A strain of Avibacterium paragallinarum, its application and vaccine

Abstract

The present invention relates to the field of biotechnology, and in particular to a strain of Haemophilus paragallinarum, which is Haemophilus paragallinarum DHN-WZN, classified as Haemophilus paragallinarum DHN-WZN, and deposited as CCTCC NO: M2023969; deposit date: June 8, 2023, deposited at: China Center for Type Culture Collection; deposited at: Wuhan, China, Wuhan University. The bacterium has good immunogenicity and high passage stability; the vaccine prepared using the Haemophilus paragallinarum DHN-WZN strain has a protection rate of more than 90% for strains of the same serotype, and a protection rate of 80% for heterologous type B serotype strains, and has a good immune cross-protection effect. The vaccine is prepared using the Haemophilus paragallinarum DHN-WZN strain with cross-protection, which can effectively reduce the required antigen content in the finished vaccine product, and can provide a high protection rate and fewer immune side effects at a lower production cost. At the same time, the present invention also provides the use of the Haemophilus paragallinarum.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an Avibacterium paragallinarum strain, application and vaccine. Background Art

[0002] Avibacterium paragallinarum (Apg), formerly known as Haemophilus paragallinarum (Hpg), is a pathogen that causes infectious coryza (IC) in chickens. IC is an acute upper respiratory tract infection and toxin-induced disease of chickens, characterized by inflammation of the nasal cavity and sinuses, sneezing, runny nose, facial swelling, etc. It is distributed worldwide and can cause a decrease in egg production in laying hens, growth retardation and increased culling rates in growing chickens, and a decrease in the quality of broiler chickens, causing great economic losses. Beach first reported the disease in 1920, and DeBlieck first isolated Haemophilus paragallinarum in 1932. Feng Wenda of my country first isolated it in 1987. At present, the disease has been widely prevalent in various breeding areas, especially affecting the production performance of laying hens.

[0003] The two serotyping schemes (Page and Kume) of the bacteria are both established based on the hemagglutination inhibition test. The classic Page typing method divides Apg into three serotypes: A, B, and C. The cross-protection of different serotypes is very weak or there is no cross-immune protection. The Kume typing scheme divides nine serotypes, A-1, A-2, A-3, A-4, B-1, C-1, C-2, C-3, and C-4, through the hemagglutination inhibition test. However, due to the cumbersome process and harsh experimental conditions of the Kume typing scheme, the Page serotyping scheme is still the main one. Among them, type B Apg is the main epidemic strain, but Zhao Yi et al. and Chen Xiufang pointed out through investigation that since 2019, the epidemic situation of Apg has changed compared with the past. At present, all three serotypes are still prevalent, but the incidence of type B Apg, which has been dominant before, has decreased, while the incidence of type C Apg has gradually increased.

[0004] At present, the vaccines used to prevent infectious coryza in chickens are all inactivated vaccines, which are divided into monovalent vaccines, bivalent vaccines and trivalent vaccines according to the different pathogens used, and no successful attenuated vaccines or other types of live vaccines have been put into use. Due to the poor cross-protection effect between different serotypes, and the three serotypes are currently popular, the farms mostly use trivalent inactivated vaccines containing three serotypes A, B and C for immunization. Chinese patent CN108624522B discloses Avibacterium paragallinarum type B HN5 strain and its prepared vaccine composition and application, the vaccine composition comprises inactivated whole bacteria antigen of Avibacterium paragallinarum type B HN5 strain, inactivated whole bacteria antigen of Avibacterium paragallinarum type A HN3 strain, and inactivated whole bacteria antigen of Avibacterium paragallinarum type C SD3 strain. Chinese patent CN110448690B discloses a two-unit inactivated vaccine for infectious rhinitis (type A+type B+type C) and rhinotracheal ornithobacterium disease (type A) and a preparation method thereof, wherein the vaccine contains inactivated antigens of Avian bacillus paragallinarum type A QL-Apg-3 strain, Avian bacillus paragallinarum type B QL-Apg-26 strain, Avian bacillus paragallinarum type C QL Apg-15 strain and serum type A rhinotracheal ornithobacterium GD05 strain. In the above-mentioned prior art, the vaccine needs to contain inactivated antigens of three serotypes, type A, type B and type C. However, when preparing the antigen, serum and coenzyme need to be added to Hpg culture, and the culture cost is high. Formaldehyde needs to be added for inactivation during harvest, resulting in the presence of bacterial proteins, endotoxins and other toxic substances in the finished vaccine, which is easy to cause large side effects after inoculation, and focal necrotic spots will also be formed at the inoculation site. In addition, the current commercial inactivated vaccine is not ideal for the protection of epidemic strains. Therefore, it is of great significance to develop an infectious coryza vaccine with a broad immune protection spectrum and good safety. Summary of the invention

[0005] The present invention aims to provide a strain of Avibacterium paragallinarum, which can be used for infection caused by Avibacterium paragallinarum serotype B and C. The vaccine prepared by the strain has a protection rate of more than 90% against strains of the same serotype and an 80% protection rate against heterologous B-type serotype strains, and has a good immune cross-protection effect.

[0006] Meanwhile, the invention also provides the application of the Avibacterium paragallinarum.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] A strain of Haemophilus paragallinarum, wherein the Haemophilus paragallinarum is Haemophilus paragallinarum DHN-WZN, classified as Haemophilus paragallinarum DHN-WZN, with a preservation number of CCTCC NO: M2023969; preservation date: June 8, 2023, preservation unit: China Center for Type Culture Collection; preservation address: Wuhan University, Wuhan, China.

[0009] In addition, the present invention also discloses the use of the Avibacterium paragallinarum described above in preparing a vaccine.

[0010] Finally, the present invention also discloses a vaccine, wherein the vaccine contains the inactivated Avibacterium paragallinarum as described above.

[0011] Preferably, the vaccine is a vaccine for providing cross protection against A. paragallinarum type C and A. paragallinarum type B.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The Avibacterium paragallinarum DHN-WZN provided by the present invention has good immunogenicity and high passage stability; a vaccine prepared by using the Avibacterium paragallinarum DHN-WZN strain has a protection rate of more than 90% against strains of the same serotype and an 80% protection rate against heterologous type B serotype strains, and has a good immune cross-protection effect; the vaccine prepared by using the Avibacterium paragallinarum DHN-WZN strain with cross-protection can effectively reduce the antigen content required in the finished vaccine product, and can provide a high protection rate and fewer immune side effects at a lower production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the PCR identification result diagram (Note: from left to right in the diagram are 2000bpMarker, parallel 1, parallel 2, positive control, and negative control);

[0015] Figure 2 This is a genetic evolutionary tree diagram. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0017] Example 1: Isolation and identification of Avibacterium paragallinarum DHN-WZN strain

[0018] 1. Isolation of Avian Bacterium paragallinarum DHN-WZN strain

[0019] Sample collection: Purulent secretions from the sinuses of sick chickens from a large laying hen farm in Yunfu, Guangdong in February 2021.

[0020] Preparation of finished liquid culture medium: tryptic soy broth (TSB) medium + 10% standard chicken serum + 0.1 g / L final concentration of coenzyme I (NAD).

[0021] Preparation of finished solid culture medium: tryptic soy agar (TSA) medium + 10% standard chicken serum + 0.1 g / L final concentration of coenzyme I (NAD).

[0022] The strains were cultured under 5% carbon dioxide conditions.

[0023] Isolation and culture of strains:

[0024] The clinically ill chickens were collected and sent to the laboratory for bacterial identification. Its clinical symptoms are inflammation of the nasal cavity and sinuses, sneezing, runny nose, facial swelling, etc. On a sterile clean bench, cut open the sinuses, and inoculate the sinus contents with a sterile cotton swab or a sterile inoculation loop into a solid plate for 16 to 24 hours. Small colonies with translucent dew drops and neat edges can be formed. The suspected colonies in the above plate were subcultured and purified. After purification, a single colony was inoculated into an appropriate amount of liquid culture medium, 150r / min, cultured for 6 to 8 hours, and the bacterial liquid was harvested for subsequent identification.

[0025] 2. Identification of Avibacterium paragallinarum DHN-WZN strain

[0026] 2.1 PCR identification

[0027] According to the universal primers for identifying infectious rhinitis in chickens published in "NY / T538-2015 Diagnostic Technology for Infectious Rhinitis in Chickens", the primer sequences are as follows:

[0028] Upstream primer: 5'-TGAGGGTAGTCTTGCACGCGAAT-3' (SEQ ID NO: 1);

[0029] Downstream primer: 5'-CAAGGTATCGATCGTCTCTCTACT-3' (SEQ ID NO: 2);

[0030] The expected amplified fragment size is 500 bp.

[0031] The bacterial genome was extracted using a commercial extraction kit, and double distilled water was used as a negative control. Universal primers were used for PCR amplification. The reaction system was 25 μL. The PCR reaction system is shown in Table 1. The PCR reaction procedure was as follows: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 45 seconds, annealing at 56°C for 45 seconds, and extension at 72°C for 1 minute, for a total of 30 cycles; and extension at 72°C for another 10 minutes.

[0032] Table 1 PCR reaction system

[0033]

[0034] Electrophoresis analysis: Take 5 μL of PCR product and perform detection by 1% agarose gel electrophoresis. Judgment: If the sample to be tested amplifies a band of about 500 bp, it is judged as positive; otherwise, it is judged as negative.

[0035] The identification results are as follows Figure 1 As shown, a band of about 500 bp was amplified from the sample to be tested, which was judged to be positive for chicken infectious coryza, indicating that the isolated strain was Avibacterium paragallinarum, named Avibacterium paragallinarum DHN-WZN strain.

[0036] 2.2 Serotyping

[0037] Preparation of highly immune serum: Positive serum was prepared from clean experimental rabbits immune to standard strains and stored at -20°C. Haemophilus paragallinarum type A Hpg-221, type B CCM6075, and type C Modesto standard strains were purchased from the China Veterinary Drug Administration.

[0038] Preparation of antigen to be tested: The isolated strain to be tested (Avibacterium paragallinarum DHN-WZN strain) was inoculated into TSA solid medium at 37°C with 5% CO 2 Incubate in the incubator for 18-24 hours. Pick 2-3 single colonies and inoculate them into 50mL TSB liquid medium for expansion culture. Incubate at 37℃ for 6-8 hours. Centrifuge at 8000g for 15 minutes to collect the bacteria. Resuspend the bacterial precipitate in 0.01mmol / L PBS and wash it three times. Resuspend the bacterial precipitate in a solution containing 0.5M KSCN and 0.425M NaCl at pH 6.3. Finally adjust the OD 650 The concentration of the antigen was 1.6, incubated at 4°C for 2 hours, and then ultrasonically lysed. Finally, the antigen was washed three times with PBS containing 0.001% thimerosal, suspended in 2 mL of PBS, and stored at 4°C to obtain the antigen to be tested.

[0039] HI determination test:

[0040] Prepare 50 μL of 1:10 to 1:1280 dilution antiserum in PBSS (PBS containing 0.1% BSA and 0.001% gelatin). Add 50 μL of antigen containing 4 hemagglutination units to each well and incubate at room temperature for 40 minutes. Then add 50ul of 1% aldehyde-treated red blood cells and incubate at room temperature for 30 to 40 minutes before reading. Judgment standard: The highest serum dilution that can completely inhibit hemagglutination of red blood cells is the HI titer of this type of serum. If the antigen being tested has agglutination inhibition with more than one type of positive serum, the one with the highest HI titer is that serum type. HI titers <10 are judged as negative.

[0041] The results are shown in Table 2. The DHN- strain of Haemophilus paragallinarum can neutralize the antibodies in the serum of the type C reference strain Modesto, so that the red blood cells cannot agglutinate, indicating that the DHN-WZN strain of Haemophilus paragallinarum is type C Haemophilus paragallinarum. At the same time, it is interesting that the DHN-WZN strain of Haemophilus paragallinarum can partially neutralize the antibodies in the serum of the type B reference strain CCM6075. The DHN-WZN strain of Haemophilus paragallinarum (Haemophilus paragallinarum DHN-WZN) was submitted to the China Center for Type Culture Collection for preservation on June 8, 2023, with a preservation number of CCTCC NO: M2023969.

[0042] Table 2 HI determination test results

[0043]

[0044] Example 2: Sequence analysis of the hypervariable region (HVR) of the HMTp-210 gene of Avibacterium paragallinarum DHN-WZN strain

[0045] Primers were designed to amplify the highly variable region of the HMTp-210 gene of Avibacterium paragallinarum.

[0046] The sequences of the serotype B amplification primers are: 5'-AACGAACCTTATGTTGAGCAAAGTG-3' (SEQ ID NO: 3) 5'-ATTTTGCCTTCTCAGGCTTATTTGA-3' (SEQ ID NO: 4).

[0047] The primer sequences for amplifying serotype C were: 5'-CCATTAACGGCTCACAGCTTTATGC-3' (SEQ ID NO: 5), 5'-CTCAATAGAAATATTTGGCTGTAATG-3' (SEQ ID NO: 6). The target fragments were amplified and sent for testing, and the gene sequences of A. paragallinarum serotype B and C were downloaded from the NCBI (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) database. Sequence alignment was performed using ClustalX 2.0 and MEGA software, respectively, and finally the phylogenetic tree was constructed using the Neighbor-Joining (NJ) method.

[0048] The analysis results showed that the 11 clinically isolated strains identified as type B by serum separation were clustered in an independent branch with type B standard serum strains 0222, 2671, and 48 on the phylogenetic tree. The 11 clinically isolated strains identified as type C by serum separation were clustered in another independent branch with type C standard serum strains GD85 and GD1861 on the phylogenetic tree. The DHN-WZN strain of Avian bacterium paragallinarum was serotyped as type C, but was classified in the branch of serotype B on the phylogenetic tree, such as Figure 2 As shown. The results of sequence alignment analysis showed that the HVR sequence identity of the Avian Bacterium paragallinarum DHN-WZN strain and the C-type standard serotype strain GD485 strain was only 81.8%, while the HVR sequence identity with the B-type standard serotype strain 0222 strain was 99.2%. It is speculated that the Avian Bacterium paragallinarum DHN-WZN strain is a strain recombined by serotype B and serotype C. This has also been reported in the literature. Taiwanese scholars Tan et al. isolated and obtained multiple local epidemic strains. The serotyping results showed that the TW 16-4 isolate had a high HI titer with the positive sera of the serotype B and serotype C standard strains. The full-length evolution analysis of the HMTp210 gene showed that the gene of the TW 16-4 isolate was a chimeric type of serotype B and serotype C, and it was speculated that the strain was a strain produced by the recombination of serotype B and serotype C.

[0049] Example 3: Virulence determination and immunogenicity study of Avibacterium paragallinarum DHN-WZN strain

[0050] 1. Toxicity Assay

[0051] The freshly cultured bacterial solution was diluted with TSB medium, and each dilution of bacterial solution was inoculated into the infraorbital sinus of 5 60-day-old SPF chickens. The control was inoculated with PBS, and 0.2 mL was injected into each infraorbital sinus. The disease was observed within 7 days (the face had swelling of the infraorbital sinus and surrounding areas on one or both sides and runny nose or tears). After the challenge, the viable bacteria of the challenge bacterial solution were counted to determine the actual challenge dose.

[0052] The results are shown in Table 3. 60-day-old SPF chickens were injected with 1.1×10 5 CFU and above, all chickens developed swelling of the infraorbital sinus and surrounding areas on one or both sides of the face or runny nose within 24 to 60 hours. Avibacterium paragallinarum could be isolated again from the infraorbital sinus of the infected chickens; 5.5×10 4 CFU bacteria were inoculated, and only one chicken did not fall ill during the 7-day observation period, indicating that the DHN-WZN strain of Avibacterium paragallinarum is highly virulent.

[0053] Table 3 Toxicity test results

[0054] The amount of virus / CFU Number of attacks Number of cases Incidence / % 8.4×105CFU 5 5 100 4.2×105CFU 5 5 100 2.1×105CFU 5 5 100 1.1×105CFU 5 5 100 5.5×104CFU 5 4 80

[0055] 2. Immunogenicity Assay

[0056] 2.1 Preparation of DHN-WZN strain immunogen

[0057] Dissolve the strain in sterile PBS according to the freeze-dried volume, pick up a small amount of bacterial solution with a bacterial loop, and streak inoculate on a TSA agar plate at 37°C and 5% CO 2 Culture in an incubator for about 24 hours. Pick 3 to 5 single colonies from the plate and inoculate them on TSA agar slant at 37°C and 5% CO 2 Cultivate in an incubator for about 24 hours as primary seeds.

[0058] Wash the first-level seeds on the slope with TSB liquid culture medium, inoculate TSB culture medium at a ratio of 2% to 3%, incubate at 37°C and 150-200 r / min for 5-8 hours, conduct purity inspection according to the appendix of the current Chinese Pharmacopoeia of Veterinary Medicine, and use them as second-level seeds after passing the inspection.

[0059] Preparation of bacterial liquid: Use a fermenter for cultivation, prepare TSB liquid culture medium according to 35% to 70% of the volume of the fermenter. Inoculate secondary seeds at a ratio of 2% of the total volume of the culture medium, cultivate at 37°C for 8 to 10 hours, and stir at a speed of 150 to 200 r / min.

[0060] Inactivation: Slowly add formaldehyde solution at 0.15% of the total bacterial solution, mix thoroughly, and inactivate at 37±0.2℃ for 24 to 48 hours.

[0061] Semi-finished product inspection: Sterility test shall be carried out according to the relevant methods of the veterinary pharmacopoeia, and sterile growth shall be observed.

[0062] Preparation of aqueous phase: 96 mL of bacterial solution, 4 mL of sterile Tween-80, mix thoroughly in a sterile container.

[0063] Preparation of oil phase: 94 mL of white oil for injection and 9 mL of Siben-80, mix and heat until completely dissolved, and sterilize at 115°C for 40 minutes.

[0064] Emulsification: Transfer all the sterilized oil phase into the emulsification tank. After it is completely cooled, slowly transfer the water phase into the emulsification tank. During this period, emulsify at 3000 rpm for 90 minutes under refrigerated water circulation to obtain a milky white oil emulsion inactivated vaccine (DHN-WZN strain vaccine).

[0065] 2.2 Immunogenicity assay

[0066] The prepared DHN-WZN strain vaccine has an antigen content of 10.0×10 8CFU / mL. 60 SPF chickens aged 60 days were used for immunization, and 0.5mL, 0.4mL, 0.3mL, 0.2mL, and 0.1mL of DHN-WZN vaccine were injected subcutaneously in the neck. Ten chickens in the control group were injected subcutaneously in the neck with 0.5mL PBS. 30 days after vaccination, 0.2mL (1.0×10 5 CFU) and observed for 7 days.

[0067] The results are shown in Table 4. 60-day-old SPF chickens were challenged with the virus 30 days after immunization. All the chickens in the control group became ill. The immunization dose of the DHN-WZN strain vaccine was 0.2 mL, that is, the antigen content in the finished product was 2.0×10 8 CFU / mL, it can still provide a 90% protection rate, which shows that the strain has good immunogenicity.

[0068] Table 4 Immunogenicity determination results

[0069] Group Number of immunizations Number of cases Protection rate / % 0.5mL immune 10 0 100 0.4mL immune 10 0 100 0.3mL immune 10 0 100 0.2mL immune 10 1 90 0.1mL immune 10 4 60 PBS control group 10 10 0

[0070] Example 4: Determination of cell stability

[0071] This example is used to evaluate the passage stability of the Avibacterium paragallinarum DHN-WZN strain isolated in Example 1.

[0072] Subculture and enumeration of the isolated strain (Avibacterium paragallinarum DHN-WZN strain):

[0073] The frozen strain of Avibacterium paragallinarum DHN-WZN was streaked onto a TSA agar plate containing 10% chicken serum and 0.01% NAD, and colonies were picked and inoculated into TSB liquid medium with 5% CO 2 The culture was cultured at 37°C in a shaking incubator for 8 to 12 hours. This method was repeated for 30 generations, and live bacteria were counted every 10 generations.

[0074] Virulence determination of subculture strains:

[0075] According to the method described above, the cultures of passages 10, 20, and 30 were taken and the culture medium was diluted at 1.0 × 10 5 CFU / chicken, 0.5×10 5 Five 60-day-old SPF chickens were inoculated with CFU / chicken, and the disease development was observed within 7 days.

[0076] Immunogenicity determination of subculture strains:

[0077] The 10th, 20th and 30th generation strains were used to prepare immunogens and vaccines according to the method described in Example 3. Before inactivation, each mL of vaccine contained 4×10 8CFU / mL. Forty 60-day-old SPF chickens were used, and the vaccine prepared by the 10th, 20th, and 30th generation strains was injected subcutaneously in the neck of 10 chickens, with an injection dose of 0.5 mL. A PBS injection blank control group was also set up. Thirty days after vaccination, each chicken in each group was injected with 0.2 mL (1.0×10 5 CFU), observed for 7 days, and calculated the incidence rate.

[0078] result:

[0079] The results of subculture counting showed that colonies of Avibacterium paragallinarum DHN-WZN strain were picked every 10 generations and incubated in TSB liquid medium with 5% CO 2 The cells were cultured at 37°C in a shaking incubator for 8 to 12 hours, and the count results were 2.5×10 9 CFU / mL, 2.2×10 9 CFU / mL, 2.5×10 9 CFU / mL, indicating that the A. paragallinarum DHN-WZN strain grew stably during the subculture process.

[0080] The 10th, 20th, and 30th generation cultures of Avibacterium paragallinarum DHN-WZN strain were cultured at 1.0 × 10 5 CFU / chicken, 0.5×10 5 CFU / chicken 5 SPF chickens aged 60 days were inoculated to determine the virulence of A. paragallinarum DHN-QD strain cultures of different generations. The results are shown in Table 5. When the culture was subcultured to the 30th generation, 1.0×10 5 CFU / chicken inoculation, there was still 100% incidence rate, indicating that the virulence of DHN-WZN strain was stable after subculture and did not change with in vitro culture.

[0081] The 10th, 20th and 30th generation strains were used to prepare immunogens, and the vaccines were used to immunize 6-day-old SPF chickens. The results of the immune protection test are shown in Table 6. The results showed that 100% of the chickens immunized with the antigens prepared from each generation strain were protected, indicating that the antigens prepared from the 30th generation of the bacteria still have good immunogenicity and can resist the attack of this bacteria.

[0082] Table 5 Virulence test results of subculture strains

[0083]

[0084] Table 6 Immunogenicity test results of subculture strains

[0085] Culture generation Number of immunizations Number of cases Protection rate / % 10th Generation 10 0 100 20th Generation 10 0 100 30th Generation 10 0 100 PBS control group 10 10 0

[0086] Example 5: Cross-protection of Avibacterium paragallinarum DHN-WZN strain against serotypes B and C

[0087] According to the vaccine formula of Example 3, monovalent vaccines of type C Hpg-668 strain and type C DHN-WZN strain were prepared. Non-immune SPF chickens were immunized with the corresponding vaccine at 60 days of age, 0.5 mL / chicken; a non-immune blank control was set up at the same time. The chickens were challenged with 0.2 mL of bacterial solution in the infraorbital sinus 30 days after immunization, and gradient challenge controls for each strain were set up at the same time. After the challenge, the chickens were observed for 7 days to see if they had swelling around the eyelids, tears, runny nose, and symptoms of poisoning. An autopsy could be performed to observe whether there was pericardial effusion, and bacterial isolation of the diseased material could be performed if necessary.

[0088] The results of immune attack protection are shown in Table 7. The results show that the vaccine prepared by serotype C Hpg-668 strain can provide good immune protection to strains of the same serotype, while the protection rate for heterologous B-type serotype strains is only 10%, which is basically no protective effect. The vaccine prepared by serotype C Avian bacillus paragallinarum DHN-WZN strain can provide good immune protection to strains of the same serotype, and the protection rate for heterologous B-type serotype strains is 70%. It is suggested that the vaccine prepared by Avian bacillus paragallinarum DHN-WZN strain can provide certain cross protection and have a better immune protection spectrum, which is of great significance for the development of chicken infectious coryza vaccine with a wide immune protection spectrum and good safety.

[0089] Table 7 Immune challenge results

[0090]

[0091] Note: DHN-XAXM and DHN-SCXN in the table were isolated by the inventors and the corresponding serotypes were identified (serum isolation and identification + HMTp-210 gene hypervariable region analysis), HPG-Beijing was donated by the Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agriculture and Forestry Sciences, and HPG-668 was purchased from the National Institute of Veterinary Drug Administration.

[0092] Example 6: Comparison of vaccines prepared from Avibacterium paragallinarum DHN-WZN strain with existing vaccines

[0093] 1. Preparation and testing of A. paragallinarum DHN-WZN strain vaccine

[0094] Two groups of vaccines were prepared according to the method of Example 3. Vaccine 1 was a single vaccine of Avibacterium paragallinarum DHN-WZN strain. Before inactivation, each mL of vaccine contained 4×10 8 CFU / mL. Vaccine 2 is a dual inactivated vaccine of serotype B Avian bacterium paragallinarum Hpg-Beijing strain and serotype C Avian bacterium paragallinarum DHN-WZN strain. Before inactivation, each mL of vaccine contains 2×10 serotype B Hpg-Beijing strains. 8 CFU / mL, DHN-WZN strain containing serotype C was 4×10 8 CFU / mL.

[0095] The appearance, dosage form, viscosity, sterility and safety of the above two groups of vaccines were tested, and the verification results are as follows: the appearance of the finished products of the two groups of vaccines is a milky white uniform emulsion, the dosage form is oil-in-water type (take a clean pipette, draw a small amount of vaccine and drop it into cold water, except for the first drop, it does not spread), good stability (draw 10mL of vaccine, add it to a centrifuge tube, centrifuge at 3000rpm / min for 15 minutes, and no water phase precipitates at the bottom of the tube), high safety (8 healthy susceptible chickens of 60 days old were used, each was subcutaneously injected with 1mL of vaccine, and observed for 14 days, and the results showed no abnormal reactions); the viscosity test was carried out according to Appendix 3102 of the Chinese Pharmacopoeia of Veterinary Medicine, and the results were all 60.1Cp; the sterility test showed that at 37℃ and CO 2 The thiocyanate (TG) cultured in the incubator and the TSA agar slants containing 10% chicken serum and 0.01% NAD, as well as the TSB, TG and TSA agar slants containing 10% chicken serum and 0.01% NAD cultured at 25°C all showed sterile growth, indicating that the antigens of the two groups of vaccines were not contaminated and were completely inactivated.

[0096] 2. Comparison with existing inactivated vaccines for infectious coryza

[0097] The prepared vaccines 1 and 2 were mixed with Mycoplasma gallisepticum and infectious coryza (type A and C) bivalent inactivated vaccine (referred to as vaccine 3, Zhaoqing Dahuanong Biopharmaceutical Co., Ltd., production batch number: 2233003), and imported inactivated vaccine for infectious coryza (containing three serotypes of inactivated Avian Bacillus paragallinarum type A 083 strain, type B Spross strain, and type C H-18 strain, with the content of each strain before inactivation at least 3×10 8.0 CFU / feather, recorded as vaccine 4). 60-day-old SPF chickens were immunized by subcutaneous injection in the neck, 0.5 mL / chicken, and a non-immunized blank control was set up. The chickens were challenged 30 days after immunization, and the incidence of SPF chickens after challenge was observed.

[0098] The results are shown in Table 8. The immunogen of A. paragallinarum DHN-WZN strain is good, and it can provide more than 90% high protection against the same serotype strain. Vaccine 3 has poor protection against serotype B strains (less than 30%), while vaccine 1 prepared from serotype C A. paragallinarum DHN-WZN strain has a protection rate of 80% against heterologous serotype B strains, and has good immune cross protection. The antigen of A. paragallinarum DHN-WZN strain and the antigen of serotype B A. paragallinarum DHN-Beijing strain with low antigen amount (each 1ml of vaccine contains 2×10 serotype B A. paragallinarum DHN-Beijing strain before inactivation) 8CFU / mL) to prepare vaccine 2, which can provide more than 90% high protection against type B and type C strains, and the protection effects of vaccine 1 and vaccine 2 are significantly better than those of imported vaccines. The use of the cross-protective Avian Bacillus paragallinarum DHN-WZN strain to prepare vaccines can effectively reduce the antigen content required in the finished vaccine, and can provide high protection rate and fewer immune side effects at a lower production cost.

[0099] Table 8 Immune effects of different inactivated vaccines

[0100]

[0101] In summary, the Avibacterium paragallinarum DHN-WZN provided by the present invention has good immunogenicity and high stability in passage; the vaccine prepared using the Avibacterium paragallinarum DHN-WZN strain has a protection rate of more than 90% against strains of the same serotype, and a protection rate of 80% against heterologous type B serotype strains, and has a good immune cross-protection effect. The use of the Avibacterium paragallinarum DHN-WZN strain with cross-protection to prepare a vaccine can effectively reduce the required antigen content in the finished vaccine product, and can provide a high protection rate and fewer immune side effects at a lower production cost.

[0102] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and scope of the equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A strain of Avibacterium paragallinarum, characterized in that The Haemophilus paragallinarum is Haemophilus paragallinarum DHN-WZN, classified as Haemophilus paragallinarum DHN-WZN, with a preservation number of CCTCC NO: M2023969; preservation date: June 8, 2023, preservation unit: China Center for Type Culture Collection; preservation address: Wuhan University, Wuhan, China.

2. Use of the Avibacterium paragallinarum as claimed in claim 1 in preparing a vaccine.

3. A vaccine, characterized in that The vaccine contains the inactivated Avibacterium paragallinarum as claimed in claim 1.

4. The vaccine according to claim 3, characterized in that The vaccine is a vaccine for providing cross protection against Avibacterium paragallinarum type C and Avibacterium paragallinarum type B.

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