CN118267459A - Trivalent inactivated vaccine for infectious rhinitis of chicken and preparation method thereof - Google Patents
Trivalent inactivated vaccine for infectious rhinitis of chicken and preparation method thereof Download PDFInfo
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- CN118267459A CN118267459A CN202310632694.XA CN202310632694A CN118267459A CN 118267459 A CN118267459 A CN 118267459A CN 202310632694 A CN202310632694 A CN 202310632694A CN 118267459 A CN118267459 A CN 118267459A
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Abstract
The invention discloses a trivalent inactivated vaccine for infectious rhinitis of chickens and a preparation method thereof, and belongs to the field of biological products for animals. The invention aims to solve the technical problems that: how to effectively prevent infectious rhinitis of chickens. In order to solve the technical problem, the invention provides a trivalent inactivated vaccine for infectious rhinitis of chickens, which comprises inactivated A serotype paragallibacterium, inactivated B serotype paragallibacterium and inactivated C serotype paragallibacterium. The vaccine provided by the invention can effectively prevent infectious rhinitis of chickens caused by A-type, B-type and C-type avian bacillus paragallinarum.
Description
Technical Field
The invention belongs to the field of biological products for animals, relates to the technical field of veterinary vaccines, and in particular relates to a trivalent inactivated vaccine for infectious rhinitis of chickens and a preparation method thereof.
Background
Infectious rhinitis (Infectious coryza, IC) is an acute respiratory infectious disease in chickens caused by avibacterium paragallinarum (Avibacterium paragallinarum, a. Paragallinarum). The disease can occur in the bred chicken and laying hen groups, the economic loss caused by the disease comprises the growth stagnation of chicken, the increase of elimination rate, the significant reduction of egg yield (10% -40%), 70% of the reduction of the egg yield in the onset of the peak period, and once the disease occurs, the quality of the egg is reduced and the egg cannot be reserved for breeding. The grown chickens are bad in growth, the feed intake is reduced by 10% -50%, the diseases are difficult to radically cure, the diseases are easy to recur, the medicine cost is increased, and serious economic loss is caused for poultry farmers.
The clinical symptoms of infectious rhinitis in chickens are mainly mucous secretion outflow from the nasal cavity, facial edema, conjunctivitis, increased tear secretion and sometimes swelling of the meat drop of the cock. Diarrhea, feeding and water consumption of the sick chickens can be reduced, and egg production of the laying hens is reduced. The onset is characterized by short latency and rapid onset. High morbidity but low mortality, which can be increased by secondary or mixed infections with other pathogens.
As early as 1920, beacon reported first infectious rhinitis in chickens, which was isolated for the first time in 1932 De Blieck and named bacillus haemophilus parasuis for a long time. Blaclall has been renamed as avian paragallinarum in 2005. According to the antigenic structure, page first uses a slide agglutination reaction to divide avian paragallinarum into A, B, C serotypes.
The development of the chicken raising industry in China is rapid, the intensive raising degree is rapidly increased, centralized management of chicken farms is realized in most areas, the chicken farms are high in raising density, and the severe bacterial disease infection pressure is faced. Therefore, the research on the domestic epidemic strain vaccine is particularly important for preventing and controlling infectious rhinitis of chickens.
Disclosure of Invention
The application aims to solve the technical problems that: how to effectively prevent infectious rhinitis of chickens.
In order to solve the technical problem, in a first aspect, the application provides a trivalent inactivated vaccine for infectious rhinitis of chickens, wherein the trivalent inactivated vaccine for infectious rhinitis of chickens comprises active components of inactivated A serotype parachicken bacillus, inactivated B serotype parachicken bacillus and inactivated C serotype parachicken bacillus.
Further, in the trivalent inactivated vaccine for infectious coryza of chicken, the a serotype parachicken bacillus is a serotype parachicken bacillus cctccc M20221926, the a serotype parachicken bacillus cctccc M20221926 is a serotype parachicken bacillus preserved in the China center for type culture collection, and a preservation number is: cctccc M20221926.
Further, in the trivalent inactivated vaccine for infectious coryza of chicken, the B serotype parachicken bacillus is an inactivated B serotype parachicken bacillus cctccc M20221927, the B serotype parachicken bacillus cctccc M20221927 is a B serotype parachicken bacillus preserved in the China center for type culture collection, and a preservation number is: cctccc M20221927.
Further, in the trivalent inactivated vaccine for infectious coryza of chicken, the C-serotype parachicken bacillus is an inactivated C-serotype parachicken bacillus cctccc M20221928, and the C-serotype parachicken bacillus cctccc M20221928 is a C-serotype parachicken bacillus preserved in the China center for type culture collection, and the preservation number is: cctccc M20221928.
Further, in the trivalent inactivated vaccine for infectious coryza of chicken, the ratio of the A serotype paragallinarum bacillus, the B serotype paragallinarum bacillus and the C serotype paragallinarum bacillus is 1:2:3 (counted by colony forming units before inactivation).
Further, in the trivalent inactivated vaccine for infectious coryza of chicken, the trivalent inactivated vaccine for infectious coryza of chicken consists of the active ingredient and an adjuvant.
The adjuvant may be an aqueous adjuvant or/and an oily adjuvant.
Such oily adjuvants include, but are not limited to, white oil, squalane or squalene, drek oil, and other animal, vegetable or mineral oils. The above-mentioned oily adjuvant may be either of natural origin or obtained by artificial synthesis.
The vaccine further comprises a suspending agent, a surfactant, an antigen inactivating agent, or a preservative. The suspending agent may include, for example, aluminum stearate, as well as other suspending agents available in the art.
The surfactant may include, for example, sorbitan monooleate (TWEE series), SPAN (SPAN), and other surfactants useful in the art. Such antigen inactivating agents include, but are not limited to, for example, formalin, beta-propiolactone, and the like. The preservative includes, for example, thimerosal. Methods of using and amounts of such materials are well known to those skilled in the art. Further, the preparation method of the trivalent inactivated vaccine for infectious rhinitis of chicken comprises the following steps:
S1) respectively culturing the A-serotype parachicken bacillus CCTCC M20221926, the B-serotype parachicken bacillus CCTCC M20221927 and the C-serotype parachicken bacillus CCTCC M20221928 to obtain three kinds of culture solutions of the parachicken bacillus;
s2) inactivating the three secondary chicken avian bacteria culture fluid prepared in the step S1) to obtain an inactivated secondary chicken avian bacteria culture fluid;
S3) mixing the inactivated three secondary chicken avian bacillus culture fluids prepared in the step S2) with an adjuvant to obtain the trivalent inactivated vaccine for the infectious rhinitis of the chicken.
In a second aspect, the application provides a preparation method of the trivalent inactivated vaccine for infectious rhinitis of chicken, which comprises the following steps:
S1) respectively culturing the A-serotype parachicken bacillus CCTCC M20221926, the B-serotype parachicken bacillus CCTCC M20221927 and the C-serotype parachicken bacillus CCTCC M20221928 to obtain three kinds of culture solutions of the parachicken bacillus;
s2) inactivating the three secondary chicken avian bacteria culture fluid prepared in the step S1) to obtain an inactivated secondary chicken avian bacteria culture fluid;
S3) mixing the inactivated three secondary chicken avian bacillus culture fluids prepared in the step S2) with an adjuvant to obtain the trivalent inactivated vaccine for the infectious rhinitis of the chicken.
In a third aspect, the application provides a avian secondary bacillus selected from any one, two or three of the following:
a1 A) the a serotype avibacterium paragallinarum cctccc M20221926;
A2 B serotype parachicken bacillus cctccc M20221927;
A3 C serotype avibacterium paragallinarum cctccc M20221928).
In a fourth aspect, the application provides an application of the avian bacillus paragallinarum in preparing infectious rhinitis vaccine.
The beneficial technical effects obtained by the application are as follows:
The trivalent vaccine for the infectious rhinitis of the chicken has an immune effect and safety, and has a good protection effect on the current epidemic strains. Provides experimental basis and solution for preventing and treating infectious rhinitis of chickens. The application also grasps related technologies such as separation and identification of the avian bacterial strain of parachicken, fermentation tank culture process, vaccine preparation, immune effect evaluation and the like, and lays a foundation for further exploring etiology and prevention and control of the disease.
Preservation description
Strain name: a serotype parachicken bacillus
Latin name: avibacterium paragallinarum A
Strain number: ZZ01
Preservation mechanism: china center for type culture Collection
The preservation organization is abbreviated as: CCTCC (cctccc)
Address: chinese university of armed chinese, postal code: 430072
Preservation date: 2022, 12 months and 12 days
Accession numbers of the preservation center: cctccc M20221926;
strain name: b serotype avibacterium paragallinarum
Latin name: avibacterium paragallinarum A
Strain number: HX05
Preservation mechanism: china center for type culture Collection
The preservation organization is abbreviated as: CCTCC (cctccc)
Address: chinese university of armed chinese, postal code: 430072
Preservation date: 2022, 12 months and 12 days
Accession numbers of the preservation center: cctccc M20221927;
strain name: c serotype parachicken bacillus
Latin name: avibacterium paragallinarum A
Strain number: ZN02
Preservation mechanism: china center for type culture Collection
The preservation organization is abbreviated as: CCTCC (cctccc)
Address: chinese university of armed chinese, postal code: 430072
Preservation date: 2022, 12 months and 12 days
Accession numbers of the preservation center: cctccc M20221928.
Drawings
FIG. 1 shows the result of gram staining of an avian secondary strain isolate;
FIG. 2 shows satellite phenomenon results of avian secondary strains;
FIG. 3 shows the result of PCR detection and identification of avian bacillus paragallinarum;
FIG. 4 shows the results of an indigo-blue test of avian secondary strain isolates;
FIG. 5 shows the results of a hydrogen peroxide test of avian paragallinarum isolates;
FIG. 6 shows the results of a hydrogen sulfide test on avian secondary strain isolates;
FIG. 7 shows the results of a glucose fermentation test of an avian secondary strain isolate;
FIG. 8 shows the results of a lactose fermentation test of the avian secondary strain;
FIG. 9 shows the results of a fermentation test of avibacterium paragallinarum isolate sucrose;
FIG. 10 shows the results of a nitrate reduction test of avian secondary strain isolates;
FIG. 11 shows the results of a urease assay of avian secondary strain isolates;
FIG. 12 shows the results of clinical symptoms of SPF-infected chickens with avian bacteria of Paramygdalin;
FIG. 13 is a Hemagglutination (HA) assay of avian Paramygdalina;
FIG. 14 is a hemagglutination inhibition assay (identification of the serotype of avian paragallinarum);
FIG. 15 shows pathological anatomy of SPF-infected chickens with avian bacteria from Paramygdalin.
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The study was carried out by separating 10 strains of bacteria from the infraorbital sinus of suspected chicken infectious rhinitis disease chickens in Hangzhou Zhejiang, jinhua Zhejiang, anhua Xuancheng, anhui Huangshan, zhejiang Zhoushan, hangzhou Xiaoshan, jiangsu Changzhou and Zhejiang Ningbo and the like through a laboratory of 5 months 2016 to 2017, and determining the bacterial strain as a secondary chicken avian bacillus through morphological, satellite phenomenon test and PCR identification detection, and further carrying out typing by utilizing a serum plate agglutination test, and the result is that: type 3 strain, type 4 strain, type 3 strain. Further determining the A type ZZ01 strain, the B type HX05 strain and the C type ZN02 strain of the production and test strains through a virulence test, culturing the strains in a proper culture medium, collecting bacterial liquid for pure test, further concentrating and purifying, emulsifying an antigen and an oil adjuvant according to a certain proportion to prepare a vaccine, and carrying out animal challenge-protection experiments, safety tests and minimum immune dose tests to prove that the safety and immune effects are obvious. The vaccine developed therefore contains 3 serotypes of strains that are now prevalent in chicken infectious rhinitis to prevent it.
Some reagent formulas mentioned in the technical scheme of the invention:
1. TSA flat plate
According to the requirements of the culture medium specification, weighing TSA culture medium into a triangular flask, adding distilled water to a specified scale, fully stirring, adjusting the pH value to 7.2, and sterilizing at 121 ℃ for 20 minutes. When the culture medium is cooled to about 50 ℃, chicken serum with the final concentration of 5-10% and NADH with the final concentration of 0.001% are added, evenly mixed, poured into a sterilization plate, and the thickness of the plate is about 4mm. Cooling and solidifying, making marks, packaging with newspaper or self-sealing bags, and storing at 2-8deg.C for use. Wherein TSA medium was purchased from Qingdao sea Bo biotechnology Co.
2. Blood agar plate
According to the requirements of the culture medium specification, weighing the nutrient agar culture medium into a triangular flask, adding distilled water to a specified scale, fully stirring, adjusting the pH value to 7.2, and sterilizing at 121 ℃ for 20 minutes. And (3) adding defibrinated sheep blood with a final concentration of 5% when the culture medium is cooled to about 50 ℃, uniformly mixing, and pouring into a sterilization plate with a thickness of about 4mm. Cooling and solidifying, making marks, packaging with newspaper or self-sealing bags, and storing at 2-8deg.C for use. Wherein the nutrient agar medium is purchased from Qingdao sea Bo biotechnology Co.
3. TSB medium
According to the requirements of the culture medium specification, weighing TSB culture medium into a triangular flask, adding distilled water to a specified scale, fully stirring, adjusting the pH value to 7.2, and sterilizing at 121 ℃ for 20 minutes. After the culture medium is cooled, chicken serum with the final concentration of 5-10% and NADH with the final concentration of 0.001% are added, evenly mixed and kept at 2-8 ℃ for standby. Wherein TSB medium was purchased from Qingdao sea Bo biotechnology Co.
4. Fermentation medium
4.1, Self-partitioning fermentation Medium
5G of polypeptone, 5g of casein peptone, 5g of sodium glutamate, 5g of sodium chloride, 3g of yeast powder and distilled water to 1000mL. Adjusting the pH value to 7.2-7.4, sterilizing for 20 minutes at 121 ℃, and preserving at room temperature for standby. Before use, glucose with the final concentration of 0.1 percent, chicken serum with the final concentration of 5 to 10 percent and NADH with the final concentration of 0.003 percent are added and fully and uniformly mixed.
4.2 Commercial fermentation Medium
Weighing commercial fermentation culture medium according to the requirements of the culture medium specification, adding distilled water to a specified scale, fully stirring, adjusting the pH value to 7.2-7.4, sterilizing at 121 ℃ for 20 minutes, and preserving at room temperature for later use. Glucose with the final concentration of 0.1 percent, chicken serum with the final concentration of 5 to 10 percent and NADH with the final concentration of 0.003 percent are added before use and fully and uniformly mixed. Wherein the commercial fermentation medium is purchased from Qingdao sea Bo biotechnology Co.
4.3 Fermentation Medium containing 1.5% agar
Preparing a liquid fermentation medium according to 4.1 or 4.2, adding 1.5% agar, adjusting pH to 7.2-7.4, and sterilizing at 121deg.C for 20 min. When the culture medium is cooled to about 50 ℃, chicken serum with the final concentration of 5-10% and NADH with the final concentration of 0.001% are added and fully and uniformly mixed. Pouring the mixture into a sterilization plate or a test tube, solidifying, packaging, and storing at 2-8 ℃ for later use.
5. Feed medium
10G of glucose is weighed, 60mL of distilled water is added for complete dissolution, then 100mL of distilled water is added, sterilization is carried out for 30 minutes at 116 ℃, and the mixture is preserved at room temperature for standby.
Wherein chicken serum was purchased from beijing solebone technologies limited;
NADH was purchased from Shanghai Biotechnology Co., ltd;
Polypeptone was purchased from Qingdao sea Bo biotechnology Co., ltd;
casein was purchased from Qingdao sea Bo biotechnology Co., ltd;
Sodium glutamate was purchased from beijing solebao technologies limited;
sodium chloride was purchased from national pharmaceutical group chemical company, inc;
yeast powder was purchased from OXOID company, uk;
tween-80 was purchased from Beijing Yili pharmaceutical Co., ltd;
white oil for injection in the following examples was purchased from Zhejiang zheng arsenicum oil technologies limited;
aluminum stearate is purchased from Shanghai long distance reagent factories;
Span-80 was purchased from Shanghai Shen Yu pharmaceutical chemical Co., ltd;
The chickens used in the examples below were SPF grade white legend obtained from the self-incubation of hatching eggs from Experimental animal technology, inc. of Beijing Meili.
EXAMPLE 1 isolation and identification of avian Proteus species
1.1 Sources of disease Material
The study was taken from the infraorbital sinus of suspected chicken infectious rhinitis disease chickens in the areas of Hangzhou Zhejiang, jinhua Zhejiang, xuancheng Anhua, huangshan Anhui, zhejiang Zhoushan, hangzhou Xiaoshan, jiangsu Changzhou and Zhejiang Ningbo, etc., by laboratory at 5 months 2016 to 7 months 2017, respectively. After the head of the dead chicken is removed or the infraorbital Dou Mian swab is directly prepared, the dead chicken is refrigerated and sent to a laboratory for the separation and identification of nasal transfer clinical isolates.
1.2 Bacterial isolation and morphology
After the dead chicken head is disinfected, the infraorbital sinus is cut off under the aseptic condition, liquid in the infraorbital sinus is taken out by an inoculating loop which is sterilized by flame and cooled, or a Dou Mian swab is streaked on a TSA flat plate, a 5% -10% CO 2 incubator is used for culturing for 24-48 hours at 37 ℃, round, smooth, gray white and semitransparent dew-like colony streaks with the diameter of about 0.3mm are selected and inoculated on a TSB flat plate, a 5% CO 2 incubator is used for culturing for 24-48 hours at 37 ℃, and the colony morphology is observed and gram staining microscopic examination is carried out. The microscopic examination results are shown in FIG. 1. In FIG. 1, strain ZZ01, HX05 and ZN02 are shown as 1-3, respectively, and the results in FIG. 1 show that: the gram staining of 3 strains was in the form of a club or a small rod, which was gram-negative bacillus.
1.3 Satellite phenomenon test
And (3) respectively picking a small amount of pure cultures of suspected strains, picking colonies, coating the colonies on a blood agar plate without NADH, using staphylococcus aureus producing NADH as a vertical cross streak, culturing for 24-48 hours at 37 ℃ in a 5-10% CO 2 incubator, and observing whether satellite phenomenon occurs (the closer to the staphylococcus colony is, the larger the distance from the staphylococcus colony is, and the smaller the colony is). The results are shown in FIG. 2. In FIG. 2, strain ZZ01, HX05 and ZN02 are shown as 1 to 3, respectively, and the results in FIG. 2 show that: obvious satellite phenomenon appears in all 3 strains.
1.4 PCR identification
1.4.1 Template preparation
For colony samples, individual colonies were picked, added to sterilized ultrapure water containing 20. Mu.L, boiled for 10 minutes, ice-cooled for 10 minutes, centrifuged at 8000r/min for 1 minute, and the supernatant was taken as a sample to be examined. Wherein the positive reference substance is A-type avian bacillus paragallinarum standard bacterium (CVCC 3007, china veterinary medicine inspection institute), and the negative reference substance is H 2 O.
1.4.2 PCR primers
Primer design was synthesized by biological engineering (Shanghai) Inc. The nucleotide sequence of the primer is as follows:
the primer 5'-TGAGGGTAGTCTTGCACGCGAAT-3' for the upstream side is provided,
And a downstream primer 5'-CAAGGTATCGATCGTCTCTCTACT-3'.
1.4.3 PCR amplification System
2 XPCR Buffer 12.5. Mu.L, 2.5mmol/L dNTPs 2. Mu.L, 10. Mu. Mol/L upstream and downstream primers 0.5. Mu.L each, rTaqDNA polymerization (5U/. Mu.L) 0.5. Mu.L, template 4. Mu.L, and addition of sterile ultra-pure water to 25. Mu.L.
1.4.4 PCR amplification procedure
Pre-denaturation at 94 ℃ for 5 min; cycling 30 times at 94 ℃ for 45 seconds, 56 ℃ for 45 seconds and 72 ℃ for 1 minute; extension was carried out at 72℃for 10 minutes.
1.4.5 Electrophoresis detection
Mu.L of the PCR product was taken and electrophoretically detected on a 1% agarose gel.
1.4.6, Result determination
The positive control showed a band of about 500bp, the negative control showed no band, and 10 avian secondary strains were each isolated with 500bp bands, and the results were shown in FIG. 3. In fig. 3, M: DNA molecular mass Marker; lane 1: a negative control; lane 2: a positive control; lanes 3-5: strain ZJ09, ZZ01, ZN 07; lanes 6-9: HX05, ZH02, JC03, AX07 strain; lanes 10-12: AX10, AH10 and ZN02 strains. The results in fig. 3 show that: the 10 isolates showed a band at 500bp, consistent with the positive control band, indicated as avian paragallinarum.
1.5 Identification of Biochemical and culture Properties
And (3) inoculating each suspected colony on the TSA culture medium to a biochemical identification tube, and carrying out saccharide fermentation experiments (glucose, lactose and sucrose), indole (indigo substrate) experiments, nitrate reduction experiments, catalase identification experiments, urease experiments and hydrogen sulfide experiments. According to the specification (bacterial trace biochemical identification tube, qingdao sea Bo Biotechnology Co., ltd.) the following operations are required:
Glucose fermentation experiments: taking 10 glucose agar fermentation medium test tubes, and taking a small number of single colonies by an inoculating needle to puncture the single colonies in the glucose agar; taking 3 test tubes to access escherichia coli as positive control; taking 3 test tubes without inoculation as blank control, placing the test tubes in an incubator containing 5% CO 2, culturing for 24-36 hours at 37 ℃, and judging positive reaction if the content in the test tubes changes from purple to yellow; if the color is purple and does not change, the color is negative.
Sucrose fermentation experiment: culturing bacterial liquid for 12 hours by a shaking table, measuring the OD value of the bacterial liquid by an enzyme-labeled instrument, adjusting the OD value to 0.08-0.1, namely, the concentration of bacterial suspension is 10 8 CFU/mL, taking 100 mu L of bacterial suspension to be detected, and adding the bacterial suspension into a sucrose trace biochemical identification tube (containing 0.01% NAD); taking 3 identification tubes to be connected with escherichia coli as a positive control; and taking 3 other identification tubes as blank control without inoculation, placing the tubes in an incubator containing 5% CO 2, and culturing at 37 ℃ for 24-36 hours to observe the phenomenon. If the color changes from purple to yellow, judging positive reaction; if the color is purple and does not change, the color is negative.
Nitrate reduction experiments: taking 10 test tubes of a nitrate culture medium, and taking a few single colonies by an inoculating needle, and inoculating the single colonies into the nitrate broth culture medium; taking 3 test tubes to access escherichia coli as positive control; taking 3 test tubes without inoculation as blank control, placing the test tubes in an incubator containing 5% CO 2, culturing for 24-36 hours at 37 ℃, adding 2-3 drops of each of the nitrate reducing reagent solution A and the solution B, and uniformly mixing. The results were observed at room temperature for 2 to 5 minutes. If the solution turns red, the reaction is positive; a negative reaction is obtained if the solution does not change color (no red color appears).
Lactose fermentation experiment: taking 10 test tubes of lactose fermentation medium, and taking a few single colonies by an inoculating needle, and inoculating the single colonies into the lactose fermentation medium; taking 3 test tubes to access escherichia coli as positive control; taking 3 test tubes without inoculation as blank control, placing the test tubes in a culture box containing 5% CO 2, culturing for 24-36 hours at 37 ℃, and judging positive reaction if the test tubes turn from purple to yellow; if the color is purple and does not change, the color is negative.
Catalase assay: 3% hydrogen peroxide solution, is prepared for use. 1 clean glass slide is taken, 10 single colonies are respectively picked up on one side of different glass slides by a sterilization inoculation needle, escherichia coli is picked up on the other side as positive control, 3% hydrogen peroxide solution is dripped for a plurality of drops, and the result is immediately observed. If a large number of bubbles appear immediately, the result is positive; negative if no bubbles are generated.
Hydrogen sulfide experiments: 10 test tubes of the agar culture medium for the rapid hydrogen sulfide test are taken, a small number of single colonies are picked by an inoculating needle, and the inoculating needle is punctured into the agar for the rapid hydrogen sulfide test; taking 3 test tubes to be connected with salmonella as positive control; taking 3 test tubes without inoculation as blank control, placing the test tubes in a culture box containing 5% CO 2, culturing for 24-36 hours at 37 ℃, and judging positive reaction if the test culture medium turns black; if the color is not changed, the result is negative.
Urease experiment: cooling the sterilized urease agar basic culture medium to 50-55 ℃, adding 5mL of sterile 40% urea water, uniformly mixing, and sub-packaging into test tubes for later use. The inoculating needle picks a few single colonies and punctures the single colonies into urease agar; taking 3 test tubes to access colibacillus as positive control; 3 tubes were taken and not inoculated as a blank. Positive is yellow; the negative is light orange red.
Indigo substrate experiment: culturing bacterial liquid for 12 hours by a shaking table, measuring the OD value of the bacterial liquid by an enzyme-labeled instrument, adjusting the OD value to 0.08-0.1, namely, the concentration of bacterial suspension is 10 8 CFU/mL, taking 100 mu L of bacterial suspension to be detected, and adding peptone water culture medium (containing 0.01% NAD); taking 3 identification tubes to be connected with escherichia coli as a positive control; taking 3 other identification tubes as blank control without inoculation, placing the tubes in an incubator containing 5% CO 2, culturing for 24-36 hours at 37 ℃, adding 2-4 drops of indigo substrate reagent, and placing the tubes at room temperature for 5-10 minutes to observe results. The positive reaction is represented by a red ring on the upper part of the culture medium; the negative reaction appears yellow, orange or brown.
The results are shown in FIGS. 4 to 11. The results in FIG. 4 show that none of the 10 isolates discoloured, indicating that no indigo reaction occurred, and that they were negative compared to the positive control. The results in FIG. 5 show that none of the 10 isolates reacted hydrogen peroxide and all were negative compared to the positive control. The results in FIG. 6 show that none of the 10 isolates color compared to the positive control, indicating that no hydrogen sulfide reaction occurred, all negative. The results in FIG. 7 show that all 10 isolates turned purple to yellow compared to the positive control, indicating that glucose fermentation was positive. The results in FIG. 8 show that none of the 10 isolates color compared to the positive control, and none of the unfermented lactose was negative. The results in FIG. 9 show that all 10 isolates turned purple to yellow compared to the positive control, indicating that the fermented sucrose was positive. The results in FIG. 10 show that the 10 isolates all changed color from colorless to orange, indicating that nitrate reduction reactions occurred, and were positive, as compared to the positive control. The results in FIG. 11 show that none of the 10 isolates showed color change, none had urease reaction, and all were negative compared to the positive control.
1.6 Serotype identification
1.6.1 Preparation of antigen to be examined
Picking colonies from the pure culture plates, inoculating 100mL of TSB culture medium, culturing for 10-16 hours at 37 ℃, centrifuging 100mL of culture at 8000g for 10 minutes, and washing the precipitate with PBS for 3 times; re-suspending with 20-30 mL KSCN/NaCl solution, stirring at 4deg.C for 2 hr, centrifuging at 8000g for 10 min, and washing the precipitate with PBS for 3 times; then resuspended in 5mL PBS, sonicated in an ice-water bath at 300W for 5 seconds, intermittently for 5 seconds, and total lysing for 15 minutes; and centrifuging 8000g for 10 minutes, and re-suspending the precipitate with 2ml PBS (containing 0.01% merthiolate) to obtain the antigen required by the hemagglutination inhibition test. And (5) storing at-20 ℃ for standby.
The preparation method of the PBS comprises the following steps: 8g of NaCl, 0.2g of KCl, 0.245g of Na 2HPO4 1.44g,KH2PO4 g, dissolved in 900mL of deionized water, and adjusted to pH 7.2 to a volume of 1000mL (also called 0.01M PBS).
1.6.2 Measurement of the hemagglutination value of the antigen to be examined
Taking 96-well V-shaped hemagglutination plates, performing 2-time serial dilution on the antigen from 2 times by using PBSS, and adding the antigen into 1 st to 11 th wells (namely, 1 st well is 1:2, 2 nd well is 1:4, and the same is done to 11 th well), wherein the final liquid amount of each well is 25 mu L, and only 25 mu L of PBSS is added into 12 th well to serve as an hydroformylation red blood cell control well; add 25. Mu.L of PBSS per well; 25 μl of 1% hydroformylation red blood cell suspension prepared by adding PBSS to each well; after full shaking and mixing, standing for 40-60 minutes at 20-25 ℃ or for 40 minutes at 37 ℃ until the red blood cells in the control hole (12 th hole) completely sink; the maximum dilution of antigen that causes complete agglutination of erythrocytes is taken as the hemagglutination titer (HA titer) of the antigen. The detection steps and the results of antigen detection are shown in FIG. 13 (in FIG. 13, "-" indicates that the red blood cells are not aggregated at all or partially aggregated, and means that the red blood cells are concentrated at the center of the well bottom or are concentrated at the center of the well bottom, and red blood cells are spread around the well bottom; "+" indicates that the red blood cells are completely aggregated, and means that the red blood cells are spread at the well bottom).
Wherein the composition of the PBSS solution is: naCl:8g, KCl:0.2g, na 2HPO4：1.44g,KH2PO4: 0.24g of the solution is dissolved in 800mL of deionized water, the pH value of the solution is regulated to 7.4 by HCl, and finally the volume is fixed to 1L, and the solution is sterilized for standby. Before use, 1% N, O-bis (trimethylsilyl) acetamide is added to obtain PBSS.
1.6.3 Identification of avian paragallinarum serotypes by hemagglutination inhibition assay
1.6.3.1, Adding type A, type B and type C control antigens according to the quantity of antigens to be detected, diluting type A, type B and type C type serum by using PBSS to 1:100, and each antigen sample needs 25 mu L of each type serum diluted by 1:100. (e.g., 3 test antigens plus control antigens of type A, type B, and type C, 150. Mu.L of each type of serum at a 1:100 dilution is required).
1.6.3.2, 96-Well V-type blood clotting plates were collected, 25. Mu.L of PBSS was added to the 1 st column to 10 th column wells, and 50. Mu.L of PBSS was added to the 11 th column wells.
1.6.3.3, Adding 25 μl of 1:100 diluted type A, type B, type C serum into 1 st, 4 th and 7 th columns of wells respectively, and sequentially performing 2-fold gradient dilution to the two latter wells (namely 1:200 for 1 st, 4 th and 7 th columns of wells, 1:400 for 2 nd, 5 th and 8 th columns of wells, and 1:800 for 3 rd, 6 th and 9 th columns of wells), wherein the final liquid amount per well is 25 μl. Column 10 plus PBSS, antigen as antigen control wells, column 11 plus PBSS alone as red blood cell control wells.
1.6.3.4 Antigen was diluted to 4 HA titer units with PBSS (if HA titer was 1:256, antigen was diluted 64-fold), 25. Mu.L was added to each well (except column 11).
1.6.3.5, Fully oscillating and mixing, and then standing for 20-30 minutes at 20-25 ℃ or 20 minutes at 37 ℃.
1.6.3.6 1% Of the hydroformylation erythrocytes prepared by adding PBSS to each well were 25. Mu.L.
1.6.3.7, After fully shaking and mixing, standing for 40-60 minutes at 20-25 ℃ or 40 minutes at 37 ℃ (until the red blood cells in the red blood cell control hole completely subside).
1.6.3.8 HI result determination
The highest serum dilution that can completely inhibit the agglutination of erythrocytes is the HI titer of the serum of that type, and if the antigen to be tested has agglutination inhibition with more than one type of positive serum, the HI titer of the antigen with which type of positive serum is the highest, the antigen is determined as which serotype. The detection steps and the detection results of the type A, type B and type C antigens are shown in FIG. 14 (in FIG. 14, "-" indicates complete agglutination inhibition, meaning that red blood cells are concentrated at the center of the well bottom and dot-shaped; "+" indicates complete agglutination or partial agglutination of red blood cells, meaning that red blood cells are spread at the well bottom or red blood cells are concentrated at the center of the well bottom and red blood cells are spread at the periphery of the well bottom).
The serotype of the isolated strain was detected by a hemagglutination inhibition test, and 3 strains of ZJ09, ZZ01 and ZN07 were isolated, 4 strains of avibacterium paragallinarum type B HX05, ZH02, JC03 and AX07, 3 strains of avibacterium paragallinarum type C AX10, AH10 and ZN02 were isolated, and the results are shown in Table 1.
The pure cultures of suspected strains are subjected to morphological, satellite phenomenon test, biochemical identification, PCR identification and serological identification, and 10 isolated strains are proved to be avian paragallinarum.
Note that: 1-3 are respectively a avian bacterium paragallinarum A type ZZ01 strain, a B type HX05 strain and a C type ZN02 strain
TABLE 1 serotype identification results for 10 isolates
Note that: "-" indicates negative.
Example 2 determination of vaccine strains
2.1 Culturing the bacterial liquid
3 Strains A type ZJ09, ZZ01 and ZN07, 4 strains B type HX05, ZH02, JC03 and AX07 of the avibacterium paragallinarum, and total 10 strains F1 generation freeze-dried strains of the AX10, AH10 and ZN02 of the avibacterium paragallinarum are taken. Respectively carrying out back thawing by using 2mL of TSB culture medium without chicken serum and NADH, respectively streaking and inoculating to a TSA flat plate, culturing for 24-48 hours at 37 ℃ in a CO 2 incubator with 5-10%, respectively picking 3-5 typical colonies and inoculating to 5mL of TSB culture medium, and culturing for 10-12 hours at 37 ℃ and 180r/min to obtain bacterial liquid.
2.2 Dilution of bacterial solution
1 Bottle of freeze-dried bacteria for A type, B type and C type virus attack are randomly extracted on the same day of virus attack, each bottle is re-melted by using 2mL of TSB culture medium without chicken serum and NADH, and the A type bacteria liquid, the B type bacteria liquid and the C type bacteria liquid are diluted to 1X 10 5 CFU/mL by using the TSB culture medium without chicken serum and NADH, and the diluted bacteria liquid is the bacteria liquid for virus attack.
2.3 Attack of toxin
3 Rounds of toxicity test were performed with SPF chickens of 6 to 7 weeks of age, respectively. The first round of challenge test was carried out using the 10 strains described above (3 strains A type ZJ09, ZZ01, ZN07, 4 strains B type HX05, ZH02, JC03, AX07, 3 strains B type AX10, AH10, ZN 02) respectively, and preparing a total challenge dose of 1X 10 5 CFU/strain, 5 chickens, 0.2 mL/strain, leg intramuscular injection, and 0.2mL pure water as a control. Taking the day of the challenge as0 day, observing the symptoms of the chicken within 7 days of the challenge, and taking any lesion of facial swelling or nasal fluid flow as the pathogenesis. Screening more than 4/5 of the pathogenic strains of the chickens subjected to the toxicity attack experiment to carry out second round of toxicity attack. The results of the first round of challenge experiments are shown in table 2. Taking 3A-type ZJ09, ZZ01 and ZN07, 3B-type avian bacillus paragallinarum HX05, ZH02 and AX07, and continuing the second round of virus attack by 3C-type avian bacillus paragallinarum AX10, AH10 and ZN 02.
TABLE 2 results of round 1 challenge for different serotypes of strains
Note that: "-" indicates that this was not done.
In the second round of challenge experiments, 4 total challenge doses, namely 4×10 4 CFU/strain, 2×10 4 CFU/strain, 1×10 4 CFU/strain and 5×10 3 CFU/strain, were set for each strain, and each dose of challenge was 5 chickens, with a challenge volume of 0.2 mL/strain, and 0.2mL of purified water was used as a control. Taking the day of the challenge as0 day, observing the symptoms of the chicken within 7 days of the challenge, and taking any lesion of facial swelling or nasal fluid flow as the pathogenesis. Screening strains with the pathogenesis of more than 4/5 (2 strains A type ZJ09 and ZZ01,2 strains B type HX05 and AX07 of the avian secondary bacillus and 2 strains C type AX10 and ZN02 of the avian secondary bacillus) for third round of virus attack.
In the third round of toxicity attack experiment, the toxicity attack strains are 2A type ZJ09 and ZZ01, 2B type HX05 and AX07 strains of the avibacterium paragallinarum and 2C type AX10 and ZN02 strains of the avibacterium paragallinarum. Each strain was given 3 total challenge doses of 2X 10 4 CFU/1X 10 4 CFU/5X 10 3 CFU/5, each strain per dose of challenge to 10 chickens, with a challenge volume of 0.2 mL/2 mL and 0.2mL pure water as controls. Taking the day of the challenge as 0 day, observing the symptoms of the chicken within 7 days of the challenge, wherein any lesion of facial swelling or nasal fluid flow is the onset, screening strains with onset of more than 8/10 as the final challenge dose, and the results are shown in a table 3, wherein the results show that the onset of more than 9/10 can be stably achieved by 1 x10 4 CFU/1 of the A type ZZ01 strain, and the onset of more than 8/10 can be stably achieved by 5 x10 3 CFU/1 of the B type HX05 strain and the C type ZN02 strain. The strain A ZZ01 (2.0X10 4 CFU/min), strain B HX05 (1.0X10 4 CFU/min) and strain C ZN02 (1.0X10 4 CFU/min) were finally determined as production and test strains and final challenge doses. Clinical manifestations after toxin attack and pathological anatomy results are shown in fig. 12 and 15.
TABLE 3 strains of different serotypes, different challenge doses, round 3 challenge results
Note that: "-" indicates that this was not done.
FIG. 12 shows the results of clinical symptoms of SPF-infected chickens with avian secondary bacteria, and the left-hand graphs 1-3 in FIG. 12 show the clinical symptoms of chickens after challenge with avian secondary bacteria A-type ZZ01 strain (2.0X10 4 CFU/d), B-type HX05 strain (1.0X10 4 CFU/d) and C-type ZN02 strain (1.0X10 4 CFU/d), respectively; the left-hand figure 4 of fig. 12 shows the clinical symptoms of control chickens. The results in fig. 12 show that: compared with the control group, the test group chickens only show symptoms within 24 hours, the sick chickens have sleepiness and swelling of the face, even can not open eyes, have dyspnea, and have open mouth breathing. Clear nasal liquid continuously flows out of the nasal cavity of the sick chicken, and then gradually solidifies to be yellow brown, and plugs are formed around the nasal cavity, so that the sick chicken breathes unsmoothly and has a pungent and foul smell. And then the sick chicken starts to show respiratory symptoms with different degrees, such as cough, sneeze, snore, increased secretion of eyes, lacrimation and the like. When the disease of some sick chickens is serious, eyes cannot be opened due to the swelling of eyeboxes.
FIG. 15 shows pathological anatomy results of SPF chickens infected with avian bacteria of the secondary chickens, wherein 1 to 3 in FIG. 15 are respectively the infraorbital sinus, the lung and the throat of a type A ZZ01 strain, a type B HX05 strain and a type C ZN02 strain of the virus-infected chickens; 4-6 are the infraorbital sinus, lung and throat of the control group of chickens. The results of fig. 15 show that: compared with the control group, the test group chicken only has main lesions on the upper respiratory tract, especially the nasal cavity, the trachea and the throat, and can be seen to have cheese necrosis or tissue exudates in the upper respiratory tract to block the cavity. Some sick chickens develop varying degrees of oedema, eyelid swelling in the face and subcutaneous tissue. The sick chicken secretions form a blockage after solidification and have strong malodorous smell along with the emission. The lung of a partially ill dead chicken develops blood stasis.
Example 3 vaccine preparation
3.1 Preparation of bacterial liquid for seedling preparation
Filling a commercial fermentation culture medium into a culture tank, adding a proper amount of defoamer, adjusting the pH value to 7.2-7.4, and sterilizing for 30 minutes at 121 ℃. Under the conditions that the temperature is 37 ℃ and the pH value is 7.2, 0.1 percent of glucose, 0.5 percent of yeast powder and 5 to 10 percent of chicken serum are added, and 0.003 percent of NADH aqueous solution is aseptically added into the seed liquid according to the inoculation proportion of 2 percent. In the fermentation process, the pH value is controlled to 7.2-7.4 by an acid and alkali supplementing device, the ventilation and the rotating speed are regulated according to DO (dissolved oxygen value), and the feeding is carried out in a 10% glucose feeding mode. A type ZZ01 strain is fermented for 7 hours to obtain bacterial liquid, B type HX05 strain is fermented for 6 hours to obtain bacterial liquid, C type ZN02 strain is fermented for 9 hours to obtain bacterial liquid, and sampling is carried out for viable count and pure inspection.
3.2, Concentration and inactivation of bacterial liquid for preparing seedlings
Removing the supernatant by adopting a centrifugal concentration method, re-suspending the supernatant to 1/2 of the volume of the original bacterial liquid by using PBS (0.01 mol/L pH value 7.2), adding formaldehyde solution with the final concentration of 0.5% according to the bacterial liquid volume, inactivating the mixture at 2-8 ℃ for 5 days, sampling and inoculating the mixture to 2 blocks of chicken soup agar plates, 0.2 mL/block, 5% CO 2 incubator, culturing the mixture at 37 ℃ for 7 days, and observing the aseptic growth of the plates.
3.3 Antigen modulation
Mixing the inactivated ZZ01 strain antigen, HX05 strain antigen and ZN02 strain antigen according to a certain proportion, and according to the counting result of live bacteria before inactivation, adjusting the number of live bacteria before inactivation of the ZZ01 strain in the mixed antigen liquid of three bacterial liquids to be 2.6X10 9 CFU/mL, the number of live bacteria before inactivation of the HX05 strain to be 5.2X10 9 CFU/mL and the number of live bacteria before inactivation of the ZN02 strain to be 7.8X10 9 CFU/mL by using PBS (pH value of 7.2), thus obtaining the antigen for preparing the seedlings.
3.4 Vaccine preparation
3.4.1 Aqueous phase preparation
And (3) taking 4 parts of Tween-80, placing into a liquid preparation tank, sterilizing and cooling. 96 parts of the adjusted antigen solution is added into the liquid preparation tank, and stirring is carried out while adding until the Tween-80 is completely dissolved.
3.4.2 Preparation of oil phase
94 Parts of white oil for injection and 1 part of aluminum stearate are taken, placed in an oil phase preparation tank, heated to 80 ℃, added with 80-80 parts of span, maintained for 30 minutes when the temperature reaches 116 ℃, and cooled for standby. Such oily adjuvants include, but are not limited to, white oil, squalane or squalene, drek oil, and other animal, vegetable or mineral oils. The above-mentioned oily adjuvant may be either of natural origin or obtained by artificial synthesis. The vaccine further comprises a suspending agent, a surfactant, an antigen inactivating agent, or a preservative. The suspending agent may include, for example, aluminum stearate, as well as other suspending agents available in the art. The surfactant may include, for example, sorbitan monooleate (TWEE series), SPAN (SPAN), and other surfactants useful in the art. Such antigen inactivating agents include, but are not limited to, for example, formalin, beta-propiolactone, and the like. The preservative includes, for example, thimerosal. Methods of using and amounts of such materials are well known to those skilled in the art.
3.5, Emulsifying and sub-packaging
The oil phase and the water phase are mixed according to the volume ratio of 1.5:1, the method is that firstly the oil phase is poured into a tissue triturator, the water phase is slowly added under low-speed stirring, then the mixture is emulsified for 30 minutes at 2800-3200 r/min, and 1% merthiolate solution is added before the stirring is stopped, so that the final concentration is 0.01%. Emulsifying to obtain milky oil emulsion inactivated vaccine, and quantitatively detecting.
The split charging specification is (1) 100 mL/bottle; (2) 250 mL/bottle; (3) 300 mL/bottle; (4) 500 mL/bottle.
Example 4 animal challenge-protection test
4.1 Immunization
60 SPF chickens with the age of 6-7 weeks are randomly divided into 2 groups, 30 chickens are arranged in each group, trivalent inactivated vaccine immunization of infectious rhinitis of the chickens is carried out on the first group, 0.5mL of vaccine is injected into the muscle of each chicken leg, and the 2 nd group is a non-immune control group.
4.2, Attack toxin
After 30 days of immunization, 10 chickens are respectively taken from a test group and a control group to attack A, B, C avian bacillus paragallinarum virus-attacking strains, the virus-attacking dose of each chicken is 2.0×10 4 CFU/0.2mL of A type ZZ01 strain, 1.0×10 4 CFU/0.2mL of B type HX05 strain, 1.0×10 4 CFU/0.2mL of C type ZN02 strain, and the virus-attacking site is intraorbital sinus injection. After detoxification, observation is carried out for 7 days. The results are shown in Table 4, on day 2 after toxin challenge, the chickens in the A control group all have 10/10 of the disease, the swelling of the infraorbital sinus is obvious, the nasal fluid flows, and the immunization group all have 9/10 of the protection; type B controls had 10/10 onset and vaccinated groups were 8/10 protection. The C-type control group is 10/10 of the disease, and the immune group is 10/10 of the protection, which proves that the vaccine has more than 8/10 of the protection efficacy on homologous strains.
TABLE 4 results of animal challenge-protection test for trivalent inactivated vaccine against infectious coryza in chickens
Example 5 safety test
The trivalent inactivated vaccine for infectious rhinitis of chicken is used for carrying out single dose (leg intramuscular injection of 0.5 mL), single dose repetition (leg intramuscular injection of 0.5mL and repeated inoculation with the same dose after 14 days after the first immunization) and one over-injection safety test (leg intramuscular injection of 1.0 mL) on SPF chicken of 6 weeks of age, and each dose is injected into 10 chickens. The chicken mental state was observed on days 14, 21 and 28, respectively, with or without abnormalities at the injection site, following 28 consecutive days of observation. The results in Table 5 show that no abnormalities in the spirit and diet of the tested chickens were observed 28 days after injection of 3 different doses. The results in table 6 show that there is a small amount of unabsorbed vaccine 14 days after a single dose injection and that absorption is complete 21 days after injection; the single-dose repeated dose groups still have a small amount of unabsorbed vaccine at the injection site of each group of immunized chickens at 14 days and 21 days after the second injection, but the vaccine at the injection site is completely absorbed at 28 days after the injection; there were still small amounts of unabsorbed vaccine at the injection site for each of the immunized chickens in the one excess group, 14 and 21 days after injection, but the vaccine at the injection site was completely absorbed at 28 days after injection.
TABLE 5 systemic reaction observations following different doses of trivalent inactivated vaccine against infectious coryza in chickens
TABLE 6 observations at injection sites after different doses of trivalent inactivated vaccine against infectious coryza in chickens
Example 6, minimum immune dose test
The trivalent inactivated vaccine for infectious rhinitis of chicken is immunized by leg muscles according to dosages of 0.13mL, 0.25mL and 0.5mL, and a toxicity attack protection test is carried out 21-28 days after immunization. The results in Table 7 show that the protection ratio of 0.13 mL/immunized group is 6/10 to 7/10, the protection ratio of 0.25 mL/immunized group and 0.5 mL/immunized group is 8/10 to 10/10, and the results are shown in Table 7. The minimum immunization dose was determined to be 0.25 mL/min.
TABLE 7 minimum dose test results of trivalent inactivated vaccine against infectious coryza in chickens
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains.
Claims (10)
1. The trivalent inactivated vaccine for the infectious coryza comprises active ingredients of inactivated A-serotype paragallinarum, inactivated B-serotype paragallinarum and inactivated C-serotype paragallinarum.
2. The trivalent inactivated vaccine for infectious coryza of chicken according to claim 1, wherein: the A serotype parachicken bacillus is A serotype parachicken bacillus CCTCC M20221926, the A serotype parachicken bacillus CCTCC M20221926 is A serotype parachicken bacillus preserved in China center for type culture collection, and the preservation number is: cctccc M20221926.
3. The trivalent inactivated vaccine for infectious coryza of chicken according to claim 1 or 2, wherein: the B serotype parachicken bacillus is inactivated B serotype parachicken bacillus CCTCC M20221927, the C serotype parachicken bacillus is inactivated C serotype parachicken bacillus CCTCC M20221928, the B serotype parachicken bacillus CCTCC M20221927 is the B serotype parachicken bacillus preserved in China center for type culture collection, and the preservation number is: cctccc M20221927.
4. A trivalent inactivated vaccine for infectious coryza of chickens according to any one of claims 1-3, wherein: the C serotype parachicken bacillus is inactivated C serotype parachicken bacillus CCTCC M20221928, and the C serotype parachicken bacillus CCTCC M20221928 is the C serotype parachicken bacillus preserved in China center for type culture collection, and the preservation number is as follows: cctccc M20221928.
5. The trivalent inactivated vaccine for infectious coryza of chickens according to claims 1-4, wherein: the ratio of the A serotype paragallibacterium to the B serotype paragallibacterium to the C serotype paragallibacterium is 1:2:3 (counted by colony forming units before inactivation).
6. The trivalent inactivated vaccine for infectious coryza of chickens according to claims 1-4, wherein: the trivalent inactivated vaccine for the infectious coryza of the chicken consists of the active ingredient and an adjuvant.
7. The trivalent inactivated vaccine for infectious coryza of chickens according to any one of claims 1-6, wherein: the preparation method of the trivalent inactivated vaccine for infectious coryza of chickens comprises the following steps:
S1) respectively culturing the A-serotype parachicken bacillus CCTCC M20221926, the B-serotype parachicken bacillus CCTCC M20221927 and the C-serotype parachicken bacillus CCTCC M20221928 to obtain three kinds of culture solutions of the parachicken bacillus;
s2) inactivating the three secondary chicken avian bacteria culture fluid prepared in the step S1) to obtain an inactivated secondary chicken avian bacteria culture fluid;
S3) mixing the inactivated three secondary chicken avian bacillus culture fluids prepared in the step S2) with an adjuvant to obtain the trivalent inactivated vaccine for the infectious rhinitis of the chicken.
8. A method for preparing the trivalent inactivated vaccine for infectious coryza of chicken according to any one of claims 1 to 7, which is characterized in that: the method comprises the following steps:
S1) respectively culturing the A-serotype parachicken bacillus CCTCC M20221926, the B-serotype parachicken bacillus CCTCC M20221927 and the C-serotype parachicken bacillus CCTCC M20221928 to obtain three kinds of culture solutions of the parachicken bacillus;
s2) inactivating the three secondary chicken avian bacteria culture fluid prepared in the step S1) to obtain an inactivated secondary chicken avian bacteria culture fluid;
S3) mixing the inactivated three secondary chicken avian bacillus culture fluids prepared in the step S2) with an adjuvant to obtain the trivalent inactivated vaccine for the infectious rhinitis of the chicken.
9. The avian bacillus paragallinarum is characterized in that: the avian bacterium paragallinarum is selected from any one or more of the following:
a1 A) the a serotype avibacterium paragallinarum cctccc M20221926;
A2 B serotype parachicken bacillus cctccc M20221927;
A3 C serotype avibacterium paragallinarum cctccc M20221928).
10. Use of the avian paragallinarum of claim 9 in the preparation of a vaccine for infectious coryza of chickens.
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