rotate and stir slowly, slowly adding 5 parts of MONTANIDE GEL 02 adjuvant, and stirring and mixing uniformly to obtain the vaccine.

(3) Subpackaging, quantitatively subpackaging, covering, sealing, sticking a label, and storing at 2-8 ℃.

1.4 vaccine product inspection

1.4.1 Properties

Appearance: a translucent white suspension with a little precipitate at the bottom.

1.4.2 sterility test: according to the appendix of the existing Chinese animal pharmacopoeia, the result conforms to the regulations.

1.4.3 safety inspection: no local and systemic adverse reactions caused by the vaccine occur. 10 SPF chickens and 10 healthy rabbits with the age of 28 days are injected with 1.0ml of vaccine subcutaneously in each neck, 5 controls are respectively arranged at the same time, the chickens and the rabbits are raised under the same condition, the feeding, drinking and clinical conditions of the test chickens and the rabbits are continuously observed for 14 days, and the feeding, drinking and clinical conditions of the test chickens and the rabbits are recorded. Any local and systemic adverse reactions caused by the vaccine should not occur.

The results show that the chicken and the rabbit in the test group and the control group develop normally and are in good mental state, and the vaccine at the injection part is well absorbed by the autopsy test group without inflammatory reactions such as red swelling, tissue necrosis and the like. The trial vaccine is safe and harmless and has no influence on the growth of animals.

1.4.4 efficacy test

25 healthy rabbits were prepared, 10 neck-derived chicken bacillus YT-1 vaccine were subcutaneously injected, and another 10 neck-derived chicken bacillus F149 vaccine were subcutaneously injected at 0.5 ml/rabbit, and 5 healthy rabbits of the same age were further collected without immunization as a control, and 28 days after immunization, blood was collected and serum was isolated, and antibodies were measured, respectively. The latex agglutination test for the immune group antibody should be "+ + + +" (indicating 100% agglutination) and the control group should be negative. Meanwhile, 25 newly born SPF hens, 10 immune duck-origin chicken bacillus YT-1 vaccines and 10 immune duck-origin chicken bacillus F149 vaccines are prepared, 0.5ml of each vaccine is obtained, 5 non-immune vaccines are taken as a control, and 0.2ml of duck-origin chicken bacillus YT-1 and F149 bacterial solutions (the living bacterial amount is 1.0 multiplied by 10) are used 28 days after immunization⁸CFU) simulating artificial insemination to attack bacteria through the reproductive tract, continuously attacking bacteria for 3 times every 5 days, and continuously observing for 30 days from the last time of bacteria attack; the control chicken had disease, and the immunized chicken had no disease.

2. Bacterial cross-neutralization assay

Preparing inactivated vaccines from the duck-origin chicken bacillus YT-1 strain and the F149 strain according to the method, immunizing healthy rabbits respectively, immunizing at an immune dose of 0.5 ml/rabbit, immunizing once every 2 weeks, immunizing for 3 times in total, collecting blood and separating serum after the three-time immunization for 2 weeks, and performing antigen-antibody cross neutralization test by using the duck-origin chicken bacillus YT-1 strain and the F149 strain latex agglutination diagnostic antigen. As a result, it was found that YT-1 diagnostic antigen and YT-1 positive serum produced distinct latex agglutinate particles, and F149 diagnostic antigen and F149 positive serum produced distinct latex agglutinate particles, but the agglutination degree of YT-1 diagnostic antigen and F149 positive serum, and F149 diagnostic antigen and YT-1 positive serum was significantly low, indicating that different strains have strong neutralizing ability to themselves, but have poor neutralizing ability to each other (Table 7).

Table 7: serum cross-neutralization test result table of duck-origin gallibacterium YT-1 strain and F149 strain

Note: denotes a virus; the R value is more than 0.8, the R value is different subtypes of the same serotype and is between 0.25 and 0.8, and the R value is less than 0.25, and the R value is different serotypes.

3. Challenge protection test

50 healthy Hailan brown laying hens aged 10 weeks are averagely divided into two groups, 10 laying hens per group, the laying hens are raised in a closed room which is provided with a ventilating duct and can provide filtered air, and a Hailan laying hen raising management scheme is strictly executed by illumination, ventilation, drinking water, feed and the like. 3 days before immunization, cloaca swab and throat swab samples of each chicken were collected for bacterial isolation, and all test chicken were negative for detection of gallibacteroides. The inactivated vaccines prepared from the duck-origin chicken bacillus YT-1 strain and F149 strain were used for immunization according to the experimental design of Table 8.

The chickens were initially tested at 135 days of age according to the test design of table 8 below, with 1 and 2 being immune YT-1, 3 and 4 being immune F149, and 5 being normal controls. The chickens were observed daily for clinical abnormalities only, and eggs from each group were collected and counted at 17:00 pm daily. Cloaca swabs and genital tract swabs (the depth of the sterile cotton swab is at least 6cm) of each group of chickens are collected on 3 rd, 5 th, 7 th, 11 th and 28 th days after bacteria attack respectively for bacteria separation, the experimental process lasts for 30 days totally, and all chickens are killed, necropsied, sampled and subjected to bacteria separation after the experiment is finished.

Table 8 experimental design

The result of the challenge protection test shows that all of the chickens in 1 group and 4 groups are normal in the whole experimental process, have no abnormal clinical symptoms, have normal laying rate and normal egg quality, are consistent with the normal control chickens in 5 groups, and do not need to discharge bacteria from cloacal swabs and genital tract swabs from 3 days to 28 days after bacteria attack; the chickens in groups 2 and 3 begin to attack diseases on the 5 th day after secondary bacteria attack, the feed intake speed is reduced firstly, the feed quantity is reduced, the laying rate is reduced rapidly, the reduction amplitude reaches more than 70%, some hens even have a phenomenon of dead birth, during the period, individual hens are depressed and thin, the quality of produced eggs is poor, the skins are thin, the quality is crisp and fragile, the egg quality is completely consistent with the natural disease condition, the cloacal swabs and the genital tract swabs can be separated from the 3 rd day to the 28 th day after bacteria attack, and the gene sequencing results are the duck-origin chicken bacillus YT-1 strain and the F149 strain. The tests show that the duck-origin chicken bacillus YT-1 strain and F149 strain inactivated vaccine immune chicken flocks only can play a good immune protection role on the strains, and do not have cross protection capability on each other.

Example 5 clinical application of Duck-origin Chicken bacillus YT-1 strain inactivated vaccine

The inactivated vaccines of the duck-origin gallibacterium YT-1 strain and the F149 strain are prepared by the method of the embodiment 3 by respectively taking the YT-1 strain and the F149 strain as vaccine seeds.

The method comprises the steps of averagely dividing 40 healthy Hailan brown laying hens aged 22 weeks into 5 groups and 8 groups, feeding the Hailan brown laying hens in a closed room which is provided with a ventilating duct and can provide filtered air, and strictly executing a Hailan laying hen feeding management scheme according to illumination, ventilation, drinking water, feed and the like. 1 week before challenge, cloaca swab and throat swab samples were collected from each chicken for bacterial isolation, and all test chicken were negative for detection of gallibacterium.

The chickens at 135 days old begin to test according to the test design in the following table 9, 1 group and 2 groups of immune YT-1 strain inactivated vaccines and 3 groups and 4 groups of immune F149 strain inactivated vaccines are subjected to twice bacterial attack as required, and 5 groups are used as a control. The chickens were observed daily for clinical abnormalities only, and eggs from each group were collected and counted at 17:00 pm daily. Collecting cloaca swabs and genital tract swabs (the depth of the sterile cotton swab is at least 6cm) of each group of chickens on 3 rd, 5 th and 7 th days after bacteria attack respectively for bacteria separation, wherein the experimental process lasts for 31 days, and all the chickens are killed, subjected to autopsy, sampled and subjected to bacteria separation after the experiment is finished.

Table 9: design of experiments

The results show that the material loss reaches the maximum 10 g/day on the 2 nd day after the secondary attack of the 2 groups and the 4 groups in the experiment, the duration is 4 weeks to 5 weeks, then the slow recovery is started, meanwhile, the wilting phenomenon starts to occur on the 5 th day after the secondary attack, individual chickens pull yellow foam-like thin manure, inflammatory secretion flows out of the reproductive tract of the chickens, and the situation is completely consistent with the actual disease occurrence situation on site and the animal regression experiment situation; in the whole test process, the test 1 group and the test 3 group have no abnormality; the control group had only normal chow.

The experiment 2 and 4 groups have severe egg drop phenomenon from the 5 th day after secondary bacteria attack, the daily laying rate is reduced from 87.5-100% to 0-25%, some chickens are even out of stock, the daily laying rate starts to recover gradually with the passage of time but cannot recover to the level before the disease, the final daily laying rate is about 50% and then wanders, the quality of egg shells of the eggs laid by individual chickens in the middle and later stages of egg laying is poor, the skins of the eggs are thin and fragile, as shown in fig. 8-A, the egg white and the egg yolk of most of eggs are normal, and the egg white of only a few eggs is thin; the test 1, test 3 and control groups were normal.

A large amount of duck-origin chicken bacilli with single colony morphology can be separated from genital tract swabs 3, 5 and 7 days after tapping bacteria in the test group 2 and the test group 4, and the duck-origin chicken bacilli in the test group 1 and the test group 3 are negative. After the test is finished, the test chickens are subjected to autopsy, and the chickens in the test groups 2 and 4 are found to have reproductive system lesions of different degrees, have the defects of follicular congestion, deformation and liquefaction, have the yolk falling into the abdominal cavity after rupture to form peritonitis, have the oviduct cyst, the effusion and the white cheese inside as shown in figures 8-B and 8-C, are consistent with the change of the on-site autopsy, have no abnormality in other organs, and have no abnormality in the test groups 1, 3 and the control group.

The post-immunization vaccine challenge protection test result shows that the prepared duck-origin chicken bacillus YT-1 strain inactivated vaccine can play a good protection role in epidemic groups of the duck-origin chicken bacillus YT-1 strains; the prepared duck source chicken bacillus F149 inactivated vaccine can play a good role in protecting epidemic duck source chicken bacillus F149 epidemic groups, but cannot play a role in protecting YT-1 epidemic groups.

In conclusion, vaccines of the two strains cannot protect each other, and in view of the fact that the breeding hens in artificial insemination in China at present mainly infect epidemic groups represented by duck-origin chicken bacillus YT-1 strains, the inactivated vaccine prepared by the duck-origin chicken bacillus YT-1 strains attacks the viruses after immunization of the breeding hens, does not drop eggs, does not expel toxins, does not have abnormal clinical symptoms, and can be used for preventing the problems of egg drop and the like caused by the bacteria clinically.

In conclusion, the inactivated vaccine of the duck-origin chicken bacillus YT-1 strain is the first vaccine aiming at egg laying reduction caused by the duck-origin chicken bacillus in China, and has great significance for the healthy development of the chicken industry in China.

Sequence listing

<110> Qingdao Yibang bioengineering Co., Ltd

<120> duck-origin gallibacterium and application thereof

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1461

<212> DNA

<213> Yayuan Chicken (Gallibacterium anatis)

<400> 1

attgaacgct ggcggcaggc ttaacacatg caagtcgaac ggtaacgggt tgaaagcttg 60

ctttcaatgc tgacgagtgg cggacgggtg agtaaggctt ggggatctgg cttttggagg 120

gggataacca ttggaaacga tggctaatac cgcatagtat cgagagatga aaggggtggg 180

aaaccacttg ccaagggatg aaccctagtg agattaggta gttggtgggg taaaggccta 240

ccaagccgac gatctctagc tggtctgaga ggatggccag ccacactggg actgagacac 300

ggcccagact cctacgggag gcagcagtgg ggaatattgc gcaatggggg gaaccctgac 360

gcagccatgc cgcgtggatg aagaaggcct tcgggttgta aagttctttc ggtggtcagg 420

aaggttagga tgttaatagc atactgattt gacgttagcc acagaagaag caccggctaa 480

ctccgtgcca gcagccgcgg taatacggag ggtgggagcg ttaatcggaa taactgggcg 540

taaagggcac gcaggcggga cgttaagtga gatgtgaaag ccccgggctt aacctgggaa 600

cagcatttca tactggcgta ctagagtact ttagggaggg gtagaattcc acgtgtagcg 660

gtgaaatgcg tagagatgtg gaggaatacc gaaggcgaag gcagcccctt gggaagatac 720

tgacgctcat gtgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc 780

tgtaaacgct gtcgatttgg gggttgtacg aaaggtatgg ctctcgaagc aaacgtgata 840

aatcgaccgc ctggggagta cggccgcaag gttaaaactc aaatgaattg acgggggccc 900

gcacaagcgg tggagcatgt ggtttaattc gatgcaacgc gaagaacctt acctactctt 960

gacatcctaa ggagaagcta gagatagctt tgtgctttcg agaacttaga gacaggtgct 1020

gcatggctgt cgtcagctcg tgttgtgaaa tgttgggtta agtcccgcaa cgagcgcaac 1080

ccttattctt tgttaccagc gggtaaagcc ggggactcaa aggagactgc cagtgacaag 1140

ctggaggaag gtggggatga cgtcaagtca tcatggccct tacgagtagg gctacacacg 1200

tgctacaatg gtgcatacag agggaggcga agtagcgata cggagcgaaa ctcagaaagt 1260

gcatcgtagt ccggattgga gtctgcaact cgactccatg aagtcggaat cgctagtaat 1320

cgcgaatcag aatgtcgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380

catgggagtg ggttgtacca gaagtagata gcttaacctt attggagggc gtttaccacg 1440

gtatgattca tgactggggt g 1461

<210> 2

<211> 1080

<212> DNA

<213> Yayuan Chicken (Gallibacterium anatis)

<400> 2

atgaaaaaga ctgcaatcgc attagcaatt tcaggattag cattcgtatc agtaccacaa 60

gcagctcctc aagcgaacac tttctacggt ggtgcggaag ctggttgggg ttgtatttgt 120

aatgatgtaa atcaatttga tgctgaaaaa aatgatggta ttggttatgg tggtcatcgc 180

aactcagtaa cttacggtgt attcggtggc ttgtaaatga ctgataactt cgctgttgaa 240

aaaaccagta acttctttgg ctagttaaaa gtggacgatg gtgatactga acgtgctggt 300

cacactgctc acggcttaaa tcttagctaa aaagcaagct ccagatgatt ttatgttgac 360

ggtttagacg tttacggtcg ttcaggtgca gcattaatcc gttctgatta cacaacgcat 420

gtagacggta tcagtaccta ttctgatcat aaattaaaag tatctcctgt attcgcaggc 480

ggtgttgaat ttgcaattct tcctgagttg gcagcacgtt tagaatacca atggattgct 540

aaagtaggta ccaaaggcgc ggtgttgaat ttgcaattct tcctgagttg gcagcacgtt 600

tagaatacca atggattgct aaagtaggta ccaaaggcgc tttcggtcaa tcagcagctc 660

ctgcgcccgt tgttgaattt gttaacaaaa aattaaataa gcaatctgat gtattgtacg 720

cattcggtaa agcaaactta aaacctgaag cagcacaatc attagataat ttacaacaag 780

aaatctctaa agttggttca ttatcttctg ttgaagttgc aggttacaca gaccgtgttg 840

gttctgacaa atataaggca aaacttacac aagaacgtgc taacacagat gctaactact 900

tagtttctga atcattatct ccagatgtga ttatagcagt tggttacggt gaagctaacc 960

cagtaactgg taacacttgt gatgcagtta aaggtcgtaa agcgtgaatc gcttgttaag 1020

caccagaccg tcgtgttgaa gtttctggaa gtggtcaaca ccaacgaaca atcgaccaat 1080

<210> 3

<211> 360

<212> PRT

<213> Yayuan Chicken (Gallibacterium anatis)

<400> 3

Met Lys Lys Thr Ala Ile Ala Leu Ala Ile Ser Gly Leu Ala Phe Val

1 5 10 15

Ser Val Ala Gln Ala Ala Pro Gln Ala Asn Thr Phe Tyr Ala Gly Ala

20 25 30

Lys Ala Gly Trp Ala Ser Phe His Asn Asp Val Asn Gln Phe Asp Ala

35 40 45

Glu Lys Asn Asp Gly Ile Gly Tyr Gly Val His Arg Asn Ser Val Thr

50 55 60

Tyr Gly Val Phe Gly Gly Tyr Gln Ile Thr Asp Asn Phe Ala Val Glu

65 70 75 80

Ala Gly Tyr Asp Phe Tyr Gly Gln Leu Lys Met His Asp Gly Asp Thr

85 90 95

Glu Arg Ala Arg His Thr Ala His Gly Leu Asn Leu Ser Leu Lys Ala

100 105 110

Ser Tyr Pro Val Ala Tyr Val Asp Gly Leu Asp Val Tyr Gly Arg Leu

115 120 125

Gly Ala Ala Leu Ile Arg Ser Asp Tyr Thr Thr His Val Asp Gly Glu

130 135 140

Lys Val Ala Ser Asp His Lys Leu Lys Val Ser Pro Val Phe Ala Ala

145 150 155 160

Gly Val Glu Tyr Ala Ile Leu Pro Glu Leu Ala Ala Arg Leu Glu Tyr

165 170 175

Gln Trp Ile Ala Lys Val Gly Lys Val Asp Gln Ile Ser Asn Glu Asn

180 185 190

Ser Ala Phe Ala Gly Ser Ser Tyr Ser Pro Ser Ile Gly Ser Val Ser

195 200 205

Leu Gly Leu Ser Tyr Arg Phe Gly Gln Ser Ala Ala Pro Ala Pro Val

210 215 220

Val Glu Val Val Asn Lys Lys Phe Ala Leu Ser Ser Asp Val Leu Phe

225 230 235 240

Ala Phe Gly Lys Ala Asn Leu Lys Pro Glu Ala Ala Gln Ser Leu Asp

245 250 255

Asn Leu Gln Gln Glu Ile Ser Lys Val Gly Ser Leu Ser Ser Val Glu

260 265 270

Val Ala Gly Tyr Thr Asp Arg Ile Gly Ser Asp Lys Tyr Asn Gln Lys

275 280 285

Leu Ser Gln Glu Arg Ala Asn Thr Val Ala Asn Tyr Leu Val Ser Lys

290 295 300

Ser Ile Ser Pro Asp Val Ile Lys Ala Val Gly Tyr Gly Glu Ala Asn

305 310 315 320

Pro Val Thr Gly Asn Thr Cys Asp Ala Val Lys Gly Arg Lys Ala Leu

325 330 335

Ile Ala Cys Leu Ala Pro Asp Arg Arg Val Glu Val Ser Val Ser Gly

340 345 350

Gln His Gln Gln Thr Ile Glu Gln

355 360

Claims

1. The duck-origin chicken bacillus is characterized in that the preservation number of the duck-origin chicken bacillus (gallibacteroides) is CCTCC NO: m2020272.

2. The use of the gallibacterium anatipestifer of claim 1 in the preparation of a vaccine.

3. The use of claim 2, wherein the vaccine is an inactivated vaccine.

4. An inactivated vaccine of duck-origin gallibacterium, characterized in that the inactivated vaccine comprises an antigen and a vaccine adjuvant, wherein the antigen comprises the inactivated gallibacterium anatum of claim 1.

5. The inactivated vaccine of claim 4, wherein the gallibacterium anatipestifer is inactivated using a formaldehyde solution.

6. The inactivated vaccine of claim 4, wherein the vaccine adjuvant is an aqueous phase adjuvant.