Bordetella bronchiseptica BAH01 and application thereof

Abstract

The invention relates to the technical field of biology, in particular to bordetella bronchiseptica BAH01 and application thereof. The invention provides an I-phase strain, namely bordetella bronchiseptica BAH01, which is high in virulence and contains seven virulence factors. At present, a strain vaccine which simultaneously meets the two conditions is not found. The strain has the following advantages: (1) a biological product prepared from an inactivated vaccine prepared from bordetella bronchiseptica BAH01 is extremely high in immune efficacy; (2) the Bordetella bronchiseptica BAH01 is high in proliferation titer on a Ginger Boehmek culture medium; (3) the bordetella bronchiseptica provided by the invention has strong pathogenicity to mice; and (4) the inactivated product of Bordetella bronchiseptica BAH01 provided by the invention has good immunogenicity, can completely block the attack of Bordetella bronchiseptica on mice, can provide higher immune protection rate, and has basic potential for developing vaccines.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a strain of Bordetella bronchiseptica BAH01 and its applications. Background Technology

[0002] Bordetella bronchiseptica (Bb) can cause pneumonia and atrophic rhinitis (AR) in pigs, and its nasal turbinate atrophy is one of the important pathogenic factors of porcine respiratory disease complex (PRDC).

[0003] Early infection with *B. b* can easily lead to secondary infections with other pathogens, thereby increasing the incidence and severity of respiratory diseases in pigs and causing serious economic losses. It can also cause synergistic effects among multiple diseases, increasing morbidity and severity; porcine bortezomibitis, represented by *Arctium lappa* (AR), is now widespread in developed pig-farming countries and has become one of the most important infectious respiratory diseases in pigs.

[0004] Bb can infect pigs of any age. Currently, the addition of medications to pig farms can reduce the continued spread of the disease, but long-term use of antibiotics can lead to the development of drug-resistant strains. For example, if the withdrawal period is not properly controlled, drug residues are more likely to remain in the pigs' bodies, which will seriously endanger human health. Therefore, there is an urgent need to find a vaccine that can effectively improve the immunity of pigs. Summary of the Invention

[0005] The purpose of this invention is to provide a strain of Bordetella bronchiseptica BAH01 and its applications, in order to solve the problems existing in the prior art.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] This invention provides a strain of Bordetella bronchiseptica BAH01, which has the accession number CCTCC NO: M 20251107.

[0008] This invention provides the application of the above-mentioned Bordetella bronchiseptica BAH01 in the preparation of Bordetella bronchiseptica inactivated vaccine.

[0009] The present invention provides an inactivated vaccine against Bordetella bronchiseptica, wherein the inactivated vaccine against Bordetella bronchiseptica comprises inactivated Bordetella bronchiseptica BAH01.

[0010] Preferably, the content of inactivated *Bordetella bronchiseptica* BAH01 in the *Bordetella bronchiseptica* inactivated vaccine is 1×10⁻⁶. 9 CFU / mL.

[0011] This invention provides a method for preparing the above-mentioned inactivated Bordetella bronchiseptica vaccine, comprising the following steps:

[0012] After inactivating Bordetella bronchiseptica BAH01 with an inactivating agent, an inactivated Bordetella bronchiseptica BAH01 bacterial solution was obtained.

[0013] The inactivated Bordetella bronchiseptica BAH01 bacterial solution was mixed with an adjuvant to obtain the inactivated Bordetella bronchiseptica vaccine.

[0014] Preferably, the content of inactivated *Bordetella bronchiseptica* BAH01 in the *Bordetella bronchiseptica* inactivated vaccine is 1×10⁻⁶. 9 CFU / mL.

[0015] Preferably, the inactivation includes the step of mixing the bacterial culture of Bordetella bronchiseptica BAH01 with the inactivating agent.

[0016] Preferably, the final concentration of the inactivating agent in the mixture of the bacterial culture of Bordetella bronchiseptica BAH01 and the inactivating agent is 0.003 wt%.

[0017] Preferably, the inactivating agent is formaldehyde; the adjuvant is an aluminum adjuvant.

[0018] Preferably, the aluminum adjuvant is aluminum hydroxide.

[0019] The present invention discloses the following technical effects:

[0020] In the prior art, the preparation of inactivated vaccines using common isolated strains is generally not very effective. This invention provides a highly virulent phase I strain containing seven virulence factors—Bordeia bacillus bronchiseptica BAH01. Currently, no strain vaccine has been found that simultaneously meets both of these conditions.

[0021] This strain has the following advantages:

[0022] (1) The biological products prepared from the inactivated vaccine made from Bordetella bronchiseptica BAH01 have extremely high immunogenicity;

[0023] (2) Bordetella bronchiseptica BAH01 showed a high proliferation titer on Bourget's medium;

[0024] (3) The Bordetella bronchiseptica provided by the present invention has strong pathogenicity to mice;

[0025] (4) The inactivated product of Bordetella bronchiseptica BAH01 provided by the present invention has good immunogenicity, can completely block the attack of Bordetella bronchiseptica on mice, can provide a high immune protection rate, and has the basic potential for vaccine development. Attached Figure Description

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is a diagram showing the growth morphology of bacterial colonies on a TSA plate.

[0028] Figure 2 The results show the toxin amplification of Bordetella bronchiseptica BAH01; the first lane is the marker, and the second to eighth lanes are Bordetella bronchiseptica BAH01.

[0029] Figure 3 The molecular weight of outer membrane proteins of Bordetella bronchiseptica BAH01 was determined; where M is the marker and 1 is Bordetella bronchiseptica BAH01.

[0030] Figure 4 The survival curves of mice in the pathogenicity experiment are shown; among them, the 10^5 group is group A, the 10^6 group is group B, and the 10^7 group is group C.

[0031] Figure 5 Anatomical diagrams of mouse lungs used in pathogenicity experiments; among them, 10 5 Group A, 10 6 Group B, 10 7 Group C; the red box shows the anatomical diagram of the lungs of the dead mouse. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Example 1: Isolation and Identification of Bordetella bronchiseptica BAH01

[0038] 1. Isolation of Bordetella bronchiseptica from porcine origin

[0039] Lung samples collected from a pig farm in Anhui Province were streaked onto TSA plates and incubated at 37°C for 12 hours. The TSA plates showed smooth, grayish-white, semi-transparent, medium-sized colonies. Figure 1 Then, a single colony was selected for purification culture. The purified strain was then propagated and subcultured.

[0040] 2. Identification of Bordetella bronchiseptica from swine

[0041] Take 300 μL of bacterial culture and extract bacterial DNA using a nucleic acid extraction or purification kit (manufacturer: Hangzhou Bori Technology Co., Ltd., catalog number: C862411043). Store the eluted DNA at -20℃.

[0042] Using the extracted DNA as a template, PCR amplification was performed using universal bacterial PCR primers (upstream primer F: 5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.1; downstream primer R: 5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.2). The PCR reaction program was 95℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, and 72℃ extension for 45 s, for 35 cycles. Afterwards, the PCR amplification products were subjected to 1% agarose gel electrophoresis, and the target band was purified by gel extraction and sent to Qingke Biotechnology Co., Ltd. for sequencing. Finally, the spliced ​​16S gene sequence was compared online with NCBIBlast (comparison website: https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The sequence showed the highest homology with *Bordetella bronchiseptica*, confirming the bacterium as *Bordetella bronchiseptica*, and named *Bordetella bronchiseptica* BAH01.

[0043]

[0044] The strain named *Bordetella bronchiseptica* BAH01 is *Bordetella bronchiseptica* and was deposited on May 19, 2025, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M 20251107.

[0045] Example 2: Toxin identification of Bordetella bronchiseptica BAH01

[0046] Using the extracted nucleic acid from Bordetella bronchiseptica BAH01 as a template, seven toxins—FLA, DNT, Bvgs, AC-Hly (AC), FHA, brfZ, and prn—were amplified by PCR, with a negative control included. The primers used for PCR are shown in Table 1. The PCR reaction mixture consisted of 25 μL: 12.5 μL 2×Phanta Max Master Mix, 1 μL upstream primer, 1 μL downstream primer, and 2 μL template, plus 8.5 μL ddH2O. The PCR program was: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, and 72℃ extension for 1 min 30 s, for 35 cycles. The amplified products were subjected to 1% agarose gel electrophoresis, as shown below. Figure 2 As shown in the figure. The results showed that Bordetella bronchiseptica BAH01 contains seven virulence factors.

[0047] Table 1 Primers

[0048]

[0049] Note: Primers were synthesized by Qingke Biotechnology Co., Ltd.

[0050] Example 3: Cladomorphism identification of Bordetella bronchiseptica BAH01

[0051] 1. Extraction of outer membrane proteins: Bordetella bronchiseptica BAH01 was inoculated onto TSA plates and incubated at 37°C for 18 hours. Colonies were washed off with PBS, and the bacterial concentration was measured and adjusted to achieve the desired OD value. 600 After the value is 1, take 1 mL, incubate in a 60℃ water bath for 30 min, centrifuge at 4℃ and 1000 r / min for 15 min, and the supernatant is the outer membrane protein sample.

[0052] 2. SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis): Mix 40 μL of sample with 10 μL of 5× loading buffer and boil in a water bath for 10 minutes to denature the proteins. Perform electrophoresis using BeyoGel Elite PAGE 12% separating gel and 5% stacking precast gel. After loading, in 1×Tris-Glycine-SDS electrophoresis buffer, first electrophoresis at a constant voltage of 80V to allow the sample to enter the separating gel, then increase the voltage to 120V and continue electrophoresis until the bromophenol blue dye front migrates to the bottom of the gel. After electrophoresis, stain the gel with BeyoGel Coomassie Brilliant Blue rapid staining solution for 30 min, then destain with deionized water until the background is clear.

[0053] The results are as follows Figure 3 As shown in the figure. The results showed that *Bordetella bronchiseptica* BAH01, as verified by SDS-PAGE, exhibited an outer membrane protein band at a molecular weight of 74 kDa. All phase I bacteria showed this band at high concentrations, while phase variant bacteria did not. Therefore, the *Bordetella bronchiseptica* BAH01 provided in this application is a highly virulent phase I bacterium.

[0054] Example 4: Determination of the proliferation titer of Bordetella bronchiseptica BAH01

[0055] After resuscitation, the bacteria were inoculated into Booz & Ginger broth at a volume ratio of 1:100 and incubated at 220 rpm in a 37°C incubator for 7 hours. The bacterial concentration was then determined using the plate count method, and the bacterial volume reached 1 × 10⁻⁶. 9 CFU / mL. This indicates that the *Bordetella bronchiseptica* BAH01 provided in this application exhibits a high proliferation titer in Bauer-Jiang medium.

[0056] Example 5: Pathogenicity test of Bordetella bronchiseptica BAH01

[0057] Six-week-old SPF female BALB / c mice were randomly divided into three groups: Group A (10 5 Group A, Group B (10) 6 Group C (10) 7 Three groups (groups A, B, and C) were formed, with three mice in each group. Groups A, B, and C were challenged with intranasal administration of Bordetella bronchiseptica BAH01 at doses of 1×10⁻⁶. 5 CFU / 0.05mL, 1×10 6 CFU / 0.05mL, 1×10 7 CFU / 0.05mL, mouse survival curve as shown Figure 4As shown in the figure. The results showed that: no mice died in group A and no obvious symptoms were observed; one mouse died in group B, and the remaining two mice had ruffled fur and were depressed; two mice died in group C; group D was a negative control, and the three mice in group D were given saline nasal drops. During the experiment, none of the mice in group D died.

[0058] The median lethal dose (LD50) of Bordetella bronchiseptica BAH01 in mice was then calculated using the Reed-Muench method. 50 10 6.5 CFU.

[0059] During the experiment, dead mice were dissected. Five days later, surviving mice were also dissected to observe their lungs and to detect bacterial load. Anatomical images of the mouse lungs are shown below. Figure 5 As shown in the figure. The results showed that the lung tissue of the dead mice in groups B and C was hemorrhage, congested, and fragile (the red box indicates the dead mice); the lungs of the mice that did not die 7 days after the challenge were shown in the figure, and no obvious lesions were found in the color or texture of the lungs. The results of the bacterial load test in the lungs of mice are shown in Table 2. The results showed that Bordetella bronchiseptica was detected in each dose group after the mice died, and the bacterial content in the tissue was high; the CT value of the lung tissue of the mice that did not die showed a lower bacterial content in the lungs.

[0060] Table 2 Results of bacterial load detection in rat lungs

[0061] Sample number Cq value Result determination <![CDATA[10 7 Group of lungs (dead)]]> 25.64 + <![CDATA[10 7 Group of lungs (dead)]]> 24.34 + <![CDATA[10 7 Group of lungs]]> 37.03 + <![CDATA[10 6 Group of lungs]]> 26.39 + <![CDATA[10 6 Group of lungs]]> 32.66 + <![CDATA[10 6 Group of lungs]]> 32.43 + <![CDATA[10 5 Group of lungs]]> 34.20 + <![CDATA[10 5 Group of lungs]]> 32.99 + <![CDATA[10 5 Group of lungs]]> 34.86 + negative control / - negative control / - negative control / -

[0062] Note: "+" indicates that the mouse lungs are positive for Bordetella bronchiseptica, and "-" indicates that the mouse lungs are negative for Bordetella bronchiseptica, meaning there is no infection.

[0063] Example 6: Preparation of inactivated vaccine against Bordetella bronchiseptica BAH01

[0064] The preparation steps for the Bordetella bronchiseptica BAH01 inactivated vaccine are as follows:

[0065] Bordetella bronchiseptica BAH01 was inoculated into Bouger-Jiang liquid medium and incubated at 37°C for 12 h. The culture was then harvested, and bacterial viability was counted on agar plates. The bacterial suspension was then centrifuged and resuspended in physiological saline to obtain a 2×10⁻⁶ Bordetella bronchiseptica BAH01 bacterial suspension. 9 (CFU / mL). Formaldehyde solution was then added to the Bordetella bronchiseptica BAH01 bacterial suspension to a final concentration of 0.003 wt%. After incubation at 37°C for 18 hours, the formaldehyde inactivation effect was assessed by streaking on Bougand agar plates. The results showed no bacterial growth on the plates, indicating good inactivation. Finally, the formaldehyde was removed by centrifugation, and the bacterial concentration was adjusted to 2 × 10⁻⁶ CFU / mL with physiological saline. 9CFU / mL, and an equal volume of aluminum hydroxide gel adjuvant (diluted 5-fold) was added. The final prepared aluminum hydroxide gel inactivated vaccine (Bordeia bronchiseptica BAH01 inactivated vaccine) contained 1×10⁻⁶ CFU / mL of inactivated Bordeia bronchiseptica BAH01. 9 CFU / mL.

[0066] Example 7: Immunopotency Experiment of Bordetella bronchiseptica BAH01 Inactivated Vaccine

[0067] Six-week-old SPF female BALB / c mice were randomly divided into five groups (group A, group B, group C, and control group; group A received Merck vaccine, group B received pre-coital vaccine, and group C received the Bordetella bronchiseptica BAH01 inactivated vaccine prepared in Example 6), with five mice in each group. First immunization: Mice in groups A, B, and C received subcutaneous injections of 1×10- of the vaccine at multiple sites. 9 CFU / 0.2mL, the control group was injected with the same volume of physiological saline; a second immunization was performed 14 days later, with the same immunization dose as the first immunization. Fourteen days after the second immunization, intranasal challenge was performed, with the challenge bacterial solution being *Bordetella bronchiseptica* BAH01, and the challenge dose being the mouse LD50. 50 Dosage, i.e., 10 6.5 CFU / 0.05mL. After challenge, the protection rate of each group was investigated daily, and the results are shown in Table 3.

[0068] Table 3 Protection rate against viral challenge in mice

[0069] Group Group A Group B Group C control group The first day after the attack - - - The day after the attack - - - 2 The third day after the attack - 1 - 1 The fourth day after the attack - - - The fifth day after the attack The sixth day after the attack Seven days after the attack Protection rate 100％ 80％ 100％ 40％

[0070] The results showed that all mice in groups A and C survived after immunization and challenge; one mouse died in group B; and three mice died in the control group. Therefore, the *Bordetella bronchiseptica* BAH01 inactivated vaccine provided by this invention has good immunogenicity, can completely block *Bordetella bronchiseptica* challenge in mice, provides a high immune protection rate (100%), and has the basic potential for vaccine development.

[0071] Simultaneously, the bacterial content in the lungs of each group of mice was detected, and the results are shown in Table 4. The results showed that positive results were detected in the lungs of groups A, B, and the control group, while no Bordetella bronchiseptica was detected in the lungs of group C mice.

[0072] Table 4. Detection of bacterial content in the lungs of different mice.

[0073] Sample number Cq value Result determination Sample number Cq value Result determination Group A 29.88 + Group C / Group A / Group C / Group A / Group C / Group A / Group C / Group A / Group C / Group B (Dead) 25.01 + Control group (deceased) 25.81 + Group B / Control group (deceased) 25.84 + Group B / Control group (deceased) 28.75 + Group B / control group 33.12 Group B / control group 33.74

[0074] Note: "+" indicates that the mouse lungs are positive for Bordetella bronchiseptica, and "-" indicates that the mouse lungs are negative for Bordetella bronchiseptica, meaning there is no infection.

[0075] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A strain of Bordetella bronchiseptica BAH01, characterized in that, The accession number of the Bordetella bronchiseptica BAH01 is CCTCC NO: M 20251107.

2. The use of Bordetella bronchiseptica BAH01 as described in claim 1 in the preparation of Bordetella bronchiseptica inactivated vaccine.

3. An inactivated vaccine against Bordetella bronchiseptica, characterized in that, The inactivated Bordetella bronchiseptica vaccine comprises inactivated Bordetella bronchiseptica BAH01 as described in claim 1.

4. The inactivated Bordetella bronchiseptica vaccine according to claim 3, characterized in that, The inactivated Bordetella bronchiseptica vaccine contains 1×10⁻⁶ inactivated Bordetella bronchiseptica BAH01. 9 CFU / mL.

5. The method for preparing the inactivated Bordetella bronchiseptica vaccine according to claim 3, characterized in that, Includes the following steps: After inactivating Bordetella bronchiseptica BAH01 with an inactivating agent, an inactivated Bordetella bronchiseptica BAH01 bacterial solution was obtained. The inactivated Bordetella bronchiseptica BAH01 bacterial solution was mixed with an adjuvant to obtain the inactivated Bordetella bronchiseptica vaccine.

6. The preparation method according to claim 5, characterized in that, The inactivated Bordetella bronchiseptica vaccine contains 1×10⁻⁶ inactivated Bordetella bronchiseptica BAH01. 9 CFU / mL.

7. The preparation method according to claim 5, characterized in that, The inactivation includes the step of mixing the bacterial culture of Bordetella bronchiseptica BAH01 with the inactivating agent.

8. The preparation method according to claim 7, characterized in that, The final concentration of the inactivating agent in the mixture of the bacterial culture of Bordetella bronchiseptica BAH01 and the inactivating agent is 0.003 wt%.

9. The preparation method according to claim 5, characterized in that, The inactivating agent is formaldehyde; the adjuvant is aluminum adjuvant.

10. The preparation method according to claim 9, characterized in that, The aluminum adjuvant is aluminum hydroxide.

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