CN117844686A - Separation, identification and application of avibacterium paragallinarum serum A strain with cross protection effect - Google Patents
Separation, identification and application of avibacterium paragallinarum serum A strain with cross protection effect Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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Abstract
The invention belongs to the technical field of microbial preparations, discloses separation, identification and application of a avibacterium paragallinarum serum A strain with a cross protection effect, and particularly discloses a avibacterium paragallinarum named as a avibacterium paragallinarum DHN-QD strain which is preserved in China Center for Type Culture Collection (CCTCC) NO: m2023968, the preservation date is 2023, 06 and 08. The avian paragallinarum DHN-QD strain provided by the invention has stronger toxicity, good immunogenicity and high passage stability, and has high immune protection effect on the strain with the same blood group, good immune protection effect on the strain with the different B type serum and better immune protection spectrum.
Description
Technical Field
The invention belongs to the technical field of microbial preparations, and particularly relates to separation, identification and application of a avibacterium paragallinarum serum A strain with a cross protection effect.
Background
The avian bacillus paragallinarum (Avibacterium paragallinarum, apg) is originally called haemophilus paragallinarum (Haemophilus paragallinarum, hpg) and is a pathogenic bacterium causing infectious rhinitis (infectious coryza, IC) of chickens. IC is an acute upper respiratory tract infection and toxin effect disease of chickens, is distributed worldwide, can cause the reduction of egg yield of laying hens, the growth and development resistance and the increase of elimination rate of bred chickens, and the reduction of meat quality of broilers, thus causing great economic loss.
The avian bacillus paragallinarum has short rod shape, no flagella, no motility, and most virulent strains have capsules. Apg is highly nutritious in vitro, and it is generally necessary to add factor V (Nicotinamide adenine dinucleotide, NAD), but it has been reported that factor V independent growth Apg was isolated in clinical cases. Classical Page typing methods split Apg into A, B, C three serotypes, with either weak or no cross-immune protection of the different serotypes. The Kume typing scheme divides nine serotypes A-1, A-2, A-3, A-4, B-1, C-2, C-3 and C-4 through a hemagglutination inhibition test, but the current Page serotyping scheme is still mainly used because of the complicated process and the severe experimental conditions of the Kume typing scheme.
Vaccination is an important means of controlling ICs. At present, the whole-bacteria inactivated vaccine is clinically applied, and divalent vaccine (A type+C type) and trivalent vaccine (A type+B type+C type) are common. Due to poor cross protection effect between different serotypes, a trivalent inactivated vaccine containing A, B, C serotypes is mostly used for immunization in a farm. Apg whole-cell inactivated vaccines have some problems: firstly, a large amount of bacteria are required to be cultivated in the production of the inactivated vaccine, and Apg has high nutrition requirement, so that the production cost of the inactivated vaccine is high; and Apg bacteria are fragile and easy to die, the endotoxin content of fermentation broth is high, and side effects caused by inoculating chicken bodies are large. In addition, the protection effect of the current commercialized inactivated vaccine on the fluidized bacterial strain is not ideal. Morales et al isolated a strain Apg (C-1 serotype) from an IC-immunized chicken house and evaluated the protective effect of four commercial trivalent inactivated IC seedlings on this isolate, as a result, it was found that only one vaccine provided a better protective effect (83%) for vaccinated chickens. Researches by researchers also find that the current common IC commercial vaccine in China has no ideal protection effect on the current epidemic strains. Therefore, the development of the chicken infectious rhinitis vaccine with wide immune protection spectrum and good safety has important significance.
Disclosure of Invention
The object of the first aspect of the invention is to provide a strain of avibacterium paragallinarum.
The second aspect of the invention aims to provide a poultry secondary chicken bacillus agent.
The object of the third aspect of the invention is to provide the use of the avian paragallinarum bacterium of the first aspect of the invention and/or the avian paragallinarum bacterium of the second aspect of the invention in the preparation of a product.
The fourth aspect of the present invention aims to provide a vaccine.
The object of the fifth aspect of the present invention is to provide a method for preparing the vaccine of the fourth aspect of the present invention.
The object of the sixth aspect of the present invention is to provide a vaccine composition.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
in a first aspect of the invention, a strain of avibacterium paragallinarum, named avibacterium paragallinarum DHN-QD strain, haemophilus paragallinarum DHN-QD, is preserved in China center for type culture collection, and has a preservation number of CCTCC NO: m2023968, the date of deposit is 2023, 06, 08, deposit unit address: chinese university of Wuhan and Wuhan.
In some embodiments of the invention, the avian paragallinarum DHN-QD strain is avian paragallinarum a.
In a second aspect of the invention, a secondary avian bacillus agent is provided, wherein the secondary avian bacillus agent comprises at least one of the secondary avian bacillus of the first aspect of the invention, the fermentation culture of the secondary avian bacillus of the first aspect of the invention and the fermentation supernatant of the secondary avian bacillus DHN-QD strain of the first aspect of the invention.
The active ingredient of the microbial inoculum can be the culture (such as fermentation product) of the avian paragallinarum strain DHN-QD and/or the avian paragallinarum strain DHN-QD, and can also contain other biological ingredients or non-biological ingredients, and the other active ingredients of the microbial inoculum can be determined by one skilled in the art according to the effect of the microbial inoculum.
The term "culture" refers to a generic term for liquid or solid products (all substances in the culture vessel, fermentation products) grown with a population of microorganisms after artificial inoculation and cultivation. I.e. the product obtained by growing and/or amplifying the microorganism, which may be a biologically pure culture of the microorganism, or may contain a certain amount of medium, metabolites or other components produced during the culture.
In the microbial inoculum, the microbial inoculum contains a carrier in addition to the active ingredient. The carrier may be a carrier commonly used in the art (e.g., microorganisms, agriculture) and which is biologically inert. The carrier may be a solid carrier or a liquid carrier; the solid carrier can be mineral material, plant material or high molecular compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of corn flour, soy flour and starch; the polymer compound may be polyvinyl alcohol and/or polyglycol; the liquid carrier may be an organic solvent or water; the organic solvent may be decane and/or dodecane.
Among the above-mentioned bacterial agents, the formulation of the bacterial agent can be various formulations, such as liquid, emulsion, suspending agent, powder, granule, wettable powder or water dispersible granule.
According to the need, the microbial inoculum can also be added with adhesive, stabilizer (such as antioxidant), pH regulator, solubilizer, excipient, slow-release agent, etc.
In some embodiments of the invention, the microbial agent further comprises: a nutrient. The nutrients include: at least one of proteins, carbohydrates, lipids, minerals, vitamins, plant extracts, amino acids, immunomodulators and milk substitutes.
In a third aspect, the invention provides the use of the avian paragallinarum bacterium of the first aspect of the invention and/or the avian paragallinarum bacterium of the second aspect of the invention in the manufacture of a product.
In some embodiments of the invention, the product has the following functions: preventing and/or treating fowl bacillus paragallinarum related diseases.
In some embodiments of the invention, the product comprises a vaccine or a medicament.
In a fourth aspect of the invention there is provided a vaccine comprising the avibacterium paragallinarum of the first aspect of the invention and/or the avibacterium paragallinarum of the second aspect of the invention.
In some embodiments of the invention, the inactivated whole-cell antigen content of the avian paragallinarum DHN-QD strain in the vaccine is not less than 1×10 before inactivation 7 CFU/mL。
In some embodiments of the invention, the inactivated whole-cell antigen content of the avian paragallinarum strain DHN-QD in the vaccine is 1×10 before inactivation 7 CFU/mL～5×10 8 CFU/mL。
In some embodiments of the invention, the vaccine further comprises a pharmaceutically acceptable adjuvant.
In some embodiments of the invention, the vaccine further comprises avian B-type, avian C-type or other avian viruses, mycoplasma.
In a fifth aspect of the invention there is provided a method of preparing a vaccine according to the fourth aspect of the invention comprising the steps of:
culturing the avian paragallinarum DHN-QD strain;
inactivating the cultured culture of the avian paragallinarum DHN-QD strain by formaldehyde, mixing the inactivated culture with auxiliary materials, and emulsifying to obtain the vaccine.
In some embodiments of the invention, the formaldehyde is used in an amount of 0.1% to 0.2% by volume of the culture of the avian paragallinarum strain DHN-QD.
In some embodiments of the invention, the inactivation conditions are 36-38 ℃ for 24-48 hours.
In some embodiments of the invention, the emulsifying conditions are 3000 to 40000 rpm for 80 to 100 minutes.
The beneficial effects of the invention are as follows:
the avian secondary strain DHN-QD provided by the invention has stronger toxicity, good immunogenicity and high passage stability, has high immune protection effect on homologous strain, has better immune protection effect on heterologous B-type serum strain, has better immune protection spectrum, and has important significance on developing chicken infectious rhinitis vaccine with broad immune protection spectrum and good safety.
The avian bacillus paragallinarum DHN-QD strain with the cross protection effect is used for preparing the vaccine, so that the content of antigen required in a vaccine finished product can be effectively reduced, the endotoxin content is low, and the high protection rate and less immune side reaction can be provided with lower production cost.
Drawings
Fig. 1 is an agarose gel electrophoresis chart, and a Marker, a positive control, a sample to be detected and a negative control are sequentially arranged from left to right.
FIG. 2 is a phylogenetic tree of clinically isolated serotype A and serotype B strains and their related exemplary strains constructed using a contiguity based on the avian paragallinarum HMTp-210 gene hypervariable region gene sequence.
Detailed Description
The following describes the present invention in further detail by way of specific examples.
It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The term "inactivated vaccine", also referred to as an inactivated vaccine, as used herein refers to a suspension of inactivated bacteria that is used as an antigen to generate immunity. Examples of inactivated vaccines include whole-cell vaccines and split vaccines. Inactivated vaccines can be readily produced using known methods. For example, a whole-cell inactivated vaccine can be obtained by treating cells with a formaldehyde solution. For example, the inactivated vaccine can be prepared by using the avian paragallinarum DHN-QD strain of the present invention through an inactivation method.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
EXAMPLE 1 isolation and identification of avian paragallinarum DHN-QD Strain
1. Isolation of avian paragallinarum DHN-QD strain
And (3) collecting the disease materials: the paradox secretion of the paradox is generated from a large-scale laying hen house in Guangdong cloud of 2021.
Preparing a finished liquid culture medium: trypticase Soy Broth (TSB) medium+10% standard chicken serum+0.1 g/L final concentration of coenzyme I (NAD).
Preparing a finished solid culture medium: trypticase Soy Agar (TSA) medium+10% standard chicken serum+0.1 g/L final concentration of coenzyme I (NAD).
During the strain culture, the strain is cultured under the condition of 5% carbon dioxide.
Isolation culture of strains:
collecting the clinical pathogenic chicken and the pathogenic materials to a laboratory for bacteria identification. The clinical symptoms are nasal cavity and nasal sinus inflammation, sneeze, nasal fluid flow, facial swelling and the like. And (3) cutting off the nasal sinuses on a sterile ultra-clean workbench, and inoculating the nasal sinuses content into a nasal delivery solid flat plate by using a sterile cotton swab or a sterile inoculating loop for culturing for 16-24 h. Translucent dew drop-like, edge-trim microcolonies can be formed. And (3) carrying out passage purification on suspected colonies in the flat plate, inoculating single colonies into a proper amount of liquid culture medium after purification, culturing for 6-8 hours at 150r/min, and obtaining bacterial liquid for subsequent identification.
2. Identification of avian paragallinarum DHN-QD strain
2.1PCR identification
According to the universal primer for identifying the infectious rhinitis of the chicken disclosed in NY/T538-2015 chicken infectious rhinitis diagnosis technology, the primer sequence is as follows:
an upstream primer: 5'-TGAGGGTAGTCTTGCACGCGAAT-3' (SEQ ID NO: 1);
a downstream primer: 5'-CAAGGTATCGATCGTCTCTCTACT-3' (SEQ ID NO: 2);
the expected amplified fragment size was 500bp.
Extracting bacterial liquid genome by using a commercial extraction kit, simultaneously setting double distilled water as negative control, carrying out PCR amplification by using a universal primer, wherein the reaction system is 25 mu L, the PCR reaction system is shown in table 1, and the PCR reaction program is as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94℃for 45 seconds, annealing at 56℃for 45 seconds, elongation at 72℃for 1 minute for 30 cycles; the extension was carried out at 72℃for another 10 minutes.
TABLE 1PCR reaction System
Electrophoresis analysis: the PCR product was taken at 5. Mu.L and detected by 1% agarose gel electrophoresis. And (3) judging: the sample to be detected is amplified to form a band with the size of about 500bp, and the band is judged to be positive; otherwise, the result is negative.
The identification result is shown in figure 1, the sample to be detected is amplified to form a band with the size of about 500bp, and the band is judged to be positive for infectious rhinitis of chickens, namely the separated strain is avian paragallinarum and named avian paragallinarum DHN-QD strain.
2.2 serotyping
Preparation of hyperimmune serum: positive serum was prepared from standard strain immune clean-grade laboratory rabbits and stored at-20 ℃. Haemophilus parasuis type a Hpg-221, type B CCM6075, model C Modesto standard strains purchased from the chinese veterinary drug administration.
Preparation of antigen to be detected: inoculating strain to be detected (avian paragallinarum DHN-QD strain) into TSA solid culture medium at 37deg.C with 5% CO 2 Culturing in an incubator for 18-24 h. 2-3 single colonies are picked up and inoculated to 50mL TSB liquid culture medium for expansion culture, the culture is carried out for 6-8 h at 37 ℃, the centrifugation is carried out for 15min at 8000g, the thalli are collected, and the thalli precipitate is resuspended and washed for 3 times by 0.01mmol/L PBS. The bacterial precipitate is suspended in a solution containing 0.5M KSCN and 0.425M NaCl with pH 6.3, and OD is finally adjusted 650 After incubation for 2h at 1.6,4 ℃, sonicated. Finally, washing the antigen with PBS containing 0.001% merthiolate for 3 times, suspending in 2mL of PBS, and preserving at 4 ℃ to obtain the antigen to be detected.
HI assay:
antisera were prepared at 50. Mu.L 1:10 to 1:1280 fold dilutions in PBSS (PBS containing 0.1% BSA and 0.001% gelatin). 50. Mu.L of antigen containing 4 hemagglutination units was added to each well and incubated for 40min at room temperature. 50ul of 1% hydroformylation red blood cells were then added and incubated at room temperature for 30-40 min before reading. Determination criteria: the highest serum dilution that resulted in complete hemagglutination inhibition of erythrocytes was the HI titer of this type of serum. If the antigen to be tested has inhibition of aggregation with more than one type of positive serum, the HI titer is the highest for that serotype. HI titers < 10 were judged negative.
As shown in Table 2, the avian secondary strain DHN-QD was able to neutralize antibodies in serum of the reference strain Hpg-221, and red blood cells were not able to agglutinate, indicating that the avian secondary strain DHN-QD was avian secondary strain A. Meanwhile, it is interesting that the avian paragallinarum DHN-QD strain can partially neutralize antibodies in the serum of the B-type reference strain CCM 6075. The avian bacterium paragallinarum DHN-QD strain (Haemophilus paragallinarum DHN-QD) is submitted to China center for type culture Collection (China center for type culture collection) for collection at the year 06-month 08, and the collection number is CCTCC NO: M2023968.
TABLE 2HI assay results
EXAMPLE 2 sequence analysis of the high variable region (hypervariable region, HVR) of the avian strain DHN-QD HMTp-210 Gene
Designing a primer to amplify HMTp-210 gene hypervariable region of avian paragallinarum, wherein the sequence of a serum A type amplification primer is as follows: 5'-GCAACGAACTTTATGTTGAGCAAAG-3' (SEQ ID NO: 3), 5'-TGTTTTCACATTATGATCTCCGTGA-3' (SEQ ID NO: 4). The sequences of the serum type B amplification primers are as follows: 5'-AACGAACCTTATGTTGAGCAAAGTG-3' (SEQ ID NO: 5), 5'-ATTTTGCCTTCTCAGGCTTATTTGA-3' (SEQ ID NO: 6). Amplifying target fragments, delivering and measuring, downloading related gene sequences of type A and type B of avian paragallinarum from NCBI (http:// blast. NCBI. Lm. Nih. Gov/blast. Cgi) database, respectively carrying out sequence comparison and analysis by ClustalX 2.0 and MEGA software, and finally constructing a phylogenetic tree by using a Neighbor-Joining method (NJ).
Analysis results show that the clinically separated 13 strains are separated and identified as A-type strains, and the A-type standard serum strains 221 and 083 strains are clustered on a phylogenetic tree to form an independent branch. The clinically isolated 6 strains are identified as B-type strains by serum separation, and are clustered on a phylogenetic tree with B-type standard serum strains Spross strain and 0222 strain. The avian paragallinarum DHN-QD strain serotypes are type a, but are categorized on the phylogenetic tree as branches of serotype B (fig. 2). The sequence alignment analysis shows that the sequence of the avian paragallinarum DHN-QD strain is only 46.79% identical with that of the HVR region of the A-type standard serum strain 221 (ON 959452), and 98.73% identical with that of the HVR region of the B-type standard serum strain Spross strain. The avian paragallinarum DHN-QD strain is presumed to be a recombinant strain of serotype a and serotype B. The present literature also reports that a plurality of local epidemic strains are obtained by separating Tan and the like of a scholars in China, and a serotyping result shows that TW 16-4 separated strains and serum B type and serum C type standard strain positive serum have higher HI titers. The full-length evolution analysis result of the HMTp210 gene shows that the TW 16-4 isolate is chimeric with serotype B and serotype C, and the strain is estimated to be a strain generated by recombination of the serotype B and the serotype C.
EXAMPLE 3 toxicity assay and immunogenicity study of avian paragallinarum DHN-QD strain
1. Toxicity determination
The freshly cultured bacterial solutions were diluted with TSB medium, 5 SPF chickens of 60 days old were inoculated to the infraorbital sinus of each diluted bacterial solution, PBS was inoculated in control, 0.2mL was injected into each infraorbital sinus, and the onset of disease was observed for 7 days (swelling of infraorbital sinus and surrounding on one or both sides of the face and runny nose or lacrimation). And (3) after the toxin is removed, viable bacteria are counted on the toxin removing bacteria liquid, and the actual toxin removing dosage is determined.
The results showed that 60 day old SPF chickens were injected with 0.8X10 5 CFU and above, all chickens showed symptoms of swelling or runny nose in and around the infraorbital sinus on one or both sides of the face only within 24-60 hours. And the avian bacterium paragallinarum can be separated from the infraorbital sinus of the sick chicken again; 0.4X10 5 CFU inoculum size inoculation, only 1 chicken did not develop disease in the 7 day observation period, suggesting that avibacterium paragallinarum DHN-QD strain was virulent (table 3).
TABLE 3 toxicity measurement results
| Toxin-counteracting bacterial load/CFU | The number of toxic materials attacking the body | The number of the diseases is only counted | Morbidity/% |
| 6.4×10 5 | 5 | 5 | 100 |
| 3.2×10 5 | 5 | 5 | 100 |
| 1.6×10 5 | 5 | 5 | 100 |
| 0.8×10 5 | 5 | 5 | 100 |
| 0.4×10 5 | 5 | 4 | 80 |
2. Immunogenicity determination
2.1 preparation of DHN-QD Strain immunogens
Dissolving strain with sterile PBS according to lyophilized volume, collecting a small amount of strain solution with a strain inoculating ring, streaking inoculating on TSA agar plate, and inoculating at 37deg.C with 5CO 2 Culturing in an incubator for about 24 hours. 3-5 single colonies are picked from the plate and inoculated on TSA agar inclined plane, 37 ℃ and containing 5CO 2 Culturing in an incubator for about 24 hours to obtain first-stage seeds.
Washing the primary seeds on the inclined plane by using a TSB liquid culture medium, inoculating the TSB culture medium according to the proportion of 2% -3%, uniformly placing the TSB culture medium at 37 ℃ and carrying out shaking culture for 5-8 hours at 150-200 r/min, carrying out pure inspection according to the current annex of Chinese veterinary drug dictionary, and taking the qualified seeds as secondary seeds.
Preparing bacterial liquid: culturing by a fermentation tank, and preparing a TSB liquid culture medium according to 35% -70% of the volume of the fermentation tank. Inoculating the secondary seeds according to the proportion of 2% of the total amount of the culture medium, culturing for 8-10 hours at 37 ℃, and stirring at the speed of 150-200 r/min.
And (3) inactivation: slowly adding formaldehyde solution according to 0.15% of the total bacterial liquid, fully and uniformly mixing, and inactivating for 24-48 hours at 37+/-0.2 ℃.
And (3) checking a semi-finished product: sterile inspection is carried out according to the relevant method of veterinary drug dictionary, and sterile growth is needed.
Preparing an aqueous phase: 96mL of bacterial liquid and 4mL of sterilizing Tween-80 are fully and uniformly mixed in a sterilizing container.
Preparing an oil phase: 94mL of white oil for injection, 80 mL of span, and the mixture is heated to be completely dissolved, and then sterilized at 115 ℃ for 40 minutes.
Emulsification: and (3) transferring all the sterilized oil phase into an emulsifying tank, slowly transferring the water phase into the emulsifying tank after complete cooling, and emulsifying for 90 minutes at 3000rpm under the freezing of chilled water circulation during the period, thus obtaining the milky oil emulsion inactivated vaccine (DHN-QD strain vaccine).
2.2 immunogenicity assays
The prepared DHN-QD strain vaccine has an antigen content of 5.0X10 in the final product 8 CFU/mL. 60 SPF chickens with 60 days of immunization are injected with 0.5mL, 0.4mL, 0.3mL, 0.2mL and 0.1mL DHN-QD strain vaccine subcutaneously in the neck, 10 chickens in the control group are injected with 0.5mL PBS subcutaneously in the neck. Each immunization group was injected with 0.2mL (1.6X10) of DHN-QD strain culture per orbital sinus 30 days after inoculation 5 CFU), 7 days of observation.
As shown in Table 4, the 60-day-old SPF chicken was immunized for 30 days to attack the virus, the control group was all ill, and the immunization dose of the DHN-QD strain vaccine was 0.2mL, i.e., the antigen content in the final product was 2.0X10 8 CFU/mL, still can provide 90% protection rate, and the strain is good in immunogenicity.
TABLE 4 immunogenicity determination results
| Group of | Immune count | The number of the diseases is only counted | Protection rate/% |
| Immunization of 0.5mL | 10 | 0 | 100 |
| 0.4mL immunity | 10 | 0 | 100 |
| 0.3mL group immunization | 10 | 0 | 100 |
| 0.2mL immunity | 10 | 1 | 90 |
| 0.1mL group immunization | 10 | 3 | 70 |
| PBS control group | 10 | 10 | 0 |
EXAMPLE 4 passage stability assay
This example was used to evaluate the passaging stability of the avian paragallinarum DHN-QD strain isolated in example 1.
Subculture and enumeration of isolates (avian paragallinarum DHN-QD strain):
streaking frozen strain of avian bacterium paragallinarum DHN-QD strain on TSA agar plate containing 10% chicken serum and 0.01% NAD, picking colony in TSB liquid culture medium, and 5% CO 2 Culturing in a shaking incubator at 37 ℃ for 8-12 hours, repeatedly subculturing for 30 generations according to the method, and counting viable bacteria every 10 generations.
Virulence determination of passaged strains:
cultures were taken 10, 20, 30 times according to the procedure described above, according to 0.8X10 5 CFU/chicken, 0.4X10 5 CFU/chicken were vaccinated with 5 SPF chickens at 60 days of age, and the onset was observed within 7 days.
Immunogenicity determination of passaged strains:
the 10 th, 20 th and 30 th generation strains were taken, the immunogen was prepared as described in example 3, and the vaccine was formulated so that the avian paragallinarum DHN-QD strain was 2X 10 per 1mL of vaccine before inactivation 8 CFU/mL. The 10 th, 20 th and 30 th generation strains were used to prepare 10 vaccine for cervical subcutaneous injection with a dose of 0.5mL using 40 SPF chickens of 60 days old. A PBS injection blank was also set. Each group was injected with 0.2mL (1.6X10) of culture of avian bacterium paragallinarum DHN-QD strain in the sinus under the orbit 30 days after inoculation 5 CFU), 7 days of observation, and the morbidity was calculated.
Results:
the passage count result shows that the avian paragallinarum DHN-QD strain colony is picked every 10 generations in the TSB liquid culture medium, and the CO content is 5% 2 Culturing in a shaking incubator at 37 ℃ for 8-12 hours, wherein the counting results are 1.8X10 respectively 9 CFU/mL、2.1×10 9 CFU/mL、2.0×10 9 CFU/mL showed stable growth of avian paragallinarum DHN-QD strain during passage.
Taking the secondary chicken poles of 10 th, 20 th and 30 th generationsBacterial DHN-QD strain cultures at 0.8X10 5 CFU/chicken, 0.4X10 5 CFU/chickens were inoculated with 5 SPF chickens at 60 days of age and the virulence of cultures of the different generations of avibacterium paragallinarum DHN-QD strain was determined. The results showed that passage to 30 generations, 0.8X10 5 CFU/chicken vaccinated, still 100% morbidity, suggesting that DHN-QD strain virulence was stable after passage and unchanged with in vitro culture (table 5).
The 10 th, 20 th and 30 th generation strains are used for preparing immunogens, vaccine is prepared for immunization of SPF chickens at the age of 6 days, and the results of immune challenge protection tests show that 100% of the chickens immunized with antigens prepared by each passage strain are protected, so that the antigens prepared by the passage of the strain to the 30 th generation are good in immunogenicity and can resist the attack of the strain (Table 6).
TABLE 5 virulence determination results for passage strains
TABLE 6 immunogenicity determination results for passage strains
| Culture generation times | Immune count | The number of the diseases is only counted | Protection rate/% |
| Generation 10 | 10 | 0 | 100 |
| Generation 20 | 10 | 0 | 100 |
| Generation 30 | 10 | 0 | 100 |
| PBS control group | 10 | 10 | 0 |
EXAMPLE 5 Cross protection of the avian paragallinarum DHN-QD strain against serum A, B type
Monovalent seedlings of type A Hpg-8 strain, type A DHN-QD strain were prepared according to the vaccine formulation of example 3. Immunization of the non-SPF-free chickens at 60 days of age corresponds to 0.5 mL/chicken; a non-immune blank was also set up. And the virus is removed 30 days later, 0.2mL of virus removing bacteria liquid is used for each infraorbital sinus, and gradient virus removing control is set for each strain. After the toxicity attack, the chicken is observed for 7 days, and the chicken is just provided with swelling around the eyelid, tearing, nasal discharge and poisoning symptoms, can be subjected to a section examination to observe whether pericardial effusion exists, and if necessary, the disease and bacteria are separated.
The immune toxicity attack protection result shows that the serum A type HPG-8 strain can be used for preparing vaccine, can provide good immune protection for the strain with the same serum type, and has no protection effect basically, and the protection rate for the heterologous B type serum strain is only 10%. The avian paragallinarum strain of serotype A, DHN-QD, produced a vaccine, provided excellent immune protection against strains of the same serotype, while the protection against heterologous B-type serum strains was 60% (Table 7). The avian bacterium paragallinarum DHN-QD strain is prompted to prepare the vaccine, a certain cross protection can be provided, the vaccine has a better immune protection spectrum, and the method has important significance for developing the chicken infectious rhinitis vaccine with wide immune protection spectrum and good safety.
TABLE 7 results of immune challenge
Note that: DHN-SDTA, DHN-AHBZ and DHN-SXXA in the tables were isolated for the inventors and subjected to corresponding serotype identifications (serological identification+HMTp-210 gene hypervariable region).
Example 6 avian paragallinarum DHN-QD strain vaccine compared to the existing vaccine
1. Preparation and inspection of avian bacillus paragallinarum DHN-QD strain vaccine
A group 2 vaccine was prepared according to the method of example 3, vaccine 1 was a single vaccine of avian paragallinarum DHN-QD strain, and each 1mL of vaccine before inactivation contained avian paragallinarum DHN-QD strain as 2X 10 8 CFU/mL. Vaccine 2 is a bivalent inactivated vaccine of a serum A type avibacterium paragallinarum DHN-OD strain and a serum B type avibacterium paragallinarum DHN-AHBZ strain, and each 1mL of vaccine before inactivation contains 2X 10 serum A type DHN-QD strain 8 CFU/mL, serum-containing type B DHN-AHBZ strain of 1X 10 8 CFU/mL。
The appearance, formulation, viscosity, sterility, safety of the two groups of vaccines were checked and the results were verified as follows: the finished product appearance of the group 2 vaccine is milky uniform emulsion, the dosage form is water-in-oil type (a clean suction tube is taken, a small amount of vaccine is dripped into cold water, and is not diffused except for the first drip), the stability is good (10 mL of vaccine is sucked, the vaccine is added into a centrifuge tube and is centrifuged at 3000rpm/min for 15 minutes, and no water phase is separated out at the bottom of the tube), the safety is high (8 healthy susceptible chickens of 60 days old are used, 1mL of vaccine is injected subcutaneously for each, 14 days are observed, and the result has no abnormal reaction); the viscosity test is carried out according to annex 3102 of Chinese animal pharmacopoeia, and the results are 62.2Cp; sterility test showed that at 37deg.C, CO 2 Sulfur salt (TG) and TSA agar slants containing 10% chicken serum and 0.01% NAD were cultured in an incubator at 25 ℃Both cultured TSB, TG and TSA agar slants containing 10% chicken serum and 0.01% NAD grew aseptically, indicating that the antigens of both sets of vaccines were uncontaminated and completely inactivated.
2. Comparison with the existing bivalent inactivated vaccine (A type+C type) for infectious coryza of chicken
The prepared vaccine 1 and vaccine 2 are respectively immunized with the existing bivalent inactivated vaccine for infectious rhinitis of chicken (marked as vaccine 3, manufactured by Luoyang Huizhong Biotechnology Co., ltd., batch number: 220405), mycoplasma gallisepticum and infectious rhinitis (A, C) bivalent inactivated vaccine (marked as vaccine 4, zhaoda Hua agricultural biopharmaceutical Co., manufactured batch number: 2233003) for 60 days of SPF chicken, injected subcutaneously in the neck, 0.5 mL/chicken, and a non-immune blank control is established. And (5) attacking toxin 30 days after immunization, and observing the disease condition of SPF chickens after the toxin is attacked.
As shown in Table 8, the avian paragallinarum DHN-QD strain has good immunogen and can provide high protection of more than 90% against the same serotype strain. And vaccine 4 (1 mL vaccine before inactivation contains type A221 strain with bacterial count of more than or equal to 5×10) 8 The strain containing C type 668 is more than or equal to 5 multiplied by 10 8 And (c)) is low in serum type A antigen content in vaccine 1 and vaccine 2 (2X 10 per 1mL vaccine before inactivation 8 CFU/mL), but the toxicity-counteracting immune protection effect is superior to that of vaccine 4, further showing that the avian paragallinarum DHN-QD strain vaccine strain has good immunogenicity. Vaccine 3 and vaccine 4 have poor protective effect (less than 30%) against serotype B strains, while vaccine 1 prepared by a serotype A parachicken bacillus DHN-QD strain has 50% protective rate against heterologous type B serum strains and has better immune cross protection. Avian secondary strain DHN-QD antigen and low antigen amount of serum type B avian secondary strain DHN-AHBZ antigen (1×10 per 1ml vaccine before inactivation 8 CFU/mL) vaccine 2, which provides high protection of more than 90% against both type a and type B strains. The avian secondary bacillus DHN-QD strain with the cross protection function is utilized to prepare the vaccine, so that the content of antigen required in a vaccine finished product can be effectively reduced, the endotoxin content is low, and the high protection rate and less immune side reaction can be provided with lower production cost.
TABLE 8 immunization efficacy of different inactivated vaccines
3. Comparison with imported trivalent inactivated vaccine against infectious coryza of chickens
Vaccine 1, vaccine 2 and chicken infectious rhinitis import inactivated vaccine (comprising inactivated avibacterium paragallinarum A083 strain, B Spross strain and C H-18 strain 3 serotypes, wherein the content of each strain before inactivation is at least 3×10) 8.0 CFU/plume, noted vaccine 5), were immunized with 60 day old SPF chickens, injected subcutaneously in the neck, 0.5 mL/mouse, while a non-immunized placebo was established. And (5) attacking toxin 30 days after immunization, and observing the morbidity of the SPF chicken.
As shown in Table 9, the avian paragallinarum DHN-QD strain is used for preparing the vaccine 1, and has certain immune cross protection on the heterologous B type serum strain. Avian pullorum strain DHN-QD antigen and low antigen amount of serum type B DHN-AHBZ antigen (1X 10 per 1mL vaccine before inactivation 8 CFU/mL) provides high protection of more than 90% against both type a and type B strains. The protection effect of the vaccine 1 and the vaccine 2 is obviously better than that of an imported vaccine. The antigen content is lower than that of imported vaccine, so that the avian paragallinarum DHN-QD strain has good immunogenicity and better cross protection.
Table 9 shows the immune effects of different inactivated vaccines
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes may be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
Claims (10)
1. The avian bacterium paragallinarum is named as avian bacterium paragallinarum DHN-QD strain, and is preserved in China Center for Type Culture Collection (CCTCC) NO: m2023968, the preservation date is 2023, 06 and 08.
2. A fowls bacillus agent, comprising at least one of fowls bacillus according to claim 1, a fermentation culture of fowls bacillus according to claim 1, and a fermentation supernatant of fowls bacillus DHN-QD strain according to claim 1.
3. Use of the avibacterium paragallinarum of claim 1 and/or the avibacterium paragallinarum of claim 2 in the preparation of a product.
4. A use according to claim 3, characterized in that the product has the following functions: preventing and/or treating fowl bacillus paragallinarum related diseases.
5. Use according to claim 3, wherein the product comprises a vaccine or a medicament.
6. A vaccine comprising the avibacterium paragallinarum of claim 1 or the avibacterium paragallinarum agent of claim 2.
7. The vaccine of claim 6, wherein said avian paragallinarum strain DHN-QD inactivated whole-cell antigen content is 1X 10 or more before inactivation 8 CFU/mL。
8. The vaccine of claim 6, wherein the vaccine isThe content of the whole inactivated antigen of the avian bacterium paragallinarum DHN-QD strain is 1 multiplied by 10 before inactivation 8 CFU/mL～5×10 8 CFU/mL。
9. The vaccine of any one of claims 6-8, further comprising a pharmaceutically acceptable adjuvant.
10. A method of preparing the vaccine of claim 9, comprising the steps of:
culturing the avian paragallinarum DHN-QD strain;
inactivating the cultured culture of the avian paragallinarum DHN-QD strain by formaldehyde, mixing the inactivated culture with auxiliary materials, and emulsifying to obtain the vaccine.
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