Avibacterium paragallinarum attenuated mutant strain and application thereof

Abstract

The application discloses a weakened mutagenic strain of Avibacterium paragallinarum and application thereof, and belongs to the fields of microorganisms and veterinary biological agents. The weakened mutagenic strain of Avibacterium paragallinarum is C-N118, which was preserved in the China Center for Type Culture Collection on July 2, 2025, and the preservation number is CCTCC NO: M 20251506. The weakened mutagenic strain of Avibacterium paragallinarum C-N118 is obtained by using 1-methyl-3-nitro-1-nitrosoguanidine mutagenesis, compared with a parent strain 2020 / JS74, the biofilm formation ability and adhesion and invasion ability of the weakened mutagenic strain of Avibacterium paragallinarum C-N118 are decreased, the pathogenicity is significantly reduced, and no clinical symptoms and lesions of SPF chickens are caused after inoculation. C-N118 is immunized by a nose drop method, the immunization dose is 10 7 CFU / each, and 14 days later, C-type Avibacterium paragallinarum 2020 / JS74 is attacked at a dose of 10 2 CFU / each, and the protection rate is 100%. It is proved that the weakened mutagenic strain of Avibacterium paragallinarum C-N118 can be used as a candidate strain of attenuated live vaccine for chicken infectious rhinitis, and is used for vaccine research and has a great application prospect.

Description

Technical Field

[0001] This invention relates to an attenuated mutant strain of Avian bacillus paragallinarum and its application, belonging to the fields of microbiology and veterinary biological agents. Background Technology

[0002] avian parafowl cholera ( Avibacterium paragallinarum Infectious coryza (ICC) is a Gram-negative bacterium that causes infectious coryza in chickens. This disease is an acute, highly contagious upper respiratory tract infection that poses a serious threat to intensive poultry farming worldwide. Characteristic clinical symptoms of ICC include facial and infraorbital sinus swelling, serous to mucopurulent nasal discharge, conjunctivitis with lacrimation, and decreased egg production in laying hens. Although mortality is usually low, the high morbidity rate leads to poor growth in pullets and decreased egg production in laying hens. Secondary infections with other pathogens can exacerbate clinical symptoms and mortality, causing significant economic losses.

[0003] *Avianobacter paragallinarum* has three serotypes: Page A, B, and C, with significant differences in antigenicity among them. Currently, the control of infectious coryza in chickens mainly relies on inactivated whole-cell vaccines, which only provide protection against homologous serotypes and suffer from problems such as long immunization cycles, significant side effects, the need for multiple vaccinations, and high preparation costs. Compared to inactivated vaccines, live attenuated vaccines can replicate to a limited extent within the host, more effectively mimicking the natural infection process, thereby inducing a more comprehensive and balanced immune response, including strong humoral immunity, cellular immunity, and crucial mucosal immunity. Secondly, they can colonize and proliferate in the respiratory mucosa, preventing the invasion and colonization of wild-type strains through competitive rejection, providing a more advanced defensive barrier. A single vaccination can induce rapid, potent, and long-lasting immunity. Administration is via nasal drops, avoiding the stress associated with injections. However, there are currently no safe and effective commercially available live attenuated vaccines for *Avianobacter paragallinarum*. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide an attenuated mutant strain of Avian bacillus paragallinarum and its application as an attenuated live vaccine for the prevention of infectious coryza in chickens, so as to provide an attenuated strain with significantly reduced biofilm formation ability, no pathogenicity, and good immunogenicity, thereby better preventing and controlling infectious coryza in chickens.

[0005] Technical solution: The attenuated mutant strain of Avianella paragallinarum described in this invention is C-N118, which was deposited at the China Center for Type Culture Collection on July 2, 2025, with accession number CCTCC NO: M20251506.

[0006] Furthermore, the C-N118 has a weak biofilm formation ability.

[0007] Furthermore, the C-N118 has weak adhesion and invasion ability to DF-1 cells.

[0008] The application of the attenuated mutant strain of *Avianobacter paragallinarum* described in this invention in the preparation of *Avianobacter paragallinarum* vaccine or vaccine for the prevention of infectious coryza in chickens.

[0009] Furthermore, the vaccine is a live attenuated avian bacillus paragallinarum vaccine.

[0010] Furthermore, the vaccine is a nasal drop.

[0011] Furthermore, the concentration of C-N118 in the vaccine is 1×10⁻⁶. 8 CFU / mL.

[0012] Furthermore, the dosage of the vaccine is 10. 7 CFU / each.

[0013] Furthermore, the vaccine also includes pharmaceutically acceptable excipients.

[0014] The infectious coryza vaccine for chickens described in this invention includes the above-mentioned attenuated mutant strain of Avian bacillus paragallinarum.

[0015] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: The attenuated mutant strain C-N118 of *Avianella paragallinarum* obtained by mutagenesis using 1-methyl-3-nitro-1-nitrosoguanidine exhibits reduced biofilm formation and adhesion invasion capabilities compared to the parent strain 2020 / JS74, resulting in significantly reduced pathogenicity. Inoculation with this strain does not induce any clinical symptoms or lesions in SPF chickens. C-N118 is immunized via nasal drops at a dose of 10... 7 CFU / bird, challenged with Avian bacillus paragallinarum type C 2020 / JS74 14 days later, dose: 10 2 CFU / bird, protection rate 100%. This demonstrates that the attenuated mutant strain C-N118 of Avian bacillus paragallinarum of this application can be used as a candidate strain for a live attenuated vaccine against infectious coryza in chickens, and has great application potential for vaccine research. Attached Figure Description

[0016] Figure 1 This is a graph showing the biofilm formation ability test results of 18 strains of type C avian bacillus isolates in this invention. Figure 2 This is a graph showing the biofilm formation ability test results of the mutant strain C-N118 in this embodiment of the invention; Figure 3 This is a graph showing the results of the adhesion and invasion ability measurement of the mutant strain C-N118 in the embodiments of the present invention; Figure 4 This is a line graph showing the clinical symptom score of the pathogenicity test of the mutant strain C-N118 in this embodiment of the invention. Figure 5 This is a line graph showing the clinical symptom score of the C-N118 mutant strain in the immunoprotection test of this invention. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0018] Example 1 Screening of isolates with strong biofilm formation ability Eighteen isolates of *Avianobacterium paragallinarum* type C were revived and streaked onto TSA plates containing 5% chicken serum and 0.01% NAD, and incubated at 37°C with 5% CO2 for 24 h. Single colonies were picked and inoculated into TSB liquid medium containing 5% chicken serum and 0.01% NAD, and incubated in 96-well plates at 37°C for 24 h. The culture medium was discarded, and the plates were washed twice with PBS buffer and fixed with 300 μL of pre-cooled methanol solution (4°C) for 30 min. 200 µL of 1% crystal violet was added to each well for staining for 15 min. The plates were washed three times with deionized water and dried at 60°C. 200 μL of 33% glacial acetic acid solution was added to each well to dissolve the dye, and the OD was measured using a microplate reader. 570 value.

[0019] With negative control well 2OD 570 OD, the critical point for judging biofilm formation ability C OD≤OD C At that time, it has no ability to form biofilms; when OD C <OD≤2OD C At that time, the biofilm formation ability is weak; when 2OD C <OD≤4OD C At this time, the biofilm formation ability is moderate; when OD ≥ 4OD C At that time, the biofilm formation ability was strong. The results were as follows: Figure 1 As shown, strain 2020 / JS74 had the strongest biofilm formation ability.

[0020] Example 2: Cultivation and Screening of Mutagenized Strains The isolate 2020 / JS74 (Guo M, Chen X, Zhang H, Liu D, Wu Y, Zhang X. Isolation, Serovar Identification, and Antimicrobial Susceptibility of Avibacterium paragallinarum from Chickens in China from 2019 to 2020. Vet Sci. 2022 Jan 12;9(1):27. doi: 10.3390 / vetsci9010027.), exhibiting the strongest biofilm formation ability, was streaked onto a TSA plate and anaerobically cultured at 37°C for 24 h. Single colonies were then picked and incubated overnight at 37°C and 180 rpm in TSB liquid medium. The next day, the colonies were inoculated into fresh TSB liquid medium and cultured until OD reached [missing value]. 600 =0.6, prepare bacterial solution for mutagenesis.

[0021] Mutagenesis was induced using 1-methyl-3-nitro-1-nitrosoguanidine (NTG) at final concentrations of 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.7 mg / mL, 0.9 mg / mL, and 1.1 mg / mL, respectively, at 37°C and 220 rpm for 30 min. Bacterial growth was observed after mutagenesis. *Avianella paragallinarum* tolerated a maximum mutagenic concentration of 0.5 mg / mL NTG, but bacterial survival was low. Therefore, 0.3 mg / mL NTG was used for mutagenesis. After 30 min, the mutagenic bacteria were inoculated into fresh TSB liquid medium at a 1% concentration and cultured until OD500. 600 =0.6, add 0.3 mg / mL NTG, and repeat the mutagenesis twice. Adjust the mutagenesis solution to 10... -4 10 -5 After dilution, the culture was spread on TSA plates and anaerobic incubated at 37°C for 24 h. 928 colonies were then selected for enrichment culture.

[0022] 928 single colonies were inoculated into TSB liquid medium and incubated in 96-well plates at 37°C for 24 h. The culture medium was discarded, and the plates were washed twice with PBS buffer. 200 µL of 1% crystal violet was added to each well for staining for 15 minutes. 200 μL of 33% glacial acetic acid was added to each well to dissolve the dye, and the OD was measured using a microplate reader. 570 Values. TSB medium without bacterial inoculation served as a control. A mutant strain C-N118 with significantly reduced biofilm formation ability was screened; staining results are shown below. Figure 2C-N118 exhibits weak biofilm formation ability, which is 92% lower than that of its parent strain.

[0023] Example 3: Determination of the adhesion and invasion ability of the mutant strain C-N118 Adhesion ability determination: 2020 / JS74 and the mutant strain C-N118 were cultured to the logarithmic growth phase (OD). 600 Add DF-1 cells (0.6-0.8 g / mL) at an MOI of 100:1 and incubate at 37°C for 2 h. Discard the supernatant and wash the cells three times with PBS to remove unattached bacteria. Add 200 μL of 1% Triton X-100 to each well to lyse the cells and incubate for 10 min. Spread the lysate on TSA plates and count the viable cells.

[0024] Invasiveness determination: 2020 / JS74 and the mutant strain C-N118 were cultured to the logarithmic growth phase (OD). 600 =0.6~0.8), add DF-1 cells at an MOI ratio of 100:1, and incubate at 37°C for 2 h. Add 100 μL of medium containing 1% penicillin-streptomycin mixture (penicillin content 10000 U / mL, streptomycin content 10 mg / mL) to each well, and incubate for 1 h to kill bacteria that have not invaded the cells. Discard the medium, wash three times with PBS, add 200 μL of 1% Triton X-100 to lyse the cells, spread on TSA plates, and count viable cells.

[0025] The adhesion results show that ( Figure 3 A) The number of adhering bacteria in the parental strain 2020 / JS74 was 3.26 × 10⁻⁶. 5 CFU / well. C-N118 adhesion ability significantly decreased ( p <0.05%. The invasiveness results indicate that ( Figure 3 (B) The number of invading bacteria in the parent strain 2020 / JS74 was 233 CFU / well, while the number of invading bacteria in C-N118 was only 56 CFU / well.

[0026] Example 4 Pathogenicity evaluation of mutant strain C-N118 Eighteen-day-old SPF chickens (purchased from Jiangsu Boehringer Ingelheim Viton Biotechnology Co., Ltd.) were randomly divided into three groups: C-N118 challenge group, 2020 / JS74 challenge group, and blank control group, with five chickens in each group. The parental strain 2020 / JS74 and the mutant strain C-N118 were inoculated onto TSA plates and cultured anaerobicly for 24 hours. Single colonies were then picked and cultured in TSB medium for 16 hours, followed by inoculation at a 1% ratio into TSB medium and cultured for 6 hours. After viable count, the bacterial concentration was adjusted to 10⁻⁶. 3CFU / mL. At 21 days of age, the infant underwent an infraorbital sinus challenge with a dose of 100 μL (10... 2 CFU / bird). The blank control group was injected with an equal volume of PBS. The clinical symptoms of the experimental chickens were continuously observed and scored for 7 days after challenge. The scoring criteria were: 0 points: no clinical symptoms; 1 point: mild facial swelling and runny nose; 2 points: moderate facial swelling; 3 points: severe facial swelling and inability to open eyes.

[0027] Clinical symptom scores are shown in Figure 4 On the second day after challenge with 2020 / JS74, all experimental chickens developed symptoms, and 3 / 5 of the chickens showed severe facial swelling with a score of 3. In contrast, the experimental chickens in the C-N118 challenge group did not show any clinical symptoms throughout the observation period.

[0028] Example 5 Evaluation of the immunogenicity of mutant strain C-N118 Single colonies of the mutant strain C-N118 were inoculated into TSB medium and cultured for 16 h until the logarithmic growth phase, then inoculated into TSB medium at a 1% ratio and cultured for 6 h (OD). 600 (Approximately 0.8), adjust the bacterial concentration to 10. 8 Live vaccines were prepared using CFU / mL.

[0029] Eighteen-day-old SPF chickens were randomly divided into two groups: a C-N118 immunization group and a challenge control group, with 10 chickens in each group. At 21 days of age, C-N118 was administered intranasally at a dose of 100 μL (10... 7 CFU / animal), and the control group was vaccinated with 100 μL PBS. Clinical manifestations were continuously observed after immunization. After 14 days, the infraorbital sinus was challenged with 2020 / JS74 virus at a dose of 100 μL (10 CFU / animal). 2 CFU / bird). Clinical symptoms were observed in the experimental chickens, and the results are shown in […]. Figure 5 The statistical protection rate is shown in Table 1.

[0030] The results showed that on the second day after challenge, 90% of the experimental chickens in the challenge control group developed the disease, and on the third day, the morbidity rate was 100%. In contrast, the experimental chickens in the immunized group showed no clinical symptoms seven days after challenge, with a morbidity rate of 0. The C-N118 immunized group showed a 100% protection rate against Avianella paragallinarum type C 2020 / JS74.

[0031] Table 1 Evaluation of Immunological Efficacy

[0032] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A strain of attenuated mutant *Avianella paragallinarum* ( Avibacterium paragallinarum ), characterized in that, The attenuated mutant strain of *Avianella paragallinarum* was C-N118, which was deposited at the China Center for Type Culture Collection on July 2, 2025, with accession number CCTCC NO: M 20251506.

2. The use of the attenuated mutant strain of *Avianobacter paragallinarum* according to claim 1 in the preparation of *Avianobacter paragallinarum* vaccine or vaccine for the prevention of infectious coryza in chickens, characterized in that, The vaccine is a live attenuated avian bacillus paragallinarum vaccine, and the vaccine is administered as nasal drops.

3. The application according to claim 2, characterized in that, The concentration of C-N118 in the vaccine is 1×10⁻⁶. 8 CFU / mL.

4. The application according to claim 2, characterized in that, The dosage of the vaccine is 10. 7 CFU / each.

5. The application according to claim 2, characterized in that, The vaccine also includes pharmaceutically acceptable excipients.

6. A vaccine for infectious coryza in chickens, characterized in that, The vaccine includes the attenuated mutant strain of *Avianella paragallinarum* as described in claim 1, and the vaccine is an attenuated live *Avianella paragallinarum* vaccine, which is a nasal drop.

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