A vaccine combination of a weakly virulent strain of Bordetella bronchiseptica for dogs and its preparation method

Abstract

The attenuated strain of Bordetella bronchiseptica (YZ01-P strain) of the present invention is genetically homologous to the pathogenic Bordetella bronchiseptica prevalent in China. After immunizing animals with the vaccine composition prepared from the attenuated strain, it can induce the animal body to rapidly produce good mucosal immunity, and its immunological efficacy can be maintained for a long time. During the period of maintaining immunological efficacy, it can effectively prevent the occurrence of canine infectious respiratory diseases caused by infection with virulent strains of Bordetella bronchiseptica. At the same time, it can also be reasonably inferred that it can greatly reduce the occurrence of canine infectious diseases characterized by respiratory diseases caused by the mixed infection of virulent strains of Bordetella bronchiseptica with canine distemper, canine parainfluenza and canine adenovirus.

Description

Technical Field

[0001] The present invention relates to a vaccine combination of a weakly virulent strain of Bordetella bronchiseptica for dogs and a preparation method thereof, belonging to the technical field of veterinary biologics. Technical Background

[0002] Canine bordetellosis is an infectious respiratory disease caused by Canine Bordetella Bronchiseptica (CBb) infecting dogs. Bordetella bronchiseptica belongs to the genus Bordetella, is Gram-negative, and is an aerobic bacterium. CBb was first isolated from the respiratory tract of dogs suffering from canine distemper by Ferry in 1910. Subsequently, he isolated pathogenic bacteria with consistent characteristics from the respiratory tracts of guinea pigs, monkeys, and humans in 1912 and 1913 respectively (Ferry, N.S. A preliminary report of the bacterial findings in canine distemper. American Veterinary Review 37:499-504. 1010; Ferry, N.S. Further studies on the Bacillus bronchicanis, the cause of canine distemper. American Veterinary Review 41:77-79. 1912).

[0003] Bordetella bronchiseptica (Bb), Bordetella pertussis, and Bordetella parapertussis belong to a group and are pathogenic bacteria that can infect various animals and cause respiratory-related diseases. Bordetella bronchiseptica is only pathogenic to mammals and occasionally infects humans. Porcine Bb can co-infect with Pasteurella multocida in pigs, resulting in atrophic rhinitis in pigs, which seriously endangers the health of the pig herd. Rabbit-derived Bb infection in rabbits can cause rabbit bordetellosis, which is extremely likely to break out at the turn of autumn and winter when the climate suddenly changes. Canine bordetellosis is an upper respiratory tract infection disease mainly characterized by coughing and bronchopneumonia, and it can be co-infected with other canine viral pathogens, such as canine adenovirus, canine parainfluenza, and canine distemper. The incidence of CBb is high and the transmission is rapid, and in severe cases, it can also cause the death of infected dogs. Whether it is pigs, rabbits, or dogs, a large amount of Bb bacteria are contained in the nasal secretions of diseased animals, which can contaminate feed, drinking water, cages, and air with cough and sneeze droplets, thus infecting other susceptible animals.

[0004] In China, around 2002, He Xingliang et al. reported on multiple occasions the isolation of Bordetella bronchiseptica from the upper respiratory tract of coughing dogs, proving that Bordetella bronchiseptica is widely prevalent in China and causing great harm to the health of dogs. At the same time, after sequencing and analyzing some antigen gene sequences of the CBb isolates, it was found that the Bordetella bronchiseptica in China has 99% similarity with the reference strains reported abroad and also 99% homology with the sequences of Bordetella bronchiseptica from rabbits (He Xingliang. Isolation and identification of Bordetella bronchiseptica. Animal Husbandry & Veterinary Medicine, Vol. 36, No. 12, 2004; Bai Xue. Isolation, identification of Bordetella bronchiseptica and cloning, expression of fimN gene and immunogenicity analysis of the expressed protein. Master's thesis of Nanjing Agricultural University, 2006).

[0005] According to foreign experience, vaccines are the main measures for preventing and controlling this disease, but currently in China, there is no commercial Bordetella bronchiseptica vaccine on the market for sale and use. Pfizer applied for a patent in China in 2006, "Canine vaccine against Bordetella bronchiseptica", with the patent publication number CN1835767A. It is a subunit vaccine prepared by mixing the p68 recombinant protein antigen of Bordetella bronchiseptica with a veterinarian-acceptable carrier (such as an adjuvant). After injection, the immune protection efficacy induced in dogs cannot meet clinical requirements: after two inoculations of experimental dogs with 15 micrograms (the optimal dose in the above patent literature) of the p68 vaccine and then a virulent challenge on the 25th day, the protection rate is only 20% (coughing continuously for 2 days is judged as non-protected). Therefore, there is an urgent need to develop an attenuated live vaccine of Bordetella bronchiseptica that matches the Bordetella bronchiseptica prevalent in China for the prevention and control of Bordetella bronchiseptica disease. Summary of the Invention

[0006] The purpose of the present invention is to solve the deficiencies of the existing technology by providing an attenuated strain of Bordetella bronchiseptica (YZ01-P strain) that is antigenically matched to the prevalent bacteria in China and has good safety and immunogenicity, as well as an attenuated live vaccine prepared using this attenuated bacterium for the prevention and control of canine infectious respiratory diseases caused by Bordetella bronchiseptica infection.

[0007] Technical Solution of the Present Invention

[0008] 1. An attenuated live vaccine combination for Bordetella bronchiseptica disease, characterized in that the vaccine combination contains the attenuated strain YZ01-P that is antigenically matched to the Bordetella bronchiseptica prevalent in China;

[0009] The total genomic sequence of the attenuated strain YZ01-P is 5,339,763 bp, encoding 5,039 genes in total, and the CG content of the genome is 68%. Compared with other publicly available Bordetella bronchiseptica strains of dogs, YZ01-P has two molecular genetic markers: (1) an 8-base (CGCCGAGG) insertion at positions 2,955,623-2,955,631 of the genome; (2) a 75,606-bp plasmid;

[0010] This strain was deposited at the General Microbiology Center of the China Microbial Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on December 26, 2022, and the deposit number is: CGMCC No. 26296.

[0011] 2. The preparation process of the live attenuated vaccine against Bordetella bronchiseptica in dogs of the present invention includes: culturing and proliferating attenuated bacteria, measuring the antigen content, adding a pharmaceutically acceptable carrier, freeze-drying after sub-packaging, and obtaining the product after vacuum capping.

[0012] Positive effects of the present invention

[0013] The attenuated strain of Bordetella bronchiseptica in dogs (YZ01-P strain) of the present invention is genetically homologous to the pathogenic Bordetella bronchiseptica in dogs prevalent in China. After immunizing animals with the vaccine composition prepared from this attenuated strain, it can induce the animal body to rapidly produce good mucosal immunity, and its immune efficacy can be maintained for a long time. During the immune efficacy maintenance period, it can effectively prevent the occurrence of canine infectious respiratory diseases caused by the infection of virulent strains of Bordetella bronchiseptica in dogs. At the same time, it can also be reasonably inferred that it can greatly reduce the occurrence of canine infectious diseases characterized by respiratory diseases caused by the mixed infection of virulent strains of Bordetella bronchiseptica with canine distemper, canine parainfluenza and canine adenovirus.

[0014] The present invention relates to microbial resource information

[0015] The microbial resource involved in the present invention is the attenuated strain YZ01-P of Bordetella bronchiseptica in dogs. This strain was obtained by the inventor through selection, domestication and purification of not less than 5 generations from the swab sample mixture of various pet dogs in the field. This strain was deposited at the General Microbiology Center of the China Microbial Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on December 26, 2022, and the deposit number is: CGMCC No. 26296. Description of the drawings

[0016] Figure 1PCR electrophoresis pattern of fimN gene of each passage of attenuated strain YZ01-P of Bordetella bronchiseptica in dogs. In the figure: M: DL2000 DNA Marker; 1-23: YZ01-P3-P25; N: negative control

[0017] Figure 2 Whole-genome sequencing map of YZ01-P

[0018] Figure 3 Whole-genome sequencing map of plasmid of YZ01-P16

[0019] Specific implementation manner of the invention

[0020] 1. Domestication and breeding of strain YZ01-P

[0021] The microbial resource involved in the present invention is attenuated strain YZ01-P of Bordetella bronchiseptica in dogs, which was obtained by the inventor through selection, domestication and purification of no less than 5 passages from the swab sample mixture of various pet dogs in the field. The process of domestication and breeding is briefly described as follows: in the animal hospital, various swabs and tissue and organ samples were collected from various pet dogs. The samples (or preserved samples) were directly spread on the modified MacConkey culture dish and cultured in an incubator at 37°C for 36-48 hours. After that, the colonies were observed visually, and the colonies that met the growth morphology of Bordetella bronchiseptica were selected for pure culture and identification, and the naturally isolated strain was named YZ01-O strain.

[0022] Using the modified MacConkey medium and culture method, YZ01-O strain was repeatedly domesticated, selected, monoclonal isolated, purified and identified for no less than 5 passages. Finally, a culture with uniform colony morphology, moderate colony size and dewdrop-like white colonies was established and named YZ01-P strain. YZ01-P strain was identified as Bordetella bronchiseptica by Gram staining and using specific PCR of Bordetella bronchiseptica in dogs. Using YZ01-P strain for animal regression test, it was found that YZ01-P strain was a non-pathogenic attenuated strain. YZ01-P was amplified and cultured using a conventional bacterial medium and serially passaged to the 25th generation, and its colony morphology, size, growth rate and FmN gene sequence all showed a stable state.

[0023] The whole genome of the representative strain of YZ01-P on the common bacterial medium was sequenced. The result showed that its whole genome sequence was 5339763 bp, encoding a total of 5039 genes, and the CG content of the genome was 68%. Compared with the whole sequences of other publicly sequenced isolates and YZ01-O, YZ01-P had two molecular genetic markers: (1) an 8-base (CGCCGAGG) insertion at positions 2955623-2955631 of the genome; (2) a 75606-bp plasmid.

[0024] 2. Preparation of Attenuated Live Vaccine

[0025] The vaccine involved in the present invention is an attenuated live vaccine against canine Bordetella bronchiseptica, which is prepared using the attenuated strain of Bordetella bronchiseptica (YZ01-P strain) established by the present inventors through domestication and breeding. The preparation process is as follows: The seed of the attenuated strain of Bordetella bronchiseptica is resuscitated and cultured in a small volume of bacterial medium in a shaker at 37 °C and 100 - 300 rpm for 8 - 24 hours, then amplified and cultured by 50 - 200 times, and then transferred to a 10 - 100 L fermenter at a ratio of 5 - 50 times for high-density bacterial fermentation culture. The fermentation culture conditions are 37 °C, the rotation speed is 100 - 500 rpm, and the culture time is generally 6 - 18 hours. After the fermentation culture broth is measured for the content of bacterial antigen, it is mixed with an appropriate amount of freeze-drying protectant, sub-packed, and finally freeze-dried.

[0026] 3. Inspection of Attenuated Live Vaccine

[0027] Safety and efficacy tests on experimental dogs were conducted on the attenuated live vaccine against canine Bordetella bronchiseptica involved in the present invention.

[0028] (1) Safety inspection of the vaccine: Five experimental dogs were inoculated with 10-fold dose of the attenuated live vaccine, and five control groups were inoculated with PBS. After observing for 14 days, no abnormal reactions were found in all vaccine-inoculated animals and control group animals. It was proved that the attenuated live vaccine is safe.

[0029] (2) Efficacy inspection of the vaccine: Ten test animals were inoculated with 1 dose of the attenuated live vaccine, and ten control groups were inoculated with PBS. After 14 days, the immunized group and the control group animals were challenged with virulent Bordetella bronchiseptica together and observed for 14 days. The results showed that all 10 dogs in the test group were protected and had no clinical symptoms including respiratory symptoms, while 100% of the control group animals had respiratory symptoms. It was proved that the attenuated live vaccine is effective.

[0030] Hereinafter, the terms involved in the present invention are explained

[0031] 1. The term "Bordetella bronchiseptica" (CBb) belongs to the genus Bordetella. The clinical symptoms caused include respiratory diseases characterized by coughing and death. Clinically, it can be mixed-infected with canine distemper, canine parainfluenza, and canine adenovirus, resulting in more complex clinical manifestations and severe clinical symptoms in animals, including animal death.

[0032] 2. The Bordetella bronchiseptica attenuated strain (YZ01-P strain) involved in the present invention has two molecular genetic markers compared with the YZ01-O strain and other CBb strains whose whole gene sequences have been determined and published: (1) an 8-base (CGCCGAGG) insertion at positions 2955623-2955631 of the genome; (2) a 75606-bp plasmid.

[0033] 3. The isolation method of the Bordetella bronchiseptica described in the present invention includes: taking animal organs, tissues and samples infected with Bordetella bronchiseptica for bacterial isolation;

[0034] 4. The animal organs, tissues and samples infected with Bordetella bronchiseptica described in the present invention include but are not limited to trachea, lungs, nasal secretions, etc.

[0035] 5. The term "pharmaceutically acceptable carrier" refers to all other components in the vaccine composition of the present invention except the Bordetella bronchiseptica attenuated strain YZ01-P strain antigen. The components are mainly freeze-drying protectants: dipotassium hydrogen phosphate, potassium dihydrogen phosphate, enzymatically hydrolyzed casein, sorbitol, skim milk powder, hydrolyzed lactalbumin, gelatin, sucrose, lactose, arginine.

[0036] 6. When the term "prevention and / or treatment" relates to the infection of virulent Bordetella bronchiseptica strains, it refers to inhibiting the replication of virulent Bordetella bronchiseptica strains, inhibiting the transmission of virulent Bordetella bronchiseptica strains, or preventing the colonization and reproduction of virulent Bordetella bronchiseptica in its host, as well as reducing the diseases or symptoms of dogs caused by the infection of virulent Bordetella bronchiseptica strains. Examples

[0037] The present invention will be further described below in conjunction with specific implementation examples, and the advantages and characteristics of the present invention will become clearer with the description. However, these examples are only exemplary and do not constitute any limitation to the scope of the present invention.

[0038] Example 1

[0039] Domestication, selection and establishment of the attenuated strain YZ01-P of Bordetella bronchiseptica in dogs

[0040] 1. Isolation, purification and identification of Bordetella bronchiseptica YZ01-O strain

[0041] In animal hospitals, various types of pet dogs were sampled for various swabs and tissue and organ samples. The collected samples were quickly inserted into sterile cryotubes containing preservation solution for storage, or directly spread on plates of modified MacConkey agar medium.

[0042] The stored samples were smeared on the plates of modified MacConkey agar medium and observed after culturing in a constant temperature incubator at 37°C for 36 - 48 hours. Pure cultures were made for single colonies suspected of being Bordetella bronchiseptica: Single colonies were picked and streaked onto the plates of modified MacConkey agar medium in the pure culture method. After culturing at 37°C for 48 hours, the colony morphology was observed and the colonies were identified by colony PCR. For single colonies identified as Bordetella bronchiseptica in dogs, aseptic plaque picking operations were carried out with an inoculation loop and inoculated into a liquid medium for expansion culture (in a test tube containing 5 mL of medium). At the same time, it was inoculated again onto the plates of modified MacConkey agar medium with an inoculation loop and cultured at 37°C for 48 hours. After Gram staining and specific PCR identification of Bordetella bronchiseptica in dogs, a primary isolate of Bordetella bronchiseptica in dogs was obtained and named YZ01 - O.

[0043] 2. Domestication, selection and establishment of Bordetella bronchiseptica YZ01 - P strain in dogs

[0044] Using the modified MacConkey selective medium and culture method, YZ01 - O was repeatedly domesticated, selected, purified and identified for no less than 5 generations, and a culture with uniform colony morphology, moderate colony size and dewdrop - like white colonies was obtained. Through Gram staining and systematic identification of this strain using specific PCR for Bordetella bronchiseptica in dogs, it was determined to be Bordetella bronchiseptica in dogs; when this strain was subcultured continuously for 25 generations using a common medium, the colony morphology and size, bacterial growth rate, Gram staining and FmN gene sequencing of each generation showed a stable state (see Figure 1 ). Further, an animal regression test was carried out using this strain: About 16 - week - old dogs negative for CBb antibody and antigen were inoculated through the respiratory tract with 10 9 PFU and observed for 14 days. There were no other respiratory symptoms such as coughing, so it was considered that this strain was a weakly virulent bacterium non - pathogenic to dogs. It was confirmed that a genetically stable weakly virulent strain of Bordetella bronchiseptica in dogs was obtained and named YZ01 - P strain. This bacterial solution could be stored at - 70°C for a long time after being mixed with 5 - 20% glycerol.

[0045] 3. Identification of each generation of serial passages of the weakly virulent strain Bordetella bronchiseptica YZ01 - P in dogs

[0046] (1) Gram staining identification: Drop 5 μL of the bacterial liquid to be tested on a clean glass slide and spread it evenly. Place the slide with the bacterial film facing up and quickly wave it back and forth 3 - 5 times above the flame of an alcohol lamp for fixation. Drop crystal violet on the bacterial film and stain for 1 min. After washing with water and air-drying, add iodine solution to cover the smear and stain for about 1 min. Wash with water, blot with absorbent paper to remove moisture, and dry. Add a few drops of 95% alcohol and gently shake for decolorization. Observe the fixed bacterial film on the glass slide against a white background until it decolorizes to colorless or does not decolorize. Wash with water, blot with absorbent paper to remove moisture, and dry. Counterstain: Counterstain with safranin staining solution for 1 min, then wash with water and blot to dry. Drop cedarwood oil and observe with an oil immersion lens. The result shows scattered Gram-negative bacilli.

[0047] (2) Specific PCR and sequencing identification: Refer to the method in the reference (Bai Xue. Isolation and identification of Bordetella bronchiseptica in dogs and cloning, expression and immunogenicity analysis of fimN gene. Master's thesis of Nanjing Agricultural University, 2006) to establish a specific PCR method for Bordetella bronchiseptica in dogs. Amplify and sequence the fimN gene from bacterial cultures of P3 to P25 generations. (Add the components in Table 1 below to a 0.2 mL PCR reaction tube, with a total reaction volume of 25 μL. Set up a blank control in addition to the test template.

[0048] The primer sequences are as follows:

[0049] FimN-F: gggaattcac gtgatcaccg 20 (Sequence 1)

[0050] FimN-R: ggctcgagga ttatccttat caagcc 26 (Sequence 2)

[0051] Table 1 fimN reaction system

[0052]

[0053] PCR program: After mixing the above system, perform PCR amplification. After pre-denaturation at 95°C for 3 min, perform the following cycles: denaturation at 95°C for 30 s, annealing at 56°C for 30 s; extension at 72°C for 40 s, for a total of 35 cycles. Incubate at 72°C for 10 min and store at 4°C.

[0054] After identification by 1.5% agarose gel electrophoresis, entrust the positive PCR products to Suzhou Hongxun Biotechnology Co., Ltd. for sequencing. (Attach the primers and reaction conditions for amplification). Figure 1PCR electrophoresis map of fim N gene of Bordetella bronchiseptica YZ01-P3 to P25 obtained by separation. The sequencing results are shown in Sequence 3 and Sequence 4. The analysis of the sequencing results confirmed that the homology of the fimN gene between P3 and P25 was 100%. The above results confirmed the establishment of a Bordetella bronchiseptica attenuated strain with stable genetic traits.

[0055] Sequencing results of the fimN gene of Bordetella bronchiseptica YZ01-P3 (Sequence 3) and P25 (Sequence 4).

[0056] YZ01-P3 (Sequence 3):

[0057]

[0058] YZ01-P25 (Sequence 4):

[0059]

[0060] (3) Whole-genome sequencing analysis

[0061] Using the second-generation sequencing method, the whole-genome sequencing of the 16th-generation culture of YZ01-P (YZ01-P16) was carried out. The results showed that the whole-genome sequence of YZ01-P16 was 5339763 bp, and the GC content of the whole genome was 68.71%; among them, 90.42% of the whole genome was gene coding region sequence, with a total length of 4828254 bp, encoding a total of 5039 genes, and the average gene length was 958 bp; the total length of the intergenic sequence was 511509 bp, accounting for 9.58% of the total genes, and the GC content was 62.13%. The whole-genome map is shown in Figure 2 .

[0062] Comparative genomic analysis was carried out on the obtained whole sequence of YZ01-P16 with the whole-genome sequences of the original isolate YZ01-O and other Bordetella bronchiseptica published by NCBI. It was found that YZ01-P16 had two molecular genetic markers: (1) At positions 2955623-2955631 of the genome, there was an insertion of 8 bases (CGCCGAGG); (2) There was a 75606-bp plasmid (see Figure 3 ).

[0063] (4) Viable antigen content in each generation of Bordetella bronchiseptica YZ01-P culture

[0064] The viable bacteria content in each generation of YZ01-P culture was determined by the viable bacteria counting method (Appendix 3405 of the Chinese Veterinary Pharmacopoeia 2020 Edition). The results showed that the viable bacteria quantity in each generation of YZ01-P culture could reach 1×10 9 CFU / mL.

[0065] Example 2

[0066] Preparation of the live attenuated vaccine against Bordetella bronchiseptica in dogs

[0067] Take 1 mL of YZ01-P seeds and resuscitate and culture them in a small volume of bacterial medium in a shaker at 37 °C and 100 - 300 rpm for 8 - 24 hours. After amplification culture at a ratio of 50 - 200 times, transfer them to a 10 - 100 L fermenter at a ratio of 5 - 50 times for high-density bacterial fermentation culture. The fermentation culture conditions are 37 °C, the rotation speed is 100 - 500 rpm, and the culture time is generally 6 - 18 hours. The OD 600 generally ranges between 2 and 10. After measuring the bacterial content of the fermentation broth, dilute it according to the formula to obtain bacterial suspensions with different bacterial contents, mix them with the lyophilization protectant, dispense them into vials, and quickly perform lyophilization treatment. After vacuum capping the samples, the vaccine composition is obtained. For the vaccine composition obtained after lyophilization, perform content and purity tests (the methods refer to Appendix 3405 and 3306 of the Chinese Veterinary Pharmacopoeia 2020 Edition). The results prove that the antigen content of the vaccine composition is qualified (not less than 10 6.5 CFU / ml / dose), and it has good purity and no contamination by other miscellaneous bacteria. The inventor continuously prepared 5 - 10 batches of experimental products, and selected one batch of products with a high dose (10 9.5 CFU / ml / dose) for safety testing, and selected one batch of products with a medium dose (10 7.4 CFU / ml / dose) for potency testing.

[0068] Example 3

[0069] Inspection of the live attenuated vaccine against Bordetella bronchiseptica in dogs

[0070] 1. Safety Test of the Attenuated Live Vaccine of Bordetella bronchiseptica for Dogs

[0071] For the safety tests of single-dose and 10-fold overdose vaccinations, 15 healthy and susceptible dogs around 2 months old were randomly divided into 3 groups, with 5 dogs in each group. Among them, group 1 was vaccinated with 1 mL of the attenuated live vaccine of Bordetella bronchiseptica for dogs, containing 10 8.5 CFU per dog; group 2 was given a 10-fold overdose vaccination, vaccinated with 4 mL of the attenuated live vaccine of Bordetella bronchiseptica for dogs, containing 10 9.5CFU per dog; Group 3 was the blank control group, and each dog was inoculated with 1 mL of PBS. After inoculation, the dogs were raised under the same conditions and observed for 14 days. Every day, the drinking water, appetite, body temperature, eyes and nose of the dogs were observed for normality, and whether there were any adverse reactions throughout the body, and whether there were coughs or other respiratory symptoms in the clinical manifestations. At the end of the observation period, the test dogs in the single-dose and 10-fold dose test groups and the control group all had good mental states, normal drinking water and appetite, normal body temperature, no eye or nose secretions, no coughs and other respiratory symptoms, no adverse reactions were seen throughout the body, and all dogs were healthy and alive (the results are shown in Table 2).

[0072] Table 2 Safety test results of attenuated live Bordetella bronchiseptica vaccine for dogs with single-dose and overdose inoculations

[0073]

[0074] The experimental results showed that it was safe to inoculate the attenuated live Bordetella bronchiseptica vaccine in 2-month-old dogs.

[0075] 2. Efficacy test of attenuated live Bordetella bronchiseptica vaccine

[0076] Twenty healthy and susceptible dogs around 2 months old were randomly divided into 2 groups, with 10 dogs in each group. Among them, Group 1 was inoculated with 1 mL of attenuated live Bordetella bronchiseptica vaccine, and the live bacteria content in the vaccine was 10 7.4 CFU / mL. The second group was the blank control group, inoculated with 1 mL of PBS. After inoculation, the dogs were raised under the same conditions and observed for 14 days. It was found that the drinking water, appetite, body temperature, eyes and nose of all test dogs were normal, there were no adverse reactions throughout the body, and there were no coughs or other respiratory symptoms in the clinical manifestations. On the 14th day after immunization, all test dogs were challenged with a virulent strain of Bordetella bronchiseptica. After challenge, the drinking water, appetite, body temperature, eyes and nose of all test dogs were observed every day for normality, whether there were any adverse reactions throughout the body, and whether there were coughs or deaths in the clinical manifestations. The results are shown in Table 3.

[0077] Table 3 Efficacy test results of attenuated live Bordetella bronchiseptica vaccine

[0078] Grouping Number of animals Dose of inoculation Number of diseased animals Number of dead animals Protection rate Vaccine 10 1 ml / animal 0 / 10 0 / 10 100％ Blank control (PBS) 10 1 ml / animal 10 / 10 0 / 10 0％

[0079] The results showed that 14 days after immunizing healthy and susceptible dogs around 2 months old with the attenuated live Bordetella bronchiseptica vaccine and then challenging them with a virulent strain of Bordetella bronchiseptica, 100% protection could be provided for the dogs. This vaccine could induce good immune protection in animals and had good protective effects against virulent challenge.

[0080] It can be seen from Table 2 and Table 3 that it is safe and effective to inoculate the attenuated live Bordetella bronchiseptica vaccine (YZ01-P strain) in healthy and susceptible dogs around 2 months old.

[0081] The above are only the preferred embodiments of the present invention, and do not impose any formal limitations on the present invention. Although the present invention has been disclosed above with the preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can, within the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments with the same number by using the above-disclosed technical content. However, as long as it does not depart from the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A live attenuated vaccine against Bordetella bronchiseptica in dogs, characterized in that, The vaccine contains the attenuated strain YZ01-P of Bordetella bronchiseptica that is antigenically matched to the strains prevalent in China; the attenuated strain YZ01-P was deposited with the General Microbiology Center of the China Microbial Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on December 26, 2022, and the deposit number is: CGMCC No. 26296.

2. The live attenuated vaccine according to claim 1, which further contains a lyophilization protectant, and the lyophilization protectant contains one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, enzymatically hydrolyzed casein, sorbitol, skim milk powder, hydrolyzed whey protein, gelatin, sucrose, lactose, and arginine.

3. A method for preparing a live attenuated vaccine against Bordetella bronchiseptica in dogs as claimed in claim 1 or 2, characterized in that, The preparation process includes culturing and proliferating attenuated bacteria, measuring the antigen content, adding a pharmaceutically acceptable carrier, freeze-drying after sub-packaging, and obtaining the product after vacuum capping.

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