CN117187118B - A strain of Bordetella bronchiseptica A1218 and its application - Google Patents

Abstract

The present invention discloses a strain of Bordetella bronchiseptica A1218 and its application, which belong to the field of biological products and preventive veterinary medicine. The Bordetella bronchiseptica A1218 was deposited in the China Center for Type Culture Collection on July 6, 2023, with a deposit number of CCTCC NO: M 20231122. The Bordetella bronchiseptica A1218 strain provided by the present invention has strong virulence, and low-dose infection can cause severe infection or even death in young dogs and cats. The inactivated product of this strain can produce a good immune response in dogs and cats immunized with it, has good immunogenicity, can provide a high immune protection rate against the attack of Bordetella bronchiseptica, and has the basic potential for developing vaccines.

Description

A strain of Bordetella bronchiseptica A1218 and its application

Technical Field

The invention relates to the field of biological products and preventive veterinary medicine, in particular to a strain of Bordetella bronchiseptica A1218 and application thereof.

Background technique

Bordetella bronchiseptica is a Gram-negative bacillus that is an important pathogen causing respiratory infections and diseases. It is widely present in the respiratory tract of many animals, including dogs, cats, pigs, rabbits, etc. Bordetella bronchiseptica is a non-collagenase-producing Gram-negative bacillus with multiple pili and flagella. The bacterium attaches to the surface of host epithelial cells through multiple pili and moves through the movement of flagella. The bacterium is about 0.5 to 1 micron wide and 1 to 3 microns long, and can grow on a medium rich in blood and nutrients. Bordetella bronchiseptica mainly causes respiratory infections and diseases. In dogs, the bacterium is one of the common pathogens causing Kennel cough, and can also cause diseases such as bronchitis and pneumonia. Sick dogs usually show respiratory symptoms such as coughing, sneezing, runny nose, and conjunctivitis. In cats, the bacteria mainly cause respiratory infections and diseases, especially in intensive breeding environments such as multi-cat households and breeding farms. Cats infected with the bacteria often show respiratory symptoms such as coughing, sneezing, runny nose, conjunctivitis and loss of appetite.

Bordetella bronchiseptica is spread through droplets, and is transmitted to other dogs and cats through contact with infected dogs and cats or environments contaminated by bacteria or direct contact with infected cats. Bordetella bronchiseptica is difficult to eradicate, and there is a certain probability of recurrence due to external environmental induction. At present, there is no vaccine related to Bordetella bronchiseptica in dogs and cats in China. Therefore, it is necessary to explore the prevalent strains of Bordetella bronchiseptica in dogs and cats in China, and develop relevant vaccines for Bordetella bronchiseptica in dogs and cats based on this.

Summary of the invention

The purpose of the present invention is to provide a strain of Bordetella bronchiseptica A1218 and its application to solve the problems existing in the above-mentioned prior art. The Bordetella bronchiseptica A1218 provided by the present invention has strong pathogenicity to dogs and cats, has good immunogenicity after inactivation, can provide a high immune protection rate against the attack of Bordetella, and has basic potential for developing vaccines.

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

The present invention provides a strain of Bordetella bronchiseptica A1218, which was deposited in China Center for Type Culture Collection on July 6, 2023, with a deposit number of CCTCC NO: M 20231122.

The present invention also provides the use of the Bordetella bronchiseptica A1218 in preparing a vaccine.

Furthermore, the vaccine is used to prevent diseases caused by Bordetella bronchiseptica.

Furthermore, the vaccine is a universal vaccine for dogs and cats.

The invention also provides a universal Bordetella bronchiseptica inactivated vaccine for dogs and cats, comprising inactivated Bordetella bronchiseptica A1218. The preservation number of the Bordetella bronchiseptica A1218 is CCTCC NO: M 20231122.

The present invention also provides a method for preparing a universal Bordetella bronchiseptica inactivated vaccine for dogs and cats, comprising the steps of preparing a bacterial solution of Bordetella bronchiseptica A1218, inactivating the bacterial solution, and adding an adjuvant to prepare the vaccine.

Furthermore, the bacterial liquid is obtained by culturing the Bordetella bronchiseptica A1218 in a TSA medium containing newborn calf serum, and the volume fraction of the newborn calf serum in the TSA medium is 4%.

Furthermore, the method for inactivating the bacterial liquid includes adding β-propiolactone to the bacterial liquid for inactivation.

Furthermore, the volume fraction of the β-propiolactone in the bacterial solution is 0.125%.

Furthermore, the inactivation is carried out at 4° C. in the dark for 36 hours.

The present invention discloses the following technical effects:

The Bordetella bronchiseptica A1218 strain provided by the present invention has strong virulence, and low-dose infection can cause severe infection or even death in young dogs and cats. The inactivated product of the strain can produce a good immune response in dogs and cats when immunized, has good immunogenicity, can provide a high immune protection rate against the attack of Bordetella bronchiseptica, and has the basic potential for developing vaccines.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

FIG1 is a graph showing the growth morphology of Bordetella bronchiseptica on a TSA plate containing 10% newborn calf serum;

FIG2 is a PCR identification result of Bordetella bronchiseptica, wherein + represents a positive control, - represents a negative control, and 1 to 9 represent collected samples;

Fig. 3 is a Gram staining microscopic examination image;

FIG4 is a graph showing the linear relationship between the aerosol challenge time and the bacterial count in the lung tissue of mice;

Figure 5 is a comparison of the immunogenicity of 8 strains of Bordetella bronchiseptica in mice, wherein A: IgG antibody levels in the serum of mice immunized with inactivated vaccines of different wild strains, and B: survival curves of mice immunized with each strain after challenge;

Figure 6 shows the results of the comparison of immunogenicity of three strains of Bordetella bronchiseptica in dogs, wherein A: serum IgG antibody levels of dogs immunized with A1203, A1218, A1224 inactivated vaccines and PBS, B: disease scores of dogs challenged with A1218 strain after being immunized with A1203, A1218, A1224 inactivated vaccines and PBS;

Figure 7 shows the efficacy of Bordetella bronchiseptica A1218 strain on dogs, wherein A: serum IgG antibody levels of dogs immunized with different doses of A1218 inactivated vaccine and PBS, B: disease scoring of dogs challenged with A1218 strain after being immunized with different doses of A1218 inactivated vaccine and PBS;

Figure 8 shows the efficacy of Bordetella bronchiseptica A1218 strain on cats, wherein A: serum IgG antibody levels of cats immunized with different doses of A1218 inactivated vaccine and PBS, B: disease scoring of cats challenged with A1218 strain after being immunized with different doses of A1218 inactivated vaccine and PBS;

Figure 9 shows the changes in body temperature of the test dogs after immunization in the inactivated vaccine overdose group, immunization control group and blank control group;

Figure 10 shows the changes in body temperature of the test cats after immunization in the inactivated vaccine overdose group, immunization control group and blank control group.

Detailed ways

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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content of this specification shall prevail.

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

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

The "dose per head" or "dose/head" in the present invention refers to the vaccine dose used per dog or cat each time. Unless otherwise specified, in the embodiments of the present invention, the "dose per head" or "dose/head" is 1 mL.

The animal experiments in the embodiments of the present invention were approved by the Ethics Committee of Huazhong Agricultural University (Ethics Number: HZAUMO-2021-0020). The experiments were carried out in accordance with the relevant provisions on experimental animal welfare and ethics in the "Regulations on Experimental Animal Management" of Hubei Province and the Ministry of Science and Technology. The animal experiments were carried out at the Experimental Animal Center of Huazhong Agricultural University.

Example 1 Isolation of strains

From September 2019 to June 2022, nasopharyngeal swabs were collected from dogs and cats in the stray dog base and pet market in Wuhan, Hubei Province with sterile cotton swabs, and lung tissue samples from dead dogs and cats in pet hospitals were collected. They were added to TSA medium containing 5% newborn calf serum and 20% glycerol, mixed and aspirated 200 μL, and the remaining samples were stored below -70°C for later use. DNA was extracted according to the OMEGAViral DNA Kit operation manual and stored below -15°C for later use. Primers for identification of Bordetella bronchiseptica were designed to amplify the conserved region of the Fla gene. The primer sequences are:

Upstream primer F: 5'-TGGCGCCTGCCCTATC-3' (SEQ ID NO. 1);

Downstream primer R: 5'-AGGCTCCCAAGAGAGAAA-3' (SEQ ID NO.2),

The primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

The reaction conditions were: 94℃ pre-denaturation for 5 min before entering the cycle, and the cycle parameters were: 94℃30s, 56℃30s, 72℃30s; after 35 cycles, 72℃ extension for 10 min. 10 μL of the amplified product was taken for 1% agarose gel electrophoresis identification. The amplified product size was 237 bp, which was consistent with the expected size (Figure 2).

The collected positive samples were diluted 3 times with sterile PBS and plated on tryptic soy agar (TSA; Becton, Dickinson and Company, MD, USA) containing 10 μg/mL nicotinamide adenine dinucleotide (NAD; Sigma, St. Louis, MO) and 10% newborn calf serum. Place in a 37°C incubator for 48 hours. Then purify and culture the isolates grown on the plate according to the Manual of Clinical Microbiology, Eleventh Edition. On each agar plate, pick 5 colonies with similar morphological characteristics to Bacillus bronchiseptica (Figure 1), i.e., small round luminous or rough colonies with a diameter of 0.5 to 1.0 mm for biochemical tests and polymerase chain reaction (PCR), and perform Gram staining microscopy (Figure 3). Finally, 12 canine Bordetella bronchiseptica isolates and 2 feline Bordetella bronchiseptica isolates were obtained, all of which were isolated and identified from nasopharyngeal swabs of dogs and cats and lung tissues of dogs and cats with respiratory disease syndrome. The strain information is listed in (Table 1).

Table 1 Information on Bordetella bronchiseptica strains in dogs and cats

Example 2 Virulence test of strains

1. Virulence test of Bordetella bronchiseptica in mice

The experimental mice were healthy female Kunming mice (SPF grade) weighing 18-22 g, obtained from the Animal Experiment Center of Huazhong Agricultural University.

145 mice were divided into 29 groups, 5 mice in each group, numbered 1 to 29. Each group numbered 1 to 14 was intraperitoneally injected with 14 strains of Bordetella bronchiseptica in Table 1 (0.2 mL/mouse), 1×10 ⁷ CFU/mouse in each group; each group numbered 15 to 28 was intraperitoneally injected with 14 strains of Bordetella bronchiseptica in Table 1 (0.2 mL/mouse), 1×10 ⁸ CFU/mouse in each group, and the 29th group was injected with an equal amount of PBS as a control. After injection, the appetite and mental state were observed every day and the death was recorded. The results showed that most of the canine and feline isolates of Bordetella bronchiseptica were lethal to mice at a dose of 1×10 ⁸ CFU/mouse, some strains were lethal to mice at a dose of 1×10 ⁷ CFU/mouse, and only two strains of Bordetella bronchiseptica were not lethal to mice at a dose of 1×10 ⁸ CFU/mouse (Table 2).

Table 2 Lethality of Bordetella bronchiseptica in dogs and cats to mice

Strain name Challenge dose Fatality Challenge dose Fatality A1201 1×10 ⁸ 5/5 1×10 ⁷ 5/5 A1218 1×10 ⁸ 5/5 1×10 ⁷ 5/5 A1206 1×10 ⁸ 5/5 1×10 ⁷ 4/5 A1224 1×10 ⁸ 5/5 1×10 ⁷ 4/5 C1811 1×10 ⁸ 5/5 1×10 ⁷ 4/5 C1816 1×10 ⁸ 5/5 1×10 ⁷ 3/5 A1212 1×10 ⁸ 5/5 1×10 ⁷ 3/5 A1203 1×10 ⁸ 5/5 1×10 ⁷ 3/5 CB1131 1×10 ⁸ 4/5 1×10 ⁷ 2/5 D1191 1×10 ⁸ 4/5 1×10 ⁷ 2/5 D1192 1×10 ⁸ 3/5 1×10 ⁷ 1/5 CB1132 1×10 ⁸ 2/5 1×10 ⁷ 0/5 A1220 1×10 ⁸ 0/5 1×10 ⁷ 0/5 C1808 1×10 ⁸ 0/5 1×10 ⁷ 0/5

2. Virulence test of A1218 strain in mice

58 SPF mice were divided into 5 groups, with 12 mice in each challenge group and 10 mice in the blank group. To simulate normal infection, the mice were challenged by aerosol. The bacterial load of A1218 strain was adjusted to 1×10 ¹² CFU/mL. The first group was challenged by aerosol for 15 minutes, the second group was challenged by aerosol for 30 minutes, the third group was challenged by aerosol for 45 minutes, and the fourth group was challenged by aerosol for 60 minutes. After the challenge, 2 mice were killed by neck dislocation in each group, and the lungs were taken for bacterial count. It was found that there was a linear relationship between the challenge time and the bacterial load in the lung tissue (Figure 4). Each group was isolated and raised, and clinical observation was performed for 14 days after the challenge. The results showed that 1/10 of the 15-minute challenge group died; 7/10 of the 30-minute challenge group died; 9/10 of the 45-minute challenge group died; 10/10 of the 60-minute challenge group died; and all the mice in the control group survived.

3. Toxicity test of A1218 strain in dogs

The A1218 strain was diluted to 1×10 ⁸ CFU/mL, 1×10 ⁹ CFU/mL and 1×10 ¹⁰ CFU/mL, respectively. Twenty 6-8 week-old test dogs with negative serum Elisa antibody values and nasopharyngeal swab antigens for Bordetella bronchiseptica were selected and divided into 4 groups. The aerosol challenge was performed in a closed space (60 cm long, 50 cm wide and 50 cm high) for 35 min. Group 1 was the 1×10 ⁸ CFU/mL challenge group, Group 2 was the 1×10 ⁹ CFU/mL challenge group, Group 3 was the 1×10 ¹⁰ CFU/mL challenge group, and Group 4 was the control group. They were kept in isolation and clinically observed for 14 days after the challenge, and the sick dogs were scored. The results showed that 3/5 dogs in the 1×10 ⁸ CFU/mL challenge group showed clinical symptoms such as depression, serous or purulent eye and nasal discharge, cough, and retching; 4/5 and 5/5 dogs in the 1×10 ⁹ CFU/mL and 1×10 ¹⁰ CFU/mL challenge groups showed clinical symptoms such as depression, serous or purulent eye and nasal discharge, cough, and retching, respectively; the control group had no obvious symptoms. This indicates that the A1218 strain has a strong pathogenicity to dogs.

4. A1218 strain cat toxicity test

The A1218 strain was diluted to 1×10 ⁷ CFU/mL, 1×10 ⁸ CFU/mL and 1×10 ⁹ CFU/mL, respectively. Twenty cats aged 10-12 weeks with negative serum Elisa antibody values and nasopharyngeal swab antigens for Bordetella bronchiseptica were selected and divided into 4 groups. The cats were challenged with aerosol in a closed space (60 cm long, 50 cm wide and 50 cm high) for 35 min. Group 1 was the 1×10 ⁷ CFU/mL challenge group, Group 2 was the 1×10 ⁸ CFU/mL challenge group, Group 3 was the 1×10 ⁹ CFU/mL challenge group and Group 4 was the control group. The cats were kept in isolation and clinically observed for 14 days after the challenge, and the sick cats were scored. The results showed that 3/5 cats in the 1×10 ⁷ CFU/mL challenge group showed clinical symptoms such as depression, serous or purulent eye and nasal secretions, cough, and retching; 4/5 cats in the 1×10 ⁸ CFU/mL challenge group showed clinical symptoms such as depression, serous or purulent eye and nasal secretions, cough, and retching; 5/5 cats in the 1×10 ⁹ CFU/mL challenge group showed clinical symptoms such as depression, serous or purulent eye and nasal secretions, cough, and retching; among them, two 10-week-old kittens in the 1×10 ⁹ CFU/mL challenge group died, and autopsy showed tracheal congestion and bleeding, severe lung lesions, whole lung hemorrhage, and edema. The control group had no obvious symptoms. This shows that the A1218 strain is more pathogenic to cats than dogs.

Example 3 Immunogenicity Study of Virus Strain

1. Comparison of immunogenicity of Bordetella bronchiseptica in mice

90 18-22g SPF mice were randomly divided into 9 groups, 10 in each group. The bacterial volume of 8 strains of Bordetella bronchiseptica (A1201, A1203, A1206, A1212, A1218, A1224, C1811 and C1816) with strong virulence was uniformly adjusted to 10 ^8.0 CFU/mL, inactivated, and the vaccine was prepared with aluminum gel adjuvant (the final concentration of aluminum gel was 0.5mg/mL). 100μL was injected into the thigh muscle of the mice; the 9th group was injected with an equal amount of PBS as a negative control. Blood was collected weekly, and the bacterial volume was adjusted to 10 ¹² CFU/mouse in the fourth week for 45 minutes. The clinical symptoms, morbidity and mortality were observed and recorded, and the dead mice were dissected and the bacteria were isolated and identified. Strains with strong immunogenicity were screened as candidate vaccine strains. The results showed that there was no significant difference in Elisa antibodies among the strains, but there were differences in the protection data against virus infection, with A1218 strain 4/5 protected, A1203 strain 3/5 protected, and A1224 strain 3/5 protected (Figure 5).

2. Comparison of immunogenicity of Bordetella bronchiseptica in dogs

Twenty healthy susceptible dogs aged 4 to 6 weeks were randomly divided into 4 groups, 5 in each group, and the dogs immunized with the A1218 strain, A1203 strain and A1224 strain with the best immunogenicity on mice were selected for immunogenicity comparison test. The inactivated vaccine was prepared for immunization of the experimental dogs. The first group was the A1218 inactivated vaccine immunization group, the second group was the A1203 inactivated vaccine immunization group, the third group was the A1224 inactivated vaccine immunization group, and the fourth group was the blank control group. The dogs were immunized subcutaneously in the neck (1×10 ⁸ CFU/dog), and the second immunization was performed in the same way 21 days later. 14 days after the second immunization, the A1218 strain was aerosolized (1×10 ¹⁰ CFU/mL was aerosolized in a closed space for 35 minutes). Blood was collected every week before the challenge, and serum was separated. Within 14 days after the challenge, the test dogs were observed for typical Bordetella bronchiseptica characteristic symptoms such as serous or purulent eye and nasal discharge, cough and retching, and upper respiratory tract swabs were collected on the 7th day after the challenge (if the test dog died, the sample was tested on the same day), and PCR was used for Bb pathogen identification. The results showed that there was no significant difference in the serum IGg antibody values of A1218, A1203 and A1224 strains (Figure 6A). From the protection data of the challenge, 5/5 of the A1218 strains were protected, 4/5 of the A1203 strains were protected, and 3/5 of the A1224 strains were protected. 5/5 of the control group developed the disease (Figure 6B), and typical Bordetella bronchiseptica characteristic symptoms such as serous or purulent eye and nasal discharge, cough and retching appeared. The score data are shown in the figure (Figure 6C), and the average score of the immune group was significantly lower than that of the control group.

This example shows that the immunogenicity of strain A1218 is the best.

Example 4 Efficacy Study of Bordetella bronchiseptica A1218 Strain

1. A1218 strain efficacy study in dogs

Twenty healthy susceptible dogs aged 4 to 6 weeks were randomly divided into 4 groups, 5 in each group. The A1218 strain was counted and inactivated, diluted with PBS, and water adjuvant was added to adjust the concentration to 1×10 ⁷ CFU/mL, 1×10 ⁸ CFU/mL, and 1×10 ⁹ CFU/mL. 1 mL/dog was injected subcutaneously in the neck; Group 1 was the 1×10 ⁷ CFU/mL immunization group, Group 2 was the 1×10 ⁸ CFU/mL immunization group, Group 3 was the 1×10 ⁹ CFU/mL immunization group, and Group 4 was injected with an equal amount of PBS as a negative control. In the third week, the second immunization was performed in the same way as the first immunization. Fourteen days after the second immunization, the virus was challenged by fog (1×10 ¹⁰ CFU/mL was atomized in a closed space for 35 minutes). Blood was collected every week before the challenge, and serum was separated. Within 14 days after the challenge, the test dogs were scored based on whether they had serous or purulent eye and nasal secretions, coughing, and retching. On the 7th day after the challenge, upper respiratory tract swabs were collected (if the test dogs died, samples were taken and tested on the same day), and PCR was used for Bb pathogen identification. The results showed that 0/5 of the 1×10 ⁷ CFU/mL immunization group were protected, 3/5 of the 1×10 ⁸ CFU/mL immunization group were protected, and 5/5 of the 1×10 ⁹ CFU/mL immunization group were protected, and all the blank control groups became ill (Figure 7 C). With the increase of antigen dose, IGg antibodies in serum increased significantly, and the antibody value increased significantly in the fourth week after the second immunization (Figure 7 A). The average score of dogs in the 1×10 ⁹ CFU/mL immunization group was 0. Although dogs in the 1×10 ⁷ CFU/mL immunization group became ill, the average score was significantly lower than that of the blank group (Figure 7 B). In addition, the Bb pathogen identification of upper respiratory tract swabs on the 7th day after the challenge was positive.

2. A1218 strain feline efficacy study

Twenty healthy susceptible cats aged 4-6 weeks were randomly divided into 4 groups, 5 in each group. The A1218 strain was counted and inactivated, diluted with PBS, and water adjuvant was added to adjust the concentration to 1×10 ⁷ CFU/mL, 1×10 ⁸ CFU/mL, and 1×10 ⁹ CFU/mL. 1 mL/cat was injected subcutaneously in the neck; Group 1 was the 1×10 ⁷ CFU/mL immunization group, Group 2 was the 1×10 ⁸ CFU/mL immunization group, Group 3 was the 1×10 ⁹ CFU/mL immunization group, and Group 4 was injected with an equal amount of PBS as a negative control. In the third week, the second immunization was performed in the same way as the first immunization. Fourteen days after the second immunization, the poison was challenged by fog (1×10 ¹⁰ CFU/mL was atomized in a closed space for 35 minutes). Blood was collected every week before the challenge, and serum was separated. Within 14 days after the challenge, the test dogs were scored based on whether they had serous or purulent eye and nasal secretions, coughing, and retching. On the 7th day after the challenge, upper respiratory tract swabs were collected (if the test cat died, samples were taken and tested on the same day), and PCR was used for Bb pathogen identification. The results showed that 2/5 of the 1×10 ⁷ CFU/mL immunization group were protected, 4/5 of the 1×10 ⁸ CFU/mL immunization group were protected, and 5/5 of the 1×10 ⁹ CFU/mL immunization group were protected. All of the blank control groups were sick. Similar to the Elisa antibody value in dogs, it increased with the increase of antigen. The average score of the immunization group was significantly lower than that of the control group, and the Bb pathogen identification of the upper respiratory tract swab samples on the 7th day after the challenge was positive (Figure 8).

In summary, the Bordetella bronchiseptica A1218 strain of the present invention has a good growth state, and the toxicity of the strain to healthy susceptible dogs and cats is relatively strong, and can cause symptoms such as cough, serous or purulent eye and nasal secretions, and bronchopneumonia; the inactivated product of the strain can produce a good immune response when immunizing mice, dogs, and cats, and has good immunogenicity. As an inactivated vaccine, it can better stimulate the body to produce high-level Elisa antibodies against Bordetella bronchiseptica, and can provide a high immune protection rate against the attack of Bordetella bronchiseptica, and has the basic potential for developing vaccines.

This strain was deposited in the China Center for Type Culture Collection on July 6, 2023, with the deposit number CCTCC NO: M 20231122, and the deposit address is Wuhan University, Wuhan, China.

Example 5 Preparation and testing of inactivated vaccines

1. Preparation of Bacterial Liquid

The A1218 strain was inoculated into TSA medium containing 4% newborn calf serum and cultured at 37°C for 24 hours. A single colony was picked and transferred for purification culture once. Then a single colony was picked and inoculated into TSB liquid medium containing 4% newborn calf serum and cultured at 37°C and 200 rpm for 16 hours as seed solution. The seed solution was transferred to TSB liquid medium containing 4% newborn calf serum at a ratio of 1:100, and cultured at 37°C for 12 hours under shaking to obtain a bacterial solution.

2. Inactivation of bacterial solution

β-propiolactone was added to the bacterial solution of Bordetella bronchiseptica A1218 at a final concentration of 0.1%, 0.2% and 0.3%, respectively, and placed on a 4°C refrigerator rotator at 60 rpm for 12h, 24h and 36h respectively; after inactivation, it was hydrolyzed in a 37°C water bath for 2h to avoid β-propiolactone residue. The inactivated bacterial solution was inoculated in TSB liquid medium containing 4% bovine serum and TSA solid medium containing 5% bovine serum, respectively, and cultured at 37°C for 24h to test the inactivation effect (Table 3).

Table 3 Inactivation of bacterial liquid

Note: “+” represents successful inactivation, and “-” represents unsuccessful inactivation.

It can be seen from Table 3 that when the final concentration of β-propiolactone is 0.125% and the inactivation time is 36h, the inactivation effect of the bacterial solution is the best.

3. Vaccine preparation and testing

The inactivated and qualified bacterial solution was added with MONTANIDE ^TM GEL-02 adjuvant purchased from SEPPIC, France, according to the instructions for use of the adjuvant to prepare the inactivated vaccine. Fifteen healthy susceptible dogs and cats aged 4 to 6 weeks with negative serum IgG antibodies and nasopharyngeal swab antigens were selected and divided into three groups. The first group was the overdose group, and each test dog or cat was subcutaneously injected with 2 doses of A1218 inactivated vaccine in the neck; the second group was the immune control group, and each test dog or cat was subcutaneously injected with 1 dose of A1218 inactivated vaccine in the neck; the third group was the blank control group, inoculated with the same amount of PBS, and clinically observed for 14 days and recorded body temperature. All were 5/5 healthy and no adverse reactions were observed (Figures 9-10).

The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims (6)

1. A strain of Bordetella bronchiseptica A1218, characterized in that it was deposited in the China Center for Type Culture Collection on July 6, 2023, with a deposit number of CCTCC NO: M 20231122. 2. Use of Bordetella bronchiseptica A1218 as claimed in claim 1 in the preparation of a Bordetella bronchiseptica vaccine, wherein the Bordetella bronchiseptica A1218 is inactivated at 4° C. for 36 hours under the condition that the final concentration volume fraction of β-propiolactone is 0.125%. 3. The use according to claim 2, characterized in that the Bordetella bronchiseptica vaccine is a universal vaccine for dogs and cats. 4. A universal inactivated Bordetella bronchiseptica vaccine for dogs and cats, characterized in that it comprises inactivated Bordetella bronchiseptica A1218, the deposit number of the Bordetella bronchiseptica A1218 is CCTCC NO: M 20231122, and the Bordetella bronchiseptica A1218 is inactivated at 4°C for 36 hours under the condition that the final concentration volume fraction of β-propiolactone is 0.125%. 5. A method for preparing a universal Bordetella bronchiseptica inactivated vaccine for dogs and cats, characterized in that it comprises the steps of preparing a bacterial solution of Bordetella bronchiseptica A1218 according to claim 1, inactivating the bacterial solution and then adding an adjuvant to prepare the vaccine; The bacterial solution was inactivated at 4° C. for 36 h under the condition that the final volume fraction of β-propiolactone was 0.125%. 6. The preparation method according to claim 5, characterized in that the bacterial liquid is obtained by culturing the Bordetella bronchiseptica A1218 in a TSA medium containing newborn calf serum, and the volume fraction of the newborn calf serum in the TSA medium is 4%.